KR100736209B1 - Method for improving the introduction of the target gene into plant cell - Google Patents

Abstract

The present invention relates to a method for transforming a gene into a plant, and to a plant manufactured using the same, and more particularly, to inoculate Agrobacterium into a target tissue capable of transformation by plant, and then to include the recombinant plasmid in Agrobacterium. The present invention relates to a method for enhancing transformation efficiency by newly improving medium composition and conditions in a co-culture process including introducing a target gene into plant cells. In addition, by using the improved transformation method of the present invention, it is possible to produce a transformed plant for a rice genotype that is difficult to transform.

Rice, Agrobacterium, coculture, medium composition and condition, transgenic plant

Description

Method for improving the introduction of the target gene into plant cell}

1 is a diagram showing the main part of the transformant carrier developed for the transformation of plants and their identification.

Figure 2 is a photograph showing the results of transforming a carrier for transformation used in the present invention in a three-base hybridization method Agrobacterium and performing colony PCR.

Figure 3 is a photograph showing the degree of expression of the GUS gene after inoculation to rice callus Agrobacterium in accordance with the method according to the present invention compared with the conventional method.

4 is a photograph showing the difference in the transformation efficiency between the method according to the present invention and the conventional method and the transformation efficiency when the method according to the present invention is applied to actual rice transformation in the regeneration step.

Figure 5 is a photograph showing the results of nucleic acid amplification reaction (PCR) of the GUS gene inserted into the genome in the transformed rice induced by the present invention and the conventional method.

Figure 6 is a photograph showing the results of the GUS assay on the leaves of the transgenic rice induced by the present invention and the conventional method.

Figure 7 is a photograph showing the resistance to herbicides when treated twice with 0.3% of the Basta Transformed rice produced by the method according to the present invention.

The present invention relates to a method for transforming a gene into a plant, and to a plant manufactured using the same, and more particularly, to inoculate Agrobacterium into a target tissue capable of transformation by plant, and then to include the recombinant plasmid in Agrobacterium. The present invention relates to a method for enhancing transformation efficiency by newly improving medium composition and conditions in a co-culture process including introducing a target gene into plant cells. In addition, by using the improved transformation method of the present invention, it is possible to produce a transformed plant for a rice genotype that is difficult to transform.

Rice is a very important crop that is used as a staple food for people in Asia. It is becoming increasingly important as it is used as a model plant to study the function of genes or to investigate the gene's action on monocots. The development of new varieties, such as varieties with improved herbicide resistance, disaster or disease resistance, is very important in terms of increased cultivation stability and supply of high value-added agricultural products. Therefore, recent efforts to develop new varieties with new traits by applying genetic engineering molecular breeding methods are actively underway.

Genetic molecular breeding method has the advantage of being able to directly introduce useful genes while maintaining the genetic trait of superior breeds compared to the traditional breeding method, and the advantage of being able to obtain useful genes from various species and transplant them into plants. have. Successful application of genetically engineered molecular breeding methods to plants requires three steps: a method of effectively organizing transformable plant tissues; Successfully transforming a gene of interest in plant cells; And methods for inducing regenerating plants from transformed cells.

 There have been several reports of successful transformation of plant genes into plant cells (Hansen and Wright, Trends Plant Sci. 4: 226-231, 1999), but recently, microparticle projection and Agrobacte The method using alium (Agrobacterium tumefaciens) is the most commonly used.

The microparticle projection method is a method of directly transferring DNA coated on microparticles to plant cells by using a gene gun, which has a large number of DNA copies inserted into plant cells, the complexity of the inserted pattern, and the DNA inserted therein. Such as transition to form (De block M. Euphytica. 71: 1-14, 1993). Due to these problems, a method of using Agrobacterium has been mainly used as an efficient plant transformation method in recent years.

Transformation method using Agrobacterium is characterized in that the T-DNA region on the Ti (tumor-inducing) plasmid or Ri (Root-inducing) plasmid possessed by the plant pathogen Agrobacterium is transferred to plant cells. The process is largely made up of four steps. Inoculation process involves contacting plant cells with Agrobacterium containing a recombinant plasmid containing the target gene, and Agrobacterium and plant cells are cultured together for several hours to several days to transfer T-DNA. Co-culture process, selection and propagation of transformed cells in a medium containing a selection marker, and finally inducing re-differentiated plants from the selected cells.

Among the four steps of the above-mentioned transformation in rice transformation, a method of improving plant transformation efficiency by improving the co-culture process with a new method is a method of inhibiting the growth of Agrobacterium (WO 01/09302 A2). For example, the method of minimizing the oxidative burst of plant cells in the co-culture process (Dey et al., Rice Genetics Newsletter 19: 82-84) is an example. International Publication No. WO 01/09302 A2 discloses the characteristics of rice, wheat and corn by suppressing the growth rate of Agrobacterium by adding one or more substances that inhibit the growth of Agrobacterium to the inoculation medium and the coculture medium. It is described that it is possible to improve the conversion efficiency and reduce the number of copies of the inserted gene. Dey et al. Reported a 30-40% increase in transformation efficiency when two antioxidants (L-cysteine, ascorbic acid) and silver nitrate were added to the co-culture medium.

The present inventors have studied the method of taking and improving the advantages of the two methods, and in addition to the addition of Agrobacterium growth inhibitory substance to the co-media, the combination of alternating temperature and filtering treatment, without co-growth of Agrobacterium In addition, it was possible to control the culture period according to the breeding conditions. In addition, by adding three antioxidants to the co-culture medium, the plant transformation efficiency was dramatically increased by minimizing the oxidative burst of plant cells. By applying this method, it was found that the development of a transgenic plant is possible even in a difficult transgenic breed, and completed the present invention.

Accordingly, it is an object of the present invention to provide an efficient method for transforming a plant using Agrobacterium and a transgenic plant obtained by the method.

In order to achieve the above object, the present invention comprises the steps of (A) preparing a plant transformation expression vector comprising a foreign gene of interest and selection factors and introducing the completed recombinant plasmid into Agrobacterium; (B) co-culturing the transformed Agrobacterium with the desired tissue of the plant; (C) selecting and propagating the tissue of the co-cultivated plant in the selection medium; And (D) regenerating the selected tissues and purifying the produced plants.

In addition, the present invention provides a transformed plant produced using the plant transformation method.

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

The present invention provides a method for effectively transforming a gene of interest in cells and tissues of a plant and a plant transformed by the method, wherein the foreign gene of interest in step (A) is a plant, an animal, a microorganism, or the like. All genes can be used, including genes from other species and artificially synthesized genes.

As a selection factor for effectively selecting the plant to which the target gene is introduced, antibiotic resistance genes such as NPTII (neomycin phosphotransferase), HPT (hygromycin phosphotransferase), CAT (chloramphenicol acetyltransferase) and gentamicin (gentamicin); herbicide resistance genes such as bar gene; And marker genes such as GUS, GFP (green fluorescent protein) and LUX (luciferase).

The expression vector for transformation to implement the gene of interest in the plant is a promoter for producing the RNA nucleotide sequence of the gene of interest in the plant cell, the structural DNA sequence having the information of the gene of interest and the RNA in the plant cell. At least one structure consisting of a 3 'non-translated DNA nucleotide sequence that functions to produce a polyadenylated nucleotide (polyadenylated nucleotide) at the 3' end of the base sequence.

In the present invention, the promoter used for the expression may be used for all kinds of functions in the plant cell, and here the promoter to be expressed at a certain time during the plant growth period, expressing only in specific parts of the plant tissue Hybrid promoters in which 'enhancer sequences' are combined to increase the expression of promoters and genes of interest are included.

In the present invention, the normally expressed OsCc1 promoter isolated from rice (Jang et al., Plant Physiol. 129: 1473-1481, 2002) and the globulin promoter for expressing the gene of interest at the time of seed development were mainly used. The type and production method of the recombinant plasmid used in the present invention will be described in more detail in Example 1.

Agrobacterium, which is used to introduce one or more genes of interest into plant cells, has disarmed Ti or Ri plasmids, which are octopine-type LBA4404 or succinamopine- All Agrobacterium strains such as the EHA101 or EHA105 strains can be used in the present invention.

The target tissue in the step (B) is monocotyledonous plants such as rice, corn, wheat and barley; And all tissues of dicotyledonous plants such as soybean, cotton, rapeseed and sunflower, and meristems such as callus, immature embryos, embryos, apex and cotyledon nodes, which are artificially induced, And stems may be used as the target tissue. In the embodiment of the present invention, callus derived from seed cultured in a dark room at 27 ° C. for 3-4 weeks was used as a target tissue.

More specific examples of the transforming plant used as the target tissue in the present invention include food crops such as rice, soybeans, barley, corn, potatoes, wheat, red beans, oats and sorghum; Vegetable crops such as tomatoes, peppers, strawberries, green onions, onions, carrots, cabbages, radishes, watermelons, cucumbers, cabbages, and pumpkins; Special crops such as ginseng, sesame, perilla, peanut, rapeseed, cotton, sugar cane, sugar beet and tobacco; Fruit trees and flower plants such as apple trees, pear trees, jujube trees, grapes, citrus fruits, persimmons, apricots, bananas, leeks, roses, chrysanthemums, lilies, gerberas, carnations, freesia, gladiolus, and tulips; Fodder crops such as orchardgrass, alfalfa, tall pescue, red clover and lygras; And other plants such as Arabidopsis.

In the present invention, Agrobacterium and plant cells are cultured together for several hours to several days to co-culture (Co-culture) process to make the transfer of T-DNA by a new method, including the growth of Agrobacterium, Optimization conditions for the stable transfer of T-DNA into plant cells, including methods for inhibiting ethylene activity and minimizing oxidative destruction of plant cells, were included.

At this time, the medium used for plant culture such as MS basic medium and N6 basic medium may be used, and additionally plant growth hormone, vitamins, amino acids, iron and trace elements, including 2,4-D, may be added. have. In addition, a treatment for covering one or more sheets of filter paper on the co-culture medium may be included. As a gelling agent, agar or gelrite (phytagel) may be used. In the embodiment of the present invention, as the co-culture medium, 1/2 N6 medium was used, and the amount of gelite (gelrite; Duchefa, cat. No. G1101) added was 2.6 to 5 g / L, and most preferably 3.0. g / L, filter paper was placed on the finished medium and the target tissue of the plant inoculated with Agrobacterium was placed thereon.

Agrobacterium growth and inhibition of ethylene activity may include a method of treating heavy metals such as silver nitrate in a co-culture medium, and the amount of silver nitrate is preferably about 1 to 10 mg / L based on the composition of the medium. Do.

The method of minimizing the oxidative destruction of plant cells may include the process of adding one or more antioxidant compounds such as L-cysteine, sodium thiosulfate and dithiothreitol in the co-culture. . The content of the antioxidant compound is preferably 5-50 mg / L of L-cysteine, 0.1-5 mM of sodium thiosulfate and 0.1-5 mM of dithiothreitol with respect to the medium composition.

Treatment temperature in the co-cultivation process was changed to 3 ℃ difference in the range of 23 ~ 28 ℃, first treated at 26 ~ 28 ℃ temperature and then changed to a temperature of 23 ~ 25 ℃ was treated for the remaining period. The co-culturing period can be treated by controlling by crop or variety, in the embodiment of the present invention 26.5 ℃ (1 day) → 23.5 ℃ (4 days-8 days) 5 days or more.

In the re-differentiated plant induction step (D), 0.1 to 5 mg / L NAphthalene Acetic Acid (NAA), 1 to 10 mg / L kinetine, 20 to 40 g / L sorbitol, and 10 to 30 g / L maltose, etc. Medium composition.

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, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention to those skilled in the art. Will be self-evident.

In the examples, the herbicide Bassta resistant bar gene was used as a foreign target gene for convenience, but it will be apparent to those skilled in the art that it is possible to apply various foreign target genes in the same manner.

Example 1 Preparation of Carrier for Transformation and Introduction to Agrobacterium

In order to compare the plant transformation efficiency of the method according to the present invention and the conventional method, four kinds of carriers including GUS expression carriers were prepared, and each of them was introduced into Agrobacterium.

The GUS gene containing intron was cloned and used in pCAMBIA 3301 vector, and the isolated gene was used as a pSB11 vector (Komari et al., Plant J. 10: Gateway system; Invitrogen, Carlsbad, CA, USA). 165-174, 1996). The transformation carrier confirmed the introduction of the gene was named pMJC-GB-GUS and was used to compare the difference between the method included in the present invention and the conventional method in the stage of plant transformation.

As the main vector, pSB11 binary vector was used, and the constant expression OsCc1 promoter isolated from rice (Jang et al., Plant Physiol. 129: 1473-1481, 2002) was used for expression of GUS gene. For selection of transformed callus and plant, Bara (Bialaphos resistance) gene controlled by CaMV 35S promoter was used, and the matrix attachment region (MAR) was added inside both borders to induce stable expression of the gene. The major gene sequence of the recombinant plasmid pMJC-GB-GUS is shown in FIG. 1. In FIG. 1, the abbreviation of each code is as follows: left border (LB), right border (RB), OsCc1 promoter (PCytC), GUS coding region with intron insertion (Intron-GUS), Pin-II terminator (TPinll), CaMV 35S promoter (P35S), Nos terminator (TNos), Bar ( bar coding region), Matrix attachment region (MAR).

In order to determine whether the method according to the present invention can be efficiently applied in actual rice transformation, two plasmids of isoflavone biosynthesis genes IFS1 (Isoflavone synthase 1) and IFS2 (Isoflavone synthase 2) and the disease-associated gene Harpin were recombined. Was produced. The preparation of the carrier was carried out in the same manner as in the GUS expressing carrier, and the promoters used were the IFS1 and IFS2 carriers, the globulin promoter strongly expressed in the seed, and the morphine carrier was the same OsCc1 promoter as the GUS carrier. To be expressed.

Transformed binary vector DNA, ie pMJC-GB-GUS, pMJC-GGB-IFS1, pMJC-GGB-IFS2 and pMJC-GB-Haffin, was isolated by Agrobacterium LBA4404 strain by trialal mating. Two antibiotics, specinomycin and tetracycline, were used to select Agrobacterium containing the recombinant plasmid. Transformation into Agrobacterium was confirmed by colony PCR.

PCR reactions were performed with 1X f-Taq buffer (Solgent, Daejeon, Korea), 50 μM of each dNTP, 1X band doctor (Solgent, Daejeon, Korea), 1.5 units of f-Taq DNA polymerase (Solgent, Daejeon, Korea) and each 25 pM of primer was added and a portion of the colonies transformed with each carrier was added to the PCR reaction solution in which the final volume was adjusted to 25 μl with sterile water. The PCR reaction was performed by MJ Research's PCT-200 instrument, and the conditions were modified for 4 minutes at 94 ° C, followed by denaturation at 94 ° C for 15 seconds, polymerization at 55 ° C for 30 seconds, and extension at 72 ° C for 60 seconds. The reaction was programmed to elongate at 72 ° C. for 7 minutes after 30 iterations. The amplified product was separated from 0.8% agar gel added with EtBr and confirmed by UV transilluminator.

Figure 2 is a photograph showing the results of transforming Agrobacterium and colony PCR using the transformation carrier pMJC-GGB-IFS2 used in the present invention in a three-step hybridization method. All 10 colonies selected showed bands at the 1949bp position, which is the expected size of the primers used, indicating that the IFS2 gene was stably transformed into Agrobacterium. In Figure 2 1-10 shows transformed colonies.

Agrobacterium colonies confirmed the transformation by the above method was used for transformation after propagation.

Example 2 Callus Induction and Agrobacterium Culture for Transformation

Callus was induced and the Agrobacterium was cultured for the introduction of the target gene into the plant.

2-1. Callus Induction and Selection

For the varieties, two varieties, black rice and ilmi rice, which are known to be transformed, and one rice, which is difficult to transform, were used. First, the brown rice with the seed shells removed was washed for 5 minutes in 70% ethanol and then sterilized twice at room temperature for 15 minutes with 50% lacx solution and then sterilized by washing at least 10 times with sterile water. Sterilized seeds were incubated in a 2N6 medium containing 2,4-D 2 mg / L (see Table 1) about 15-20 (90 × 20 cm petridish) upside down, and then in 3- Callus was induced by incubation for 4 weeks. Induced callus was selected from the callus in a good state without the change of a constant round shape of 1-2 mm in the callus was grown in 2N6 medium for 3 days and used for transformation.

2-2. Cultivation and Suspension Preparation of Agrobacterium

Four recombinant plasmids stored as glycerol stock in an ultra-cold freezer (-70 ° C) were taken out of the transformed Agrobacterium and streaked streaked on AB-ST medium (see Table 1). The day was incubated. The incubated Agrobacterium was dispensed with 15-20 ml AAM medium per petri dish (see Table 1), scraped off using a sterile loop and transferred to a falcon tube. It was then vortaxed for several seconds so that Agrobacterium could be well suspended and the final inoculation concentration was 1.5-2.0 at OD650 nm.

Type and composition of medium for each stage of transformation  Transformation stage Badge Furtherance Callus Induction and Proliferation 2N6 N6 macro, micro salts, N6 vitamins. Casamino acid 300mg / L, proline 500mg / L, sucrose 30g / L, glutamine 500mg / L, gelite 2.5g / L, 2.4-D 2mg / L, pH 5.8 Agrobacterium culture AB-ST AB buffer, salt, glucose 5 g / L, Bacto-agar 15 g / L, spectinomycin 50 mg / L, tetratracycline 10 mg / L, pH 7.0 Agrobacterium Suspension and Inoculation AAM AA macro, micro salt, amino acid stock, MS vitamin, iron stock, casamino acid 500 mg / L, sucrose 68.5 g / L, glucose 36 g / L, acetosyringone 100 uM / L, pH 5.8 Co-culture 2N6-AS 2N6 medium, acetosyringone 100uM / L, glucose 10g / L, gelite 2.5g / L, 2.4-D 2mg / L, pH 5.2 1 / 2-2N6- AS-New 1 / 2-2N6 medium, acetosyringone 100uM / L, glucose 10g / L, L-cysteine 10.5mg / L, sodium thiosulfate 1mM, dithiothreitol 1mM, silver nitrate 5mg / L, gelite 3.0g / L, 2.4 -D 2 mg / L, pH 5.2 Transformation callus selection 2N6-CP 2N6 medium, 250 mg / L cefotaxime, 6 mg / L L-phosphinotricine, 2.5 g / L gelite, 2.4-D 2 mg / L, pH 5.8 subdivision MSR-CP MS macro, micro salts, MS vitamins, Cytotaxin 250 mg / L, NAA 1 mg / L, Kinetine 5 mg / L, Sorbitol 30 g / L, Maltose 20 g / L, L-phosphinothricin 3 mg / L, Zeolite 4 g / L, pH 5.8 Root induction MS0 MS macro and micro salts, MS vitamin, sucrose 30 g / L, gelite 2.5 g / L, pH 5.8

Components and Additions of Callus Induced and Plant Regeneration Media Callus induction subdivision N6 (Chu) Badge Amount MS badge Amount N6 macro salt KNO ₃ (NH ₄ ) ₂ SO ₄ KH ₂ PO ₄ MgSO ₄ (or MgSO ₄ · 7H ₂ O) CaCl ₂ (or CaCl ₂ · 2H ₂ O)  2.83 g / l 463 mg 400 mg 90 mg (or 185 mg) 125.33 mg (or 166 mg) Macro Salt KNO ₃ NH ₄ NO ₃ KH ₂ PO ₄ MgSO ₄ CaCl ₂  1900 mg 1650 mg 170 mg 180.54 mg 332.02 mg Iron (or FeNaEDTA)  36.70mg Iron FeNaEDTA  36.70 mg N6 fine salt _{MnSO 4 · H 2 O ZnSO 4} · 7H 2 OH 3 BO 3 KI Na 2 MoO 4 · 2H 2 CuSO 4 · 5H 2 O CoCl 2 .6H 2 O  3.3mg 1.5mg 1.6mg 0.8mg--- Fine salt _{MnSO 4 · H 2 O ZnSO 4} · 7H 2 OH 3 BO 3 KI Na 2 MoO 4 · 2H 2 CuSO 4 · 5H 2 O ( or CuSO ₄₎ CoCl ₂ .6H ₂ O  16.9 mg 8.6 mg 6.20 mg 0.83 mg 0.25 mg 0.025 mg or 0.016 mg 0.025 mg N6 vitamin nicotinic acid thiamine, HCl pyridoxine, HCl glycine  0.5mg 0.1mg 0.5mg 2.0mg Vitamin nicotinic acid thiamine, HCl pyridoxine, HCl glycine myo-inositol  0.5mg 0.1mg 0.5mg 2.0mg 100mg

Example 3 Agrobacterium Inoculation and Co-Cultivation Method for Enhancing Transformation Efficiency

Agrobacterium inoculation and coculture methods were improved to improve the introduction of target genes into plants.

3-1. Agrobacterium inoculation

Callus grown for 3 days was transferred to an empty Petri dish, and then inoculated with 15-20 ml of Agrobacterium suspension for about 20-30 minutes. After inoculation, the callus was transferred to a petri dish containing sterilized filter paper to remove the remaining Agrobacterium suspension, which was then wound into a co-culture medium.

3-2. Co-culture

Coculture medium composition and conditions were altered to improve the introduction of target genes into plant cells and to increase the viability of the transformed cells (see Table 3). The difference in media composition was that three antioxidant compounds, L-cysteine 10.5, were used to reduce the content of basic medium (2N6) by 1/2 and to minimize the oxidative destruction of plant cells during coculture with Agrobacterium. mg / L, sodium thiosulfate 1 mM, dithiothritol 1 mM, and the like were added, and 5 mg / L of silver nitrate was added to inhibit excessive growth of Agrobacterium and ethylene activity. In addition, the amount of added gellite was increased to 3.0 g / L in order to prevent the nitrate from being depleted due to the addition of silver nitrate.

Unlike the conventional method in which the inoculated callus is placed directly on the co-culture, the treatment differs from the conventional method, by increasing the stability of the inoculated cells by covering one sheet of filter paper (Watman # 1) on the co-culture and placing it on the co-culture. The temperature during the temperature change treatment was about 3 ℃. Through the improved method according to the present invention, it is possible to control the growth rate of Agrobacterium, thereby extending the co-culturing period to 5 days or more (up to 9 days depending on rice varieties) and the co-culturing period to 9 days or more. No damage was observed due to excessive growth of Agrobacterium.

Differences Between Conventional and Improved Methods of the Invention in Co-Cultivation Processing content Conventional method (2N6-AS) Method of the invention (1 / 2-2N6-AS-New) Badge composition Basic Medium (2N6) Reference 1/2 of standard quantity Antioxidant compound none Three antioxidant compounds added (L-cysteine 10.5mg / L, sodium thiosulfate 1mM, dithiothritol 1mM) Agrobacterium Growth and Ethylene Activity Inhibitors none Silver Nitrate 5mg / L Gelite amount 2.5g / L 3.0 g / L  Filterpaper none Cover the filter paper (# 1) over the media Temperature treatment 26.5 ℃ fixed 26.5 ° C → 23.5 ° C Co-culture period 3 days 26.5 degrees Celsius (1 day) → 23.5 degrees Celsius (4 days-8 days) processing more than 5 days

EXAMPLE 4 Selection and Regeneration of Transformed Callus

Callus to which the target gene was introduced was selected, and the regenerated plants were grown as completely transformed plants through induction, rooting, and purification.

4-1. Selection of Transformed Callus

After the coculture process, callus was washed at least 10 times with a solution containing 250 mg / L of cefotaxim to remove Agrobacterium, and the washed callus was transferred to a Petri dish containing sterile filter paper. The remaining solution was completely removed. After this process, callus was transferred to 2N6-CP medium containing PPT (6mg / L) (see Table 1), and the transformed callus was selected first for 3 weeks in 27 ° C dark condition. Secondary selection was carried out by incubating for 2 weeks under the same conditions.

4-2. Induction of Regenerated Plants from Transformed Callus

The selected callus was MSR-CP medium containing 3 mg / L PPT, 250 mg / L Cytotaxin, 1 mg / L Naphthalene Acetic Acid (NAA), 5 mg / L Kinetine, 30 g / L Sorbitol and 20 g / L Maltose ( And then cultured in 25 ℃ bright conditions to induce the transformed plant.

4-3. Root Derivation and Purification

Replanted plants were transferred to MS0 medium without growth hormone (see Table 1) to induce roots. The root-derived plant was immersed in a 1000-fold hypoxic aqueous solution for about 7-10 days, and the purified plant was transplanted to a pot and managed in a greenhouse.

Example 5 Transformation Efficiency Comparison According to the Method According to the Present Invention

In order to determine the transformation efficiency of the co-culture method according to the present invention and the conventional method using a GUS carrier, the degree of transformation and the conversion rate of the GUS gene introduced into the plant cell after co-culture.

5-1. GUS Expression Differences in Callus Inoculated after Co-Cultivation

Agrobacterium pMJC-GB-GUS / LBA440 was inoculated into calli of black and white rice, respectively, and co-cultured by the method according to the present invention and the conventional method, and then the expression level of GUS was examined. In the callus treated with the conventional method, the expression level of GUS was very weak in both black and yellow rice, but in the callus treated with the improved method, GUS expression was very strong in 20-30% in the black rice and 40-50% in ilmi rice. 3). These results show that the co-cultivation process can be introduced with high efficiency into plant tissues for the target gene when the improved method according to the present invention is treated.

5-2. Comparison of Rice Transformation Rate Between the Method According to the Present Invention and the Conventional Method

When the co-cultivation process in the black rice was processed by the conventional method, 18 transgenic plants were induced from 500 callus used for inoculation, resulting in a transformation rate of 3.6%. On the other hand, in the test treated with the co-culture method according to the present invention, in the medium containing 2.5 g of gelite (pitagel) content, 17 transgenic plants were induced and showed a transformation rate of 9.4%, but the gelite content was 3.0. When g was added, 77 transgenic plants were induced, and the transformation rate was 27.5%, which was about 7 times higher than that of the conventional method. The difference in the rate of transformation according to the content of gelite was caused by the phenomenon of retreating when silver nitrate was added to the co-culture medium. The medium containing 2.5g of gelite was observed to generate a lot of water on the filter paper laid thereon. It is thought that this cause influenced the transformation rate. Therefore, in the present invention, the gelite content was adjusted to 3.0 g or more and used. The conversion rate in Ilmi rice was more than doubled to 6.8% in the improved method compared to 3.3% in the conventional method, and the increase rate was slightly lower than that of the black rice. In addition, in the treatment of abundant rice, which was difficult to transform, the transformed plants were not obtained by the conventional method, but the two independent transformed plants were obtained by the improved method according to the present invention. This can be overcome to some extent (see Table 4).

Comparison of Rice Transformation Rate Between the Method According to the Present Invention and the Conventional Method kind Per treatment Interculture period Pitazel (Phytagel) content Transformation Callus number Transformation Plant count (independent line) Mold quality Conversion rate Increase Black rice Conventional method 3 days 2.5 g 500 pcs All 18 3.6% 100 The present invention 7 3.0 280 77 27.5 764 The present invention 7 2.5 180 17 9.4 261 Rice Conventional method 3 2.5 240 8 3.33 100 The present invention 7 3.0 280 19 6.79 204 A la carte Conventional method 3 2.5 250 0 0 100 The present invention 7 3.0 220 2 0.9 190

Example 6 Practical Application to Rice Transformation of the Method According to the Present Invention

In order to find out whether the method according to the present invention shows the same high efficiency as the GUS carrier even in the actual rice transformation, transformation was performed in IFS1, IFS2, and harpin carriers. Transformation of IFS1 and IFS2 genes into black rice showed 20.8% and 32.1% transformation rate, which was not significantly different from 27.5% of GUS carrier. In addition, the transformation rate of the Harpin carrier was about 12.4%, which was about 2 times higher than that of the GUS carrier, 6.8%. These results indicate that the improvement method according to the present method is well applied to actual rice transformation regardless of the type of carrier (see Table 5).

Application of the method according to the invention to rice transformation kind Vector Interculture period Pitagel Content Transformation Callus number Transformation Plant count (independent line) Mold quality Conversion rate Black rice pMJC-GB-IFS1 7 days 3.0 g 240 pieces All 50 20.8% pMJC-GB-IFS2 7 3.0 280 90 32.1 Rice pMJC-GB-Haffin 7 3.0 940 117 12.4

Example 7 Molecular Biological Analysis and Bioassay of Transgenic Plants

7-1. Confirmation of Transformation of GUS Gene Using PCR

Whether the GUS gene used in the present invention was successfully inserted into the transformed rice genome was confirmed by PCR method.

First, genomic DNA was isolated from young leaves of rice T0 plants transformed with GUS gene by CTAB method (Rogers & Benich, 1994). GUS557FOR of SEQ ID NO: 1, 5'-GAA GCCGATGTCACGCCGTATGTT-3 '(24 mer) and SEQ ID NO: 2, a primer set designed to amplify DNA fragments having a size of 1449 bp from ORF 557 bp to 2005 bp of the isolated DNA as a template PCR was performed using GUS2005REV, 5'-CTAGCTT GTTTGCCTCCCTGCTGC-3 '(24 mer).

For PCR reactions, rice genomic DNA was 50 ng, 1X f-Taq buffer (Solgent, Daejeon, Korea), 50 μM of each dNTP, 1X band doctor (Solgent, Daejeon, Korea), 1.5 units of f-Taq DNA polymerase (Solgent). , Daejeon, Korea) and 25 pM of each primer were added and the final volume was adjusted to 50 μl with sterile water. The PCR reaction was performed by MJ Research's PCT-200 instrument, and the conditions were modified for 4 minutes at 94 ° C, followed by denaturation at 94 ° C for 15 seconds, polymerization at 55 ° C for 30 seconds, and extension at 72 ° C for 60 seconds. The reaction was programmed to elongate at 72 ° C. for 7 minutes after 30 iterations. The amplified product was separated from 0.8% agar gel added with EtBr and confirmed by UV transilluminator.

Figure 5 is inoculated in the GUS carrier in black and white rice callus and the co-cultivation process by the conventional method and the improved method according to the present invention (Phytagel 3.0g) five of the 99 transformed T0 plants derived from the conventional method, In the improvement method, 15 individuals were randomly selected and the results of PCR were shown. All 20 selected plants showed one band at the 1449 bp position, which is the expected size of the primers used, demonstrating that the GUS gene was stably transformed into the rice genome. In FIG. 5, the non-transformant is a control group (Con), and the redivided individual by the conventional method is 1-1 to 1-5, and the redivided individual by the improved method according to the present invention is 3-1 to 3-15. It was.

7-2. Confirmation of GUS Gene Expression

Expression of the GUS gene was confirmed in the regeneration plants mentioned in 7-1 above, where the GUS gene was inserted into the rice genome.

Cut the leaves of the regenerated plants into several pieces with scissors and remove 50mM X-Gluc solution (50mM sodium phosphate buffer pH 7.0, 0.2% Triton X-100, 3mM potassium ferricyanide, 3mM potassium ferrocyanide and 20 Expression of GUS was confirmed by treatment with% methanol overnight at 37 ° C. and then decolorized in 70% ethanol Figure 6 shows the results and in 13 out of 15 redifferentiated individuals by the improved method, In the conventional method, GUS expression was confirmed in three out of five, indicating that the GUS gene was stably expressed.

7-3. Herbicide resistant bioassay

Transgenic plants were reconfirmed by evaluating the function of herbicide resistant bar genes used as starter markers. The herbicide-resistance bioassay was performed on all 398 transgenic T0 plants mentioned in Examples 6 and 7-1 above and minimized the experimental errors by spreading the 0.3% bathaster solution evenly over the entire plant. It was. As a result of the assay, the 398 individuals obtained in the present invention were all survived after the spraying of bathaster and showed resistance to herbicides. As such, the fact that there was no individual to avoid has proved that the bar gene used in the present invention is strictly applied as a selection marker in rice transformation.

7 is a result of inoculating a GUS carrier in a la carte rice callus and confirmed herbicide resistance to the transformed plants obtained after the co-culture process by the method according to the present invention. When 0.3% Basta solution was sprayed twice on the transformants and the non-transformants, the non-transformed plants were completely killed as shown in FIG. 7, but the transformed plants were vividly living and resistant to herbicides. In FIG. 7, the non-transformer was shown as wild type.

As described above, when transforming by the method according to the present invention was able to significantly increase the transformation efficiency of the plant, the target transgenic plant can be produced in a short time to reduce the manpower and cost required during the transformation process I could save. In addition, it is possible to produce a transgenic plant even in a difficult to transform varieties will be able to develop early commercially available excellent varieties.

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Claims (10)

In the method for producing a transformed plant using Agrobacterium introduced with a foreign target gene, Food crops including rice, beans, barley, corn, potatoes, wheat, red beans, oats, and sorghum; Vegetable crops including tomatoes, peppers, strawberries, green onions, onions, carrots, cabbages, radishes, watermelons, cucumbers, cabbages, and zucchini; Special crops including ginseng, sesame seeds, perilla, peanuts, rapeseed, cotton, sugar cane, beets and tobacco; Fruit trees and flowering plants, including apple trees, pears, jujube trees, grapes, citrus fruits, persimmons, apricots, bananas, leeks, roses, chrysanthemums, lilies, gerberas, carnations, freesia, gladiolus and tulips; Fodder crops, including orchardgrass, alfalfa, tall pescue, red clover and lygragrass; And agrobacterium suspension in which the target gene has been introduced into at least one plant tissue selected from the group consisting of arabidopsis at a concentration of 1.5-2.0 at OD650nm, Here, the base medium 2N6 composition is reduced to 1/2, at least one antioxidant selected from the group consisting of 5-50 mg / L L-cysteine, 0.1-5 mM sodium thiosulfate, 0.1-5 mM dithiothritol, and 1-10 mg of nitric acid. Method for producing a transgenic plant, characterized in that the co-culture by adding / L. delete The method of claim 1, wherein the plant tissue is a callus of rice. delete delete According to claim 1, wherein the method of producing a transgenic plant, characterized in that the addition of 2.6 ~ 5g / L of gelite in the co-culture process. The method of claim 1, further comprising the step of covering one sheet of filter paper (Watman # 1) on the co-culture medium and placing callus on the co-culture medium. According to claim 1, wherein in the co-cultivation process the treatment temperature is treated at 26 ~ 28 ℃ for the first day and then the rest of the period to 23 ~ 25 ℃ to change the temperature 3 ℃ than the temperature of the first day 5 days or more Method for producing a transgenic plant, characterized in that the maintenance. delete delete

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