KR102160220B1 - Medium composition for somatic embryogenesis of Medicago sp. plant and use thereof - Google Patents

Abstract

The present invention generates a somatic embryo of a plant of the genus Medicago , including 1-Naphthaleneacetic acid (NAA) as auxin and Thidiazuron (TDZ) as cytokinin It provides a medium composition for use. The medium composition for somatic cell embryo development of a plant of the genus Medica according to the present invention contains a combination of new hormones as compared to a medium for generating somatic cell embryos of a conventional Medicago trankatula, and thereby, the somatic cell embryo generation efficiency is improved by about 10 times or more. Have. In addition, if the method for transforming a plant of the genus Medica according to the present invention is used, it is possible to stably transform the reference genotype A17 of Medicago truncula, which was very difficult to transform with the conventional method. Therefore, using the present invention, genome-based gene function studies and genetic manipulation of the reference genotype A17 of Medicago truncatula are possible.

Description

Medium composition for somatic embryogenesis of Medicago sp. plant and use thereof}

The present invention relates to a medium composition for somatic cell embryo development and a use thereof, and more particularly, to a medium composition having high somatic cell embryo generation efficiency of a plant of the genus Medica and a method for transforming a plant of genus Medica using the same.

Medicago truncatula ( Medicago truncatula ) is a legume genus (genus Medicago ) plant, alfalfa ( Medicago sativa ) is used as an important green manure and forage crops. In particular, as a model plant for genetics and genomics studies of legumes, Medicago Trunka in research for the symbiosis with nitrogen-fixing bacteria and the symbiosis of mycorrhiza and gene function to improve the value of food and fodder crops. Tula ( Medicago truncatula ) is used worldwide. In addition, the genome of Medicago truncatula was decoded using the A17 strain [Young et al (2011). The Medicago genome provides insight into the evolution of rhizobial symbioses, Nature, Nov 16, 480(7378), 520-524.], based on this, various research materials and information have been accumulated and used globally.

However, for A17, the reference genotype of Medicago truncatula, stable transformation using Agrobacterium tumefaciens is very difficult, so genetic function studies or breed development using transformation are not performed. As an alternative, a mutant group was nurtured using Medicago Truncatula R108, a cultivar that is relatively easy to regenerate plants (Trinh et al., 1998, Plant Cell Reports, 17: 345-355.), and Medicago Truncatula. Plant transformation is carried out using A17 cultivar and a related cultivar or mutant cultivar Medicago Truncatula 2HA (Rose et al., 1999, Journal of Plant Physiology 155:788-791.; Araujo et al., 2004, Plant Cell, Tissue and Organ Culture 78:123-131.), there is a problem due to the genetic mutation and the difference in the genome sequence from the reference genotype Medicago Trunkatula A17.

Stable transformation of plants of the genus Medica is a direct shoot organogenesis method that directly induces new plants around apical tissues located at the base of cotyledons, mainly by the transformation method of legumes such as soybeans (Trieu et al. ., 1996, Plant Cell Reports, Volume 16, Issue 1-2, pp 6-11) and a method of inducing indirect somatic embryogenesis after forming a callus in cotyledons, leaves, or hypocotyl tissues ( Cosson et al., Methods Mol Biol. 2006;343:115-27) have been utilized. Of these, the Medicago Truncatula A17 genotype has low somatic embryogenesis induction efficiency, and its transformation uses a direct new organ development method, but it is known that the efficiency is very low compared to time and effort.

The present invention has been derived under the conventional technical background, and an object of the present invention is to provide a medium composition having high somatic embryo generation efficiency of a plant of the genus Medica and a method for transforming a plant of genus Medica using the same.

The inventors of the present invention use various combinations of hormones added to the medium for somatic embryo development in order to dramatically increase the regeneration efficiency of the transgenic plant by the development of somatic embryos in the process of transformation of the Medicago Truncatula A17 genotype using somatic embryo development. The test was performed, and as a result, it was confirmed that the somatic embryogenic efficiency was remarkably improved at a specific combination of hormones and a predetermined range of concentrations, thereby stably transforming the Medicago Truncatula A17 genotype, and the present invention was completed.

In order to solve the above object, the present invention is a medium composition containing auxin and cytokinin, wherein the auxin is 1-Naphthaleneacetic acid (NAA) and the cytokinin is Thidiazuron (TDZ), based on the total volume of the medium composition, the concentration of the 1-naphthalic acid is 0.4 to 5 μM, and the concentration of thidiazuron is 1.5 to 5 μM, characterized in that Medicago plants It provides a medium composition for the generation of somatic embryos.

In order to solve the above object, an example of the present invention comprises the steps of co-culturing an explant of a plant of the genus Medica with Agrobacterium genus into which a transformant gene has been introduced; Culturing the co-cultured explants in a callus-forming medium to form a callus; Culturing the callus in the above-described somatic culture medium composition to generate a somatic embryo; And it provides a method for transforming a plant of the genus Medica comprising the step of developing the somatic embryo into a plant. In addition, another example of the present invention is to form a callus by culturing the explant (Explant) of a plant of the genus Medica in a callus-forming medium; Co-culturing the callus with the Agrobacterium genus into which the transgene is introduced; Culturing the co-cultured callus in the above-described somatic cell culture medium composition to generate somatic cell embryos; And it provides a method for transforming a plant of the genus Medica comprising the step of developing the somatic embryo into a plant.

The medium composition for somatic cell embryo development of a plant of the genus Medica according to the present invention contains a combination of new hormones as compared to a medium for the development of somatic cell embryos of a conventional Medicago trankatula, thereby improving the somatic cell generation efficiency by about 10 times or more. Have. In addition, if the method for transforming a plant of the genus Medica according to the present invention is used, it is possible to stably transform the reference genotype A17 of Medicago truncula, which was very difficult to transform with the conventional method. Therefore, using the present invention, genome-based gene function studies and genetic manipulation of the reference genotype A17 of Medicago truncatula are possible.

1 is a Medicago Truncatula genotype A17 ( Medicago truncatula cultivar Jemalong A17) Infecting the leaf section of a plant with Agrobacterium tumefaciens into which a vector for plant transformation has been introduced, co-culturing in a co-culture medium CCM3, and culturing in CIM3, a medium for callus formation, to form callus, It shows the proportion (%) of leaf slices in which somatic embryos occurred when the formed callus was cultured in a medium for somatic embryo development of various hormone combinations.
Figure 2 is a Medicago Truncatula genotype A17 ( Medicago truncatula cultivar Jemalong A17) Infecting the leaf section of a plant with Agrobacterium tumefaciens into which a vector for plant transformation has been introduced, co-culturing in a co-culture medium CCM3, and culturing in CIM3, a medium for callus formation, to form callus, It shows the number of somatic embryos generated per leaf section when the formed callus was cultured in a medium for somatic embryo development of various hormone combinations.
FIG. 3 shows the development of somatic embryos and plants during the production process of a Medicago truncatula A17 plant transformed by a kanamycin resistance gene.
4 is a step-by-step view of the production process of a Medicago truncatula A17 plant transformed with a kanamycin resistance gene.
5 is a result of observing the fluorescence of a green fluorescent protein (GFP) of a Medicago truncatula A17 plant transformed with a kanamycin resistance gene.
Figure 6 is a kanamycin (Kanamycin) resistance gene transformed by using a chain polymerase reaction (PCR) Medicago Trunkatula ( Medicago truncatula ) This is the result of confirming the green fluorescent protein (GFP) gene in the A17 plant.
7 is a step-by-step view of the production process of a Medicago truncatula A17 plant transformed by a basta resistance gene.
FIG. 8 is a result of observing the fluorescence of a green fluorescent protein (GFP) of a Medicago truncatula A17 plant transformed by a Basta resistance gene.
9 is a Medicago Truncatula transformed by a Basta resistance gene using a chain polymerase reaction (PCR) ( Medicago truncatula ) This is the result of confirming the green fluorescent protein (GFP) gene in the A17 plant.
Figure 10 is the present invention when producing a Medicago truncatula A17 plant transformed by a kanamycin resistance gene using Nolan et al.'s callus formation medium and somatic cell embryo development medium Medicago trunkatula transformed by kanamycin resistance gene using a medium for callus formation and a medium for somatic embryo development of truncatula ) shows the development of somatic embryos when A17 plants are produced.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a medium composition having high somatic embryo generation efficiency of plants of the genus Medica. The medium composition for the development of somatic embryos of the plant of the Medicago genus of the present invention includes 1-Naphthaleneacetic acid (NAA) as auxin and Thidiazuron (TDZ) as a cytokinin Characterized in that. In addition, the concentration of the 1-naphthaleneacetic acid (NAA) in the medium composition for somatic cell embryo development of the Medicago plant of the present invention is 0.4 to 5 μM based on the total volume of the medium composition, and When considering the generation efficiency, it is preferably 0.5 to 4 μM, and more preferably 0.8 to 2 μM based on the total volume of the medium composition. In addition, the concentration of the Thidiazuron (TDZ) in the medium composition for somatic cell embryo development of the Medicago plant of the present invention is 1.5 to 5 μM based on the total volume of the medium composition, when considering the somatic cell generation efficiency It is preferably 1.5 to 3 μM, and more preferably 1.8 to 2.5 μM, based on the total volume of the medium composition.

In addition to the combination of 1-naphthaleneacetic acid (NAA) and tidiazuron, the medium composition for the development of somatic cell embryos of plants of the Medicago genus of the present invention is a medium for the development of somatic cell embryos in plants of the known Medicago genus It may further include various ingredients included in. For example, the medium composition for the development of somatic embryos of plants of the Medicago genus of the present invention is a compound known in the art to promote the development of somatic embryos from callus, such as gibberellin, such as GA3 or GA4 and abcisic acid (ABA). Or, it may contain L-alpha-(2-aminoethoxyvinyl)glycine (AVG) at a concentration of 0.2 to 5 μM. In addition, the medium composition for the generation of somatic embryos of the plant of the Medicago genus of the present invention may contain appropriate antibiotics, such as Cefotaxime, Augmentin, Timentin, Carbenicillin, etc., to inhibit Agrobacterium when it continues to grow. It may contain Augmentin at a concentration of 100 to 900 mg/ℓ. In addition, the medium composition for generating somatic embryos of a plant of the Medicago genus of the present invention may include gums such as agar, agarose or Gelrite.

The medium composition for the development of somatic embryos of plants of the Medicago genus of the present invention is in addition to 1-Naphthaleneacetic acid (NAA) and thidiajuron (NH ₄ )H ₂ PO ₄ H ₂ O, KNO ₃ , CaCl ₂ 2H ₂ O, MgSO ₄ 7H ₂ O, Fe-EDTA 3H ₂ O, MnSO ₄ H ₂ O, H ₃ BO ₃ , KI, ZnSO ₄ 7H ₂ O , CuSO ₄ 5H ₂ O, Na ₂ MoO ₄ 2H ₂ O, CoCl ₂ 6H ₂ O, myo-inositol, nicotinic acid, pyridoxine hydrochloride (Pyridoxine HCl), thiamine hydrochloride (Thiamine HCl ), Sucrose, Casein hydrolysate, MES[2-(N-morpholino)ethanesulfonic acid], calcium gluconate, AVG[L-α-(2-aminoethoxyvinyl)glycine] , Augmentin (Augmentin; Amoxicillin clavulanate) and gellan gum (Gellan gum) may be further included. In addition, the medium composition for the generation of somatic embryos of the Medicago plant of the present invention has a concentration of 0.5 to 4 μM based on the total volume of the medium composition when considering the efficiency of somatic embryo generation, 1-Naphthaleneacetic acid (NAA). ), Thidiazuron at a concentration of 1.5~3μM, (NH ₄ )H ₂ PO ₄ ·H ₂ O at a concentration of 200~400 mg/ℓ, KNO _{3 at a} concentration of 2000~3000 mg/ℓ, 75~275 mg/ℓ concentration Of CaCl ₂ ·2H ₂ O, MgSO ₄ 7H ₂ O at a concentration of 300~500 mg/ℓ, Fe-EDTA 3H ₂ O at a concentration of 10~50 mg/ℓ, MnSO _{4 at a} concentration of 5~25 mg/ℓ H ₂ O, H ₃ BO _{3 at a} concentration of 1 to 15 mg/ℓ, KI at a concentration of 0.2 to 4 mg/ℓ, ZnSO ₄ 7H ₂ O at a concentration of 0.2 to 4 mg/ℓ, 0.05 to 1 mg/ℓ concentration CuSO ₄ ·5H ₂ O, Na ₂ MoO ₄ ·2H ₂ O at a concentration of 0.01~0.25 mg/l, CoCl ₂ ·6H ₂ O at a concentration of 0.01~0.25 mg/l, myoinositol at a concentration of 10~250 mg/l ( myo-inositol), nicotinic acid at a concentration of 0.1 to 2.5 mg/l, pyridoxine HCl at a concentration of 0.1 to 2.5 mg/l, thiamine HCl at a concentration of 1 to 25 mg/l, 10 Sucrose at a concentration of ~35 g/ℓ, Casein hydrolysate at a concentration of 0.1 to 1.5 g/L, MES[2-(N-morpholino)ethanesulfonic acid] at a concentration of 0.1 to 1.5 g/L, Calcium gluconate at a concentration of 0.1 to 1.5 g/l, AVG [L-α-(2-aminoethoxyvinyl) glycine] at a concentration of 0.2 to 2.5 μM, Augmentin at a concentration of 200 to 900 mg/l (Augmentin; Amoxicillin clavulanate) and gellan gum at a concentration of 1 to 5 g/L, preferably 1-Naphthaleneacetic acid (NAA) at a concentration of 0.8~2μM, thidiazuron at a concentration of 1.8~2.5μM, (NH ₄ )H ₂ PO ₄ ·H ₂ O at a concentration of 250~350 mg/ℓ, KNO _{3 at a} concentration of 2200 to 2800 mg/ℓ, CaCl ₂ ·2H ₂ O at a concentration of 120 to 240 mg/ℓ, MgSO ₄ 7H ₂ O at a concentration of 350 to 450 mg/ℓ, Fe at a concentration of 15 to 40 mg/ℓ -EDTA·3H ₂ O, MnSO ₄ ·H ₂ O at a concentration of 8~15 mg/ℓ, H ₃ BO _{3 at a} concentration of 2~8 mg/ℓ, KI at a concentration of 0.5~2.5 mg/ℓ, 0.5~2.5 mg/ ℓ concentration of ZnSO ₄ ·7H ₂ O, 0.1~0.5 mg/ℓ concentration of CuSO ₄ ·5H ₂ O, 0.05~0.2 mg/ℓ concentration of Na ₂ MoO ₄ ·2H ₂ O, 0.05~0.2 mg/ℓ concentration CoCl ₂ ·6H ₂ O, myo-inositol at a concentration of 50 to 200 mg/l, nicotinic acid at a concentration of 0.5 to 2 mg/l, pyridoxine hydrochloride at a concentration of 0.5 to 2 mg/l ), Thiamine HCl at a concentration of 5 to 20 mg/ℓ, Sucrose at a concentration of 15 to 30 g/L, Casein hydrolysate at a concentration of 0.2 to 1.0 g/L, 0.2 to 1.0 g/ℓ concentration of MES[2-(N-morpholino)ethanesulfonic acid], 0.2~1.0 g/ℓ concentration of calcium gluconate, 0.5~2.0 μM concentration AVG[L-α-(2-aminoethoxyvinyl) ) glycine], augmentin at a concentration of 250-800 mg/ℓ (Augmentin; Amoxicillin clavulanate) and gellan gum at a concentration of 2 to 4 g/l.

The medium composition for the development of somatic embryos of plants of the Medicago genus of the present invention further includes a compound for selection of transformation such as kanamycin, hygromycin, and Basta as a system for selecting only transformed somatic embryos. can do. It is preferable that the concentration of the compound for selection of transformation in the medium composition for the development of somatic cell embryos of the plant of Medicago genus of the present invention is selected from a concentration of 1 to 100 mg/ℓ. For example, the concentration of the kanamycin in the medium composition for the development of somatic embryos of the plant of the Medicago genus of the present invention is preferably 20 to 80 mg/l and more preferably 30 to 60 mg/l. In addition, in the medium composition for the development of somatic embryos of the Medicago plant of the present invention, the concentration of Basta is preferably 2 to 10 mg/L, and more preferably 3 to 8 mg/L.

The medium composition for the development of somatic embryos of plants of the Medicago genus of the present invention preferably contains water as a medium. The medium composition for the generation of somatic embryos of the plant of the Medicago genus of the present invention is preferably sterilized with an autoclave and then cooled to exist in a solid state by gums, one of the constituents.

Medica high speed (Medicago) somatic embryo culture medium composition for the generation of the plant of the present invention is applied to to induce somatic embryogenesis of callus formation from Medica high speed (Medicago) plant, the Medica high speed (Medicago) plants that kind is greatly It is not limited, and about 90 Species have been reported. The medium composition for the development of somatic embryos of the plant of the Medicago genus of the present invention is preferably Medicago truncatula ( Medicago truncatula ) is applied to induce the development of somatic embryos of callus formed from, more preferably Medicago truncatula genotype A17 ( Medicago truncatula genotype A17) is applied to induce somatic embryogenesis of callus.

One aspect of the present invention relates to a method for transforming a plant of the Medicago genus. A method for transforming a plant of the Medicago genus according to an embodiment of the present invention comprises the steps of co-culturing an explant of a plant of the genus Medica with the Agrobacterium genus into which the transgene is introduced; Culturing the co-cultured explants in a callus-forming medium to form a callus; Culturing the callus in the above-described somatic culture medium composition to generate a somatic embryo; And developing the somatic embryo into a plant. In addition, a method for transforming a plant of the Medicago genus according to another example of the present invention comprises the steps of culturing an explant of a plant of the genus Medica in a culture medium for callus formation to form a callus; Co-culturing the callus with the Agrobacterium genus into which the transgene is introduced; Culturing the co-cultured callus in the above-described somatic cell culture medium composition to generate somatic cell embryos; And developing the somatic embryo into a plant. The transformation method of a plant of the Medicago genus according to an embodiment of the present invention is a method of transforming an explant of a plant of the genus Medica by infecting it with a fungus of the genus Agrobacterium into which a transformant gene has been introduced. A method for transforming a plant of the Medicago genus according to another example of the invention is a method of transforming a callus formed from an explant of a plant of the genus Medica by infecting it with a fungus of the genus Agrobacterium into which a transformant gene has been introduced. . Hereinafter, a method for transforming a plant of Medicago genus according to an example of the present invention will be described in stages. A method for transforming a plant of Medicago genus according to another embodiment of the present invention has the same technical characteristics as a method for transforming a plant of Medicago genus according to an embodiment of the present invention, except for the above differences, so a detailed description is omitted. do.

Medica Express Plant Explant Transgene introduced Agro Step of co-culturing with bacteria of the genus Bacterium

In the method of transforming a plant of the Medicago genus of the present invention, the explant of the plant of the genus Medica is a fragment obtained by resecting a tissue or organ of a plant of the genus Medica, as long as it is capable of forming a callus. It can be obtained from the site, preferably leaf slices. In addition, the leaf segment used in the present invention is a leaf of a mature Medicago truncatula, more preferably a Medicago truncatula A17 plant, and also petioles, stems, petals and roots, cotyledons and hypocotyls of young plants or young plants. You can use it in the same way. In the present invention, it is recommended to use leaves grown in aseptic conditions as the optimal plant material. In addition, in general, it can be used after collecting leaves of plants grown in a greenhouse or incubator and subjected to appropriate sterilization treatment. For example, a leaf isolated from a plant is immersed in an aqueous ethanol solution of 70% concentration for 1 to 3 minutes, sterilized by immersing it in a 10 to 50% concentration of Lax solution for 10 to 30 minutes, and then washed with sterilized distilled water. Can be used as a material for sectioning. Cut into appropriate size with a scalpel and prepare leaf slices.

Transformation method of the plant of the Medicago genus of the present invention The type of the transgenic gene is not largely limited, and may be selected from a variety of known transgenes. For example, the transgene is composed of one or more selected from antibiotic resistance gene, herbicide resistance gene, disease resistance gene, virus resistance gene, stress resistance gene, enzyme biosynthesis gene, recombinant protein expression gene, or plant growth promoting gene. Can be. The antibiotic may be selected from various known antibiotics such as kanamycin and hygromycin. In addition, the herbicide may be selected from various known herbicides such as Basta. Specific examples of transgenes that can be used in the transformation method of the Medicago plant of the present invention include drought stress resistance gene, phytoene synthase (PSY) gene, RECA1 gene, peroxidase gene, E. coli Enterotoxin B subunit gene, gul1/sur2-7 gene that promotes plant growth, OsRAF gene, anthocyanin biosynthesis regulatory gene AtbHLH113, disease resistance gene CaChitIV (Capsicum annuum Chitinase class IV), disease resistance gene CaMLO2 (Capsicum annuum mildew resistance) locus O2), disease resistance gene CaGRP1 (Capsicum annuum Glycine-rich RNA-binding Protein 1), AVRBsT gene, disease resistance gene CaADC1 (Capsicum annuum Arginine Decarboxylase 1), disease resistance CaALDH1 (Capsicum annuum Dehydrogenase 1 ), shade resistance or salt resistance gene serotonin N-acetyltransferase2 gene, cold resistance protein 15A (COR15A), toxoflavin lyase (tflA), xylanase gene, tocotrienol biosynthesis Genes HGGT (homogentisic acid geranylgeranyl transferase), OsGlu2 gene, heat shock resistance gene, oxidative stress resistance gene, activin A gene, dry protein BrDST28, temperature stress resistance gene AtP3B, plant growth promoting CYP705A13 gene, heat shock transcription factor gene CaHsfB1, HOX25 gene, salt stress resistance gene BrGI (Brassica rapa GIGANTEA) gene, root development related gene CRF2 or CRF3, COMT (caffei c acid O-methyltransferase) enzyme, plant growth regulation blp chimera gene, HC-Pro (helper component-proteinase) gene derived from Soybean mosaic virus, heavy metal resistance gene Camelina HMA3 gene, heat shock protein Isolated gene (CaHSP70), cold stress resistance gene grxC gene, TTG1 gene, OsFKBP16-3 (Oryza sativa FK506-binding protein 16-3) protein coding gene, taxadiene synthase gene, CaSGT1 ( Capsicum annuum Suppressor of G Two allele of SKP1 1).

In the method for transforming a plant of the Medicago genus of the present invention, the transformant gene is introduced into the Agrobacterium spp. through a vector system including a transformant gene. The vector system used in the present invention may use a variety of materials suitable for plant transformation, and includes a promoter operating in a plant, a transgene structure, and a selection marker for selection of a transgenic plant. The structure of the transformant gene may be one of overexpression of a target gene, binding of a promoter and a gene to a fluorescent protein or uid gene, or gene silencing using microRNA, shRNA, and lhRNA. The vector system may include a kanamycin resistance gene, a hygromycin resistance gene, a herbicide resistance gene, or the like as a marker gene for plant selection. However, it is not limited to this example and may include various vector systems known in the art.

In the method of transforming a plant of the Medicago genus of the present invention, the Agrobacterium genus bacteria into which the transgene is introduced are various known Agrobacterium bacteria that can transform the explants by infecting the explants of the plant. It may be selected from, and is preferably Agrobacterium tumefaciens when considering transformation efficiency and the like.

In the method of transforming a plant of the Medicago genus of the present invention, the method of introducing a transformation vector into a leaf segment may be carried out according to a known conventional plant transformation method using Agrobacterium tumefaciens, and electroporation , Gene gun, etc. may include a gene introduction method known in the art.

In the case of co-culturing the explants with the Agrobacterium genus bacteria into which the transgene is introduced in the method for transformation of the Medicago genus of the present invention or the co-culture with the Agrobacterium genus bacteria into which the transgenes are introduced. A variety of co-culture media can be used. The co-culture medium used in the present invention may use MS medium or SH medium known in the art, and its composition is (NH ₄ ) ₂ SO ₄ , KNO ₃ , CaCl ₂ , MgSO ₄ , Fe-EDTA, MnSO ₄ , HBO ₃ , KI, ZnSO ₄ , CuSO ₄ , Na ₂ MoO ₄ , CoCl ₂ , myoinositol, nicotinic acid, pyridoxine, thiamine HCl, Glycine, casein hydrolysates, calcium gluconate, MES, acetosyringone, sugar, auxin, cytokinin It may include. In addition, the co-culture medium used in the present invention may preferably contain gums such as agar, agarose or Gelrite. To adjust the pH of the co-culture medium, MES may be added at a concentration of 0.2-2 g/ℓ, preferably 0.5 g/ℓ, and the pH of the co-culture medium may be adjusted to 5.7 to 5.8. In addition, casein hydrolysates, calcium gluconate, amino acids proline and cysteine, etc. for promoting the growth of Agrobacterium tumefaciens and callus may be added to the co-culture medium at a concentration of 0.1 to 2 g/L, respectively. In addition, acetoryringone may be added in the range of 50-200 μM to the co-culture medium to promote the transformation activity of Agrobacterium tumefaciens. In addition, the saccharide used in the co-culture medium may be one of the saccharides used for plant transformation known in the art, and preferably, sucrose can be used in a concentration range of 5 to 50 g/L. . In addition, auxin and cytokinin used as constituents of the co-culture medium may be one of auxin and cytokinin compounds known in the art for plant transformation, preferably NAA and BAP from 1 to 20 It can be used in combination in μM concentration. According to a specific embodiment of the present invention, when a leaf segment is co-cultured with Agrobacterium tumefaciens, the leaf segment is infected with Agrobacterium tumefaciens and introduced into Agrobacterium tumefaciens or a transgenic gene containing the same. The vector is introduced into the leaf segment, and the leaf segment is transformed.

Co-cultured Eating out Callus By culturing in a forming medium Callus Forming steps

In the transformation method of the plant of the Medicago genus of the present invention, the step of forming a callus is a callus capable of culturing a leaf segment infected with Agrobacterium tumefaciens in a callus-forming medium containing an appropriate auxin and cytokinin. (embryogenic callus) It is a step that induces the formation and growth of cells. The callus-forming medium may contain 2-50 μM of NAA, which is auxin, and 1-25 μM, of BAP, which is cytokinin. In addition, the callus-forming medium may contain a compound known in the art, L-alpha-(2-aminoethoxyvinyl) glycine (AVG), at a concentration of 0.2 to 5 μM in order to promote the formation of embryogenic callus. In addition, the medium for callus formation may contain at least one selected from appropriate antibiotics, such as Augmentin, Timentin, Carbenicillin, and Cefotaxime, in order to inhibit the growth of Agrobacterium tumefaciens, and preferably 400 to 800 May contain Augmentin in mg/ℓ concentration. In addition, the callus-forming medium may contain gums such as agar, agarose, or Gelrite.

In the transformation method of the plant of the Medicago genus of the present invention, the medium for callus formation is preferably 1-naphthaleneacetic acid (NAA), 6- Benzylaminopurine (6-Benzylaminopurine, BAP), (NH ₄ )H ₂ PO ₄ H ₂ O, KNO ₃ , CaCl ₂ 2H ₂ O, MgSO ₄ 7H ₂ O, Fe-EDTA 3H ₂ O, MnSO ₄ H ₂ O, H ₃ BO ₃ , KI, ZnSO ₄ 7H ₂ O, CuSO ₄ 5H ₂ O, Na ₂ MoO ₄ 2H ₂ O, CoCl ₂ 6H ₂ O, myo-inositol , Nicotinic acid, Pyridoxine HCl, Thiamine HCl, Sucrose, Casein hydrolysate, MES[2-(N-morpholino)ethanesulfonic acid], gluconic acid Calcium (Calcium gluconate), AVG [L-α-(2-aminoethoxyvinyl) glycine], Augmentin (Augmentin; Amoxicillin clavulanate), and Gellan gum. In addition, in the transformation method of the Medicago plant of the present invention, the callus-forming medium is 1-naphthaleneacetic acid (NAA), 1-Naphthaleneacetic acid (NAA) at a concentration of 2 to 20 μM based on the total volume of the medium composition. 6-Benzylaminopurine (BAP) at a concentration of 8μM, (NH ₄ )H ₂ PO ₄ ·H ₂ O at a concentration of 200~400 mg/ℓ, KNO _{3 at a} concentration of 2000~3000 mg/ℓ, 75~ CaCl ₂ ·2H ₂ O at a concentration of 275 mg/ℓ, MgSO ₄ ·7H ₂ O at a concentration of 300 to 500 mg/ℓ, Fe-EDTA·3H ₂ O at a concentration of 10 to 50 mg/ℓ, 5 to 25 mg/ℓ Concentration of MnSO ₄ ·H ₂ O, 1~15 ㎎/ℓ concentration of H ₃ BO ₃ , 0.2~4 ㎎/ℓ concentration of KI, 0.2~4 ㎎/ℓ concentration of ZnSO ₄ ·7H ₂ O, 0.05~1 CuSO ₄ ·5H ₂ O at a concentration of mg/ℓ, Na ₂ MoO ₄ ·2H ₂ O at a concentration of 0.01~0.25 mg/ℓ, CoCl ₂ ·6H ₂ O at a concentration of 0.01~0.25 mg/ℓ, 10~250 mg/ℓ Myo-inositol at a concentration, Nicotinic acid at a concentration of 0.1 to 2.5 mg/l, Pyridoxine HCl at a concentration of 0.1 to 2.5 mg/l, Thiamine hydrochloride at a concentration of 1 to 25 mg/l ( Thiamine HCl), Sucrose at a concentration of 10 to 35 g/ℓ, Casein hydrolysate at a concentration of 0.1 to 1.5 g/ℓ, MES[2-(N-morpholino) at a concentration of 0.1 to 1.5 g/L )ethanesulfonic acid], calcium gluconate at a concentration of 0.1~1.5 g/ℓ, AVG[L-α-(2-aminoethoxyvinyl)glycine] at a concentration of 0.2~2.5 μM, ogg at a concentration of 400~800 mg/ℓ Mentin (Augmentin; Amoxicillin clavulanate) and gellan gum at a concentration of 1~5 g/ℓ ), and preferably 1-Naphthaleneacetic acid (NAA) at a concentration of 4 to 15 μM, 6-benzylaminopurine (BAP) at a concentration of 2 to 6 μM, 250 to 350 mg/ ℓ concentration of (NH ₄ )H ₂ PO ₄ ·H ₂ O, 2200~2800 mg/ℓ concentration of KNO ₃ , 120~240 mg/ℓ concentration of CaCl ₂ ·2H ₂ O, 350 ~ 450 mg/ℓ concentration MgSO ₄ ·7H ₂ O, Fe-EDTA·3H ₂ O at a concentration of 15~40 mg/ℓ, MnSO ₄ ·H ₂ O at a concentration of 8~15 mg/ℓ, H ₃ BO _{3 at a} concentration of 2~8 mg/ℓ , KI at a concentration of 0.5~2.5 mg/ℓ, ZnSO ₄ ·7H ₂ O at a concentration of 0.5~2.5 mg/ℓ, CuSO ₄ ·5H ₂ O at a concentration of 0.1~0.5 mg/ℓ, Na at a concentration of 0.05~0.2 mg/ℓ ₂ MoO ₄ ·2H ₂ O, CoCl ₂ ·6H ₂ O at a concentration of 0.05~0.2 mg/l, myo-inositol at a concentration of 50~200 mg/l, Nicotinic acid at a concentration of 0.5~2 mg/l acid), Pyridoxine HCl at a concentration of 0.5 to 2 mg/ℓ, Thiamine HCl at a concentration of 5 to 20 mg/L, Sucrose at a concentration of 15 to 30 g/L, 0.2 to 1.0 Casein hydrolysate at a concentration of g/l, MES[2-(N-morpholino)ethanesulfonic acid] at a concentration of 0.2 to 1.0 g/l, calcium gluconate at a concentration of 0.2 to 1.0 g/l, AVG [L-α-(2-aminoethoxyvinyl)glycine] at a concentration of 0.5 to 2.0 μM, augmentin at a concentration of 500 to 700 mg/L (Augmentin; Amoxicillin clavulanate) and gellan gum at a concentration of 2 to 4 g/L.

In addition, in the transformation method of the plant of the Medicago genus of the present invention, the callus-forming medium is a compound for selective growth of cells in which genetic transformation has occurred, for example, kanamycin, hygromycin , Basa, etc. may include one selected from. In the callus-forming medium, the concentration of the compound for selection of transformation is preferably selected from a concentration of 1 to 100 mg/ℓ. For example, the concentration of kanamycin in the callus-forming medium is preferably 20 to 80 mg/L, and more preferably 30 to 60 mg/L. In addition, the concentration of Basta in the callus-forming medium is preferably 2 to 10 mg/L, and more preferably 3 to 8 mg/L.

In addition, in the method for transforming a plant of the Medicago genus of the present invention, the medium for callus formation preferably contains water as a medium. In the method of transforming a plant of the Medicago genus of the present invention, the callus-forming medium is preferably sterilized with an autoclave and then cooled, so that it is present in a solid state by gums, one of the constituents.

Callus Somatic embryo Cultured in the development medium composition Somatic embryos Generating steps

The step of generating somatic embryos in the method for transforming a plant of the Medicago genus of the present invention is characterized by using the above-described medium composition for generating somatic cells. Technical characteristics of the medium composition for generating somatic cell embryos refer to the above description, and detailed descriptions are omitted.

The step of generating somatic embryos in the transformation method of a plant of the Medicago genus of the present invention consists of repeating secondary somatic embryogenesis to increase the number of somatic embryos or obtain uniform plant development. I can.

Somatic embryos Plant development stage

In the transformation method of the Medicago plant of the present invention, the step of developing a somatic embryo into a plant body specifically transfers the callus or leaf section in which the somatic embryo has occurred to a plant development medium and culture to inhibit the repetition of somatic embryos and into the plant. It consists of inducing the development of. The plant development medium may be selected from a variety of known mediums, preferably does not contain auxin and cytokinin in order to suppress repetitive formation of somatic embryos, and the concentration of saccharides decreases compared to the medium composition for somatic cell embryo development. It is characterized by that. The concentration of sucrose, which is a saccharide, in the plant development medium is preferably 2 to 15 g/l and more preferably 5 to 12 g/l. The exchange of the plant development medium can be performed at intervals of 2 to 6 weeks, and preferably, by replacing at intervals of 2 to 3 weeks, the effect of rapidly attenuating the effect of the hormone absorbed in the medium composition for somatic embryo development can be obtained. have. In addition, in the method of transforming a plant of the Medicago genus of the present invention, when the somatic embryo is cultured in a plant development medium for about 2 to 4 weeks, it develops into a plant with a root, and the plant with a root is in a somatic embryo or plant cluster. Transgenic plants can be obtained by separating and planting in a pot containing basic medium or soil excluding sugars.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only intended to clearly illustrate the technical features of the present invention, and do not limit the scope of the present invention.

One. Medicago Trunkatula ( Medicago truncatula ) Of A17 Somatic embryo Identification of optimal conditions for occurrence

(1) Agrobacterium into which vector for plant transformation has been introduced Tumefacien S( Agrobacterium tumefaciens ) Of preparation

The vector for plant transformation pK7WG2D containing the kanamycin resistance gene NPT II and the green fluorescent protein (GFP) gene [Karimi et al., Trends Plant Sci. 2002 May;7(5):193-5.] was introduced into Agrobacterium tumefaciens EHA105 by electroporation. Thereafter, Agrobacterium tumefaciens EHA105, into which the vector for plant transformation was introduced, was inoculated into TY/Ca medium to which the antibiotic Spectinomycin was added at a concentration of 100 mg/ℓ and at 28°C. After culturing, an Agrobacterium suspension was prepared by diluting so that the value of OD600 (Optical Density at 600nm) was 0.5 in the co-culture medium CCM3 described later. Hereinafter, Agrobacterium tumefaciens into which a vector for plant transformation has been introduced is briefly referred to as'Agrobacterium'.

(2) Medicago Trunkatula ( Medicago truncatula ) Leaf section of A17 and Agrobacterium Tumefaciens ( Agrobacterium tumefaciens ) Co-culture

Medicago truncatula cultivar Jemalong A17 ( Medicago truncatula cultivar Jemalong A17) grown for about 2 months. Leaves are collected from plants, the collected leaves are immersed in 70% ethanol for 1 minute, and a 20% aqueous solution of lactose (effective chlorine concentration is about 1). %) for 20 minutes to disinfect the surface. Thereafter, three rectangular leaf slices were prepared from one leaf using a scalpel. Thereafter, the leaf slices were immersed in the previously prepared Agrobacterium suspension suspension and treated for 20 minutes in a vacuum of 20 InHg to induce infection. Thereafter, the Agrobacterium suspension on the leaf slices was removed by absorption with a paper towel, and the leaf slices infected with Agrobacterium were placed on a co-culture medium CCM3 and co-cultured for 3 days in dark conditions at 22°C. Table 1 below shows the components and concentrations of the co-culture medium CCM3.

Ingredients density Schenk and Hildebrandt Basal Salt 1× Sucrose 20 g/ℓ B5 vitamins solution 1× Casein hydrolysates 0.5 g/ℓ MES 0.5 g/ℓ Calcium gluconate 0.5 g/ℓ NAA 10 μM BAP 4 μM GELRITE 3 g/ℓ Acetosyringone 100 μM

* MES: 2-(N-morpholino)ethanesulfonic acid

* NAA: 1-Naphthaleneacetic acid

* BAP: 6-Benzylaminopurine

* GELRITE: Gellan gum

Table 2 below shows the composition of Schenk and Hildebrandt Basal Salt (1×) used as a component of the co-culture medium CCM3. In addition, the composition of the B5 vitamins solution (1,000×) used as a component of the co-culture medium CCM3 is shown in Table 3 below. To the co-culture medium CCM3, the B5 vitamins solution (1,000×) shown in Table 3 was added at a concentration of 1/1,000.

Ingredients density Ammonium phosphate, monobasic, H2O 300 mg/ℓ Potassium nitrate 2500 mg/ℓ Calcium chloride, 2H2O 175 mg/ℓ Magnesium sulfate, 7H2O 400 mg/ℓ Fe-EDTA, 3H2O 25 mg/ℓ Manganese sulfate, H2O 10 mg/ℓ Boric acid 5 mg/ℓ Potassium iodide 1 mg/ℓ Zinc sulfate, 7H2O 1 mg/ℓ Cupric sulfate, 5H2O 0.2 mg/ℓ Sodium molybdate, 2H2O 0.1 mg/ℓ Cobalt chloride, 6H2O 0.1 mg/ℓ

Composition density myo-inositol 100 g/ℓ Nicotinic acid 1 g/ℓ Pyridoxine HCl 1 g/ℓ Thiamine HCl 10 g/ℓ

(3) Callus Optimization of formation conditions

After the above co-cultivation, Agrobacterium formed on leaf slices was washed with sterilized distilled water, and then immersed in an antibiotic augmentin (Augmentin: Amoxicillin/clavulanic acid) solution at a concentration of 300 mg/L and stirred at room temperature for about 4 hr. I did. In addition, various callus-forming media were prepared by combining a hormone with a 40 mg/L concentration of kanamycin in a basal medium for callus formation. Subsequently, the leaf slices were drained, placed on various callus-forming media, and cultured for 4 weeks in weak light conditions. As a result, callus was formed in all combinations of hormonal conditions.

In Table 4 below, the composition and concentration of the base medium for callus formation are shown. Since the composition of Schenk and Hildebrandt Basal Salt (1×) and the composition of B5 vitamins solution (1,000×) are the same as those described in the co-culture medium, detailed descriptions are omitted.

Composition density Schenk and Hildebrandt Basal Salt 1× Sucrose 20 g/ℓ B5 vitamins solution 1× Casein hydrolysates 0.5 g/ℓ MES 0.5 g/ℓ Calcium gluconate 0.5 g/ℓ L-alpha-(2-aminoethoxyvinyl) glycine (AVG) 1 μM Augmentin 600 mg/ℓ GELRITE 3 g/ℓ

* MES: 2-(N-morpholino)ethanesulfonic acid

In addition, in Table 5 below, various combinations of hormones added to the basal medium for callus formation are divided into cytokinin and auxin. The cytokinin is a type of plant hormone and refers to a substance that plays a role in controlling plant growth and promoting cell division. Cytokinins include adenine derivatives such as kinetin, zeatin, and 6-benzylaminopurine (BAP), and diphenylurea and thidiazuron (TDZ). There is a phenylurea derivative. In addition, the auxin is one of the plant growth hormones that promote the growth of plants, and refers to indole acetic acid (IAA) in a narrow sense, but includes both natural and synthetic compounds that act like indole acetic acid in a broad sense. And, auxins in a broad sense are called Auxins. The auxins include natural compounds such as 2-phenylacetic acid (PAA), Indole-3-butyric acid (IBA), and Indole-3-propionic acid (IPA), as well as 2,4-D (2,4-Dichlorophenoxyacetic acid). ; 1-Naphthalene acetic acid (NAA); Synthetic compounds such as 2,4,5-T (2,4,5-Trichlorophenoxyacetic acid) are all included.

Cytokinin Auxins 2,4-D NAA 2.5 μM TDZ 5 μM - 2.5 μM TDZ 10 μM - 2.5 μM TDZ 20 μM - 2.5 μM TDZ 40 μM - 2.5 μM TDZ - 5 μM 2.5 μM TDZ - 10 μM 4 μM BAP 5 μM - 4 μM BAP 10 μM - 4 μM BAP 20 μM - 4 μM BAP 40 μM - 4 μM BAP - 5 μM 4 μM BAP - 10 μM 5 μM Zeatin 5 μM - 5 μM Zeatin 10 μM - 5 μM Zeatin 20 μM - 5 μM Zeatin 40 μM - 5 μM Zeatin - 5 μM 5 μM Zeatin - 10 μM

* TDZ: Thidiazuron

* BAP: 6-Benzylaminopurine

* 2,4-D: 2,4-Dichlorophenoxyacetic acid

* NAA: 1-Naphthaleneacetic acid

Callus was formed in all of the above callus formation media, but the formed callus (leaf slices with callus formed or separated callus mass) were transferred to the optimal somatic embryo development medium EID3, which will be described later, and cultured. When the callus was formed in a callus-forming medium having a hormone combination and then cultured in the somatic embryo-generating medium EID3, the highest embryogenic callus frequency was shown. Specifically, the callus formed in the callus-forming medium of various hormonal combinations was transferred to the somatic embryo-generating medium EID3 to which 40 mg/L of kanamycin was added, and cultured for 4 weeks. As a result, when callus was formed in a callus-forming medium with a combination of 10 μM NAA and 4 μM BAP, and cultured in the somatic embryo development medium EID3, the proportion of leaf slices with embryogenic callus was 38%, The number of somatic embryos generated was 2.875, which was significantly higher than that of the callus-forming medium of other hormone combinations. In Table 6 below, the ratio (%) of leaf slices in which embryogenic callus was formed according to the hormonal combination condition of the callus formation medium was shown in Table 7 below. Indicated the number.

Cytokinin Auxins (μM) 2,4-D NAA 5 10 20 40 5 10 2.5 μM TDZ 13% 0% 0% 0% 13% 6% 4 μM BAP 19% 0% 13% 0% 25% 38% 5 μM Zeatin 6% 6% 0% 0% 13% 6%

Cytokinin Auxins (μM) 2,4-D NAA 5 10 20 40 5 10 2.5 μM TDZ 0.25 0 0 0 0.375 0.25 4 μM BAP 1.375 0 0.25 0 1.875 2.875 5 μM Zeatin 0.25 0.125 0 0 0.375 0.25

From the results of Tables 6 and 7, a callus-forming medium having a hormone combination of 10 μM NAA and 4 μM BAP was selected as the optimal medium, and the medium was named CIM3. Table 8 below shows the composition and concentration of CIM3, which is an optimal callus formation medium.

Composition density Schenk and Hildebrandt Basal Salt 1× Sucrose 20 g/ℓ B5 vitamins solution 1× Casein hydrolysates 0.5 g/ℓ MES 0.5 g/ℓ Calcium gluconate 0.5 g/ℓ NAA 10 μM BAP 4 μM L-alpha-(2-aminoethoxyvinyl) glycine (AVG) 1 μM Augmentin 600 mg/ℓ GELRITE 3 g/ℓ

(4) Somatic embryo Optimization of occurrence conditions

A variety of medium for somatic embryo development was prepared by combining kanamycin and a hormone at a concentration of 40 mg/ℓ in a basic medium for somatic embryo development. Cultured for 4 weeks in callus formation medium CIM3 to induce somatic embryo development by transferring leaf slices or isolated callus masses with callus to various somatic embryo development media, and incubating for 4 weeks under light conditions and 28°C temperature conditions I did.

In Table 9 below, the components and concentrations of the basic medium for somatic embryo development are shown. Since the composition of Schenk and Hildebrandt Basal Salt (1×) and the composition of B5 vitamins solution (1,000×) are the same as those described in the co-culture medium, detailed descriptions are omitted.

Composition density Schenk and Hildebrandt Basal Salt 1× Sucrose 20 g/ℓ B5 vitamins solution 1× Casein hydrolysates 0.5 g/ℓ MES 0.5 g/ℓ Calcium gluconate 0.5 g/ℓ L-alpha-(2-aminoethoxyvinyl) glycine (AVG) 1 μM Augmentin 300 mg/ℓ GELRITE 3 g/ℓ

* MES: 2-(N-morpholino)ethanesulfonic acid

In addition, in Table 10 below, various combinations of hormones added to the basic medium for somatic embryo development are shown.

NAA TDZ BAP 0.5 μM 1 μM - 0.5 μM 2 μM - 0.5 μM 4 μM - 0.5 μM - 4 μM 0.5 μM - 10 μM 0.5 μM - 17 μM 1 μM 1 μM - 1 μM 2 μM - 1 μM 4 μM - 1 μM - 4 μM 1 μM - 10 μM 1 μM - 17 μM 4 μM 1 μM - 4 μM 2 μM - 4 μM 4 μM - 4 μM - 4 μM 4 μM - 10 μM 4 μM - 17 μM

After culturing the callus in various somatic embryo-generating media for 4 weeks, the number of leaf slices with somatic embryos developed and the number of somatic embryos per leaf slice were measured to select the optimal hormone combination. 1 is a Medicago truncatula genotype A17 ( Medicago truncatula cultivar Jemalong A17) after infecting a leaf section of a plant with Agrobacterium tumefaciens into which a vector for plant transformation was introduced, and co-cultured in a co-culture medium CCM3, callus It shows the proportion (%) of leaf slices in which somatic embryos were generated when the callus was formed by culturing in CIM3, a medium for formation, and the formed callus was cultured in a medium for somatic embryo development of various hormone combinations. Figure 2 is a Medicago Truncatula genotype A17 ( Medicago truncatula cultivar Jemalong A17) Infecting the leaf section of a plant with Agrobacterium tumefaciens into which a vector for plant transformation has been introduced, co-culturing in a co-culture medium CCM3, and culturing in CIM3, a medium for callus formation, to form callus, It shows the number of somatic embryos generated per leaf section when the formed callus was cultured in a medium for somatic embryo development of various hormone combinations. As shown in FIGS. 1 and 2, in the medium for somatic embryo development with a hormone combination of 1 μM NAA and 2 μM TDZ, the proportion of leaf slices with somatic embryos was 88%, and the number of somatic embryos per leaf slice was the highest at 5.1. This is a remarkably higher effect than the medium for somatic embryo development with the next highest frequency of 4 μM NAA and 2 μM TDZ hormone combination.

From the results shown in Figs. 1 and 2, a medium for somatic embryo development having a hormone combination of 1 μM NAA and 2 μM TDZ was selected as the optimal medium, and the medium was named EID3. Table 11 below shows the composition and concentration of EID3, which is an optimal somatic embryo development medium.

Composition density Schenk and Hildebrandt Basal Salt 1× Sucrose 20 g/ℓ B5 vitamins solution 1× Casein hydrolysates 0.5 g/ℓ MES 0.5 g/ℓ Calcium gluconate 0.5 g/ℓ NAA 1 μM TDZ 2 μM L-alpha-(2-aminoethoxyvinyl) glycine (AVG) 1 μM Augmentin 300 mg/ℓ GELRITE 3 g/ℓ

2. Kanamycin ( Kanamycin ) Transformed by a resistance gene Medicago Tron Katula( Medicago truncatula ) Production of A17 plant

(1) Agrobacterium transgenic recombinant vector and recombinant vector introduced Tumefaciens ( Agrobacterium tumefaciens ) Of preparation

As a kanamycin selection system, an overexpression vector of the GA2-oxidase 10 (MtGA2ox10) gene, which is a Medicago Gibberellin degrading enzyme, was produced, and it was introduced into Agrobacterium tumefaciens EHA105 and introduced into Medicago Trunkatula. truncatula ) was used for transformation of A17. Specifically, first amplify the MtGA2ox10 gene using Medicago's first strand cDNA as a template, and clone it into the pDONR221 plasmid according to the GatewayTM (Invitrogen, USA) experimental procedure, and then again this binary vector pK7WG2D (Karimi et al., 2002). ), the recombinant plasmid GA2ox10-OX was constructed. The pK7WG2D vector has a kanamycin resistance gene NPT II and a green fluorescent protein (GFP) gene as plant selection markers. In addition, primers and nucleotide sequences for amplifying the amplification of the MtGA2ox10 gene are as follows.

* Forward primer: 5'-AAAAAGCAGGCTtcATGATTGACTCAAATCCACC-3'

* Reverse primer: 5'-AGAAAGCTGGGTcCTAAGCCATGGTCGTTGTGT-3'

Later, Agrobacterium tumefaciens EHA105 ( Agrobacterium tumefaciens EHA105) was introduced into GA2ox10-OX plasmid by electroporation, and inoculated into TY/Ca medium to which spectinomycin, an antibiotic was added at a concentration of 100 mg/L, and cultured at 28°C, co-cultured Agrobacterium suspension was prepared by diluting the medium CCM3 so that the value of OD600 (Optical Density at 600nm) became 0.5.

(2) Medicago Trunkatula ( Medicago truncatula ) Leaf section of A17 and Agrobacterium Tumefaciens ( Agrobacterium tumefaciens ) Co-culture

Medicago Truncatula genotype A17 ( Medicago truncatula cultivar Jemalong A17) was grown in pots for about 2 months, and leaves were harvested. Thereafter, the collected leaves were stirred in 70% ethanol for 1 minute to disinfect the surface, and then added to a solution consisting of 20% aqueous Lax solution (effective chlorine concentration is about 1%) and a little Tween-20 for 20 minutes. It was sterilized during. Thereafter, the aqueous solution of Lax was removed, and the leaf was washed with sterile distilled water to remove all of the Lax component present on the leaf surface. Thereafter, moisture from the sterilized leaves was removed with a paper towel. After that, using a scalpel, three leaf slices were prepared from one leaf in a rectangular shape. Thereafter, the leaf slices were immersed in the previously prepared Agrobacterium suspension suspension, placed in a vacuum ruler, and a vacuum of about 20 InHg was applied for 20 minutes to induce infection. After releasing the vacuum, the Agrobacterium suspension on the leaf slices was absorbed on a paper towel to partially remove it, and placed on a co-culture medium CCM3, followed by co-culture for 3 days in dark conditions at 22°C.

(3) Callus Formation induction

After the above co-cultivation, Agrobacterium, which formed colonies on the surface of leaf slices, was removed, and placed on the callus-forming medium CIM3 to induce the formation of embryogenic callus. Specifically, in order to remove Agrobacterium from the surface of leaf slices, repeat the leaf slices 4 to 5 times, immerse them in sterile distilled water and stir to wash away Agrobacterium, and immerse them in a 300 mg/L aqueous agmentin sulfate solution at room temperature. The mixture was stirred for 4 hr so that antibiotics were absorbed into the leaf slices. Thereafter, the leaf slices were touched on a paper towel to remove moisture, and placed on callus-forming medium CIM3 to which 40 mg/ℓ concentration of kanamycin was added, and cultured under weak light conditions. After about 2-3 weeks, it was transferred to a new medium. When Agrobacterium grew, Agrobacterium was removed and transferred to a new medium according to the above method. Callus was formed on most leaf slices after about 4-6 weeks.

(4) Somatic embryo Induction of development and acquisition of transgenic plants

Callus-formed leaf slices or isolated callus masses were transferred to the somatic embryo development medium EID3 to which 40 mg/L of kanamycin was added and cultured. As a result, about 2 weeks after cultivation in the somatic embryo development medium EID3, the development of somatic embryos was observed, and after 6 to 8 weeks, the development of secondary embryos progressed to form clusters or clusters. Thereafter, the primary somatic embryos or the clusters or clusters of the secondary somatic embryos were transferred to the plant development medium SDM3 to which 50 mg/l concentration of kanamycin was added and cultured, and as a result, a complete plant was developed. Figure 3 is a Medicago Truncatula transformed by a kanamycin resistance gene ( Medicago truncatula ) A17 shows the development of somatic embryos and plants during the production process. Figure 3A shows the appearance of somatic embryos when the callus is transferred to the somatic embryo development medium EID3 and cultured for 4 weeks, and FIG. 3B is a primary somatic embryo generated on the somatic embryo development medium EID3 and Repetition of secondary embryogenesis is indicated by arrows, and C in FIG. 3 shows the appearance of a plant developed when transferred to the plant development medium SDM3 and cultured.

It stems and leaves are fully developed and planted to grow the plant roots have developed a planter soil Medicare cargo agent reonka Tula (Medicago truncatula ) A17 transgenic plants were obtained. Table 12 shows the composition and concentration of the plant development medium SDM3.

Composition density Schenk and Hildebrandt Basal Salt 1× Sucrose 10 g/ℓ B5 vitamins solution 1× MES 0.5 g/ℓ Calcium gluconate 0.5 g/ℓ L-alpha-(2-aminoethoxyvinyl) glycine (AVG) 1 μM Plant agar 8 g/ℓ

(5) Verification of transformation of plants

Medicago Trunkatula obtained earlier ( Medicago truncatula ) A17 transgenic plant was verified by whether or not green fluorescent protein (GFP) contained in the transformation vector was introduced into the plant. Specifically, the obtained Medicago Trunkatula ( Medicago truncatula ) A17 transgenic plants were examined with a fluorescence microscope to observe whether or not green fluorescence appeared. In addition, Medicago Truncatula obtained using polymerase chain reaction (PCR) truncatula ) A17 transgenic plants were amplified and the GFP gene was measured in the amplification product. The Medicago Trunkatula ( Medicago truncatula ) The primers and nucleotide sequences used to amplify the GFP gene from the transgenic plant of A17 are as follows.

* Forward primer: 5'-TTCATCTGCACCACCGGCAAG-3'

* Reverse primer: 5'-GATCGCGCTTCTCGTTGGGGT-3'

(6) Experiment result

4 is a step-by-step view of the production process of a Medicago truncatula A17 plant transformed with a kanamycin resistance gene. Contents of each step shown in FIG. 4 are as follows.

* Step A: Medicago Trunkatula truncatula ) A17 leaf slices of plant Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) and placed on the co-culture medium CCM3.

* Step B: Transferring the co-cultured leaf slices to callus formation medium CIM3 and inducing callus formation. The upper part is the second week of culture, and the lower part is the green fluorescent protein (GFP) fluorescence. It is the appearance of observing.

* Step C: This is the appearance of somatic embryos when the leaf section on which the callus was formed was transferred to EID3 for somatic embryo development and cultured.

* Step D: Secondary embryo generation is repeated on the somatic embryo development medium EID3 to form a somatic embryo bundle.

* Step E: Plant stem and leaves are formed on the plant development medium SDM3.

* Step F: Medicago truncatula transformed by the kanamycin resistance gene grown by planting in a pot truncatula ) A17 plant.

5 is a result of observing the fluorescence of a green fluorescent protein (GFP) of a Medicago truncatula A17 plant transformed with a kanamycin resistance gene. FIG. 5A is a view before fluorescence is irradiated, and FIG. 5B is a view observed with a fluorescence microscope. As shown in FIG. 5, the transgenic plant exhibited a distinct green fluorescence in the stem of the leaf in fluorescence microscopy.

Figure 6 is a kanamycin (Kanamycin) resistance gene transformed by using a chain polymerase reaction (PCR) Medicago Trunkatula ( Medicago truncatula ) This is the result of confirming the green fluorescent protein (GFP) gene in the A17 plant. As shown in FIG. 6, the amplified DNA of the same size as 514 bp of the vector plasmid GA2ox10-OX used as a positive control sample was shown in all of the 23 transgenic plants to be investigated. Thus, it can be seen that transformation has occurred in all plants.

3. Busta ( Basta ) Transformed by a resistance gene Medicago Trunkatula ( Medicago truncatula ) Production of A17 plant

(1) Agrobacterium tumefaciens (Agrobacterium tumefaciens) ( Agrobacterium tumefaciens ) Of preparation

As a Basta selection system, a CRISPR/Cas9 vector, pGK3304 plasmid, was introduced into Agrobacterium tumefaciens EHA105, and Medicago truncatula ( Medicago truncatula ) was used for transformation of A17. Basta is a product brand of herbicides containing glufosinate ingredients. In addition, the pGK3304 plasmid has a basa resistance gene Bar, a green fluorescent protein (GFP) gene, and a Cas9 gene as plant selection markers. Agrobacterium tumefaciens EHA105 ( Agrobacterium tumefaciens EHA105) by electroporation, inoculated into TY/Ca medium to which spectinomycin, an antibiotic was added at a concentration of 100 mg/L, and incubated at 28°C, co-culture medium CCM3 Agrobacterium suspension was prepared by diluting so that the value of OD600 (Optical Density at 600nm) was 0.5.

(2) Medicago Trunkatula ( Medicago truncatula ) Leaf slices of A17 and Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) Co-culture

Medicago Truncatula genotype A17 ( Medicago truncatula cultivar Jemalong A17) was grown in pots for about 2 months, and leaves were harvested. Thereafter, the collected leaves were stirred in 70% ethanol for 1 minute to disinfect the surface, and then added to a solution consisting of 20% aqueous Lax solution (effective chlorine concentration is about 1%) and a little Tween-20 for 20 minutes. It was sterilized during. Thereafter, the aqueous solution of Lax was removed, and the leaf was washed with sterile distilled water to remove all of the Lax component present on the leaf surface. Thereafter, moisture from the sterilized leaves was removed with a paper towel. After that, using a scalpel, three leaf slices were prepared from one leaf in a rectangular shape. Thereafter, the leaf slices were immersed in the previously prepared Agrobacterium suspension suspension, placed in a vacuum ruler, and a vacuum of about 20 InHg was applied for 20 minutes to induce infection. After releasing the vacuum, the Agrobacterium suspension on the leaf slices was absorbed on a paper towel to partially remove it, and placed on a co-culture medium CCM3, followed by co-culture for 3 days in dark conditions at 22°C.

(3) induction of callus formation

After the above co-cultivation, Agrobacterium, which formed colonies on the surface of leaf slices, was removed and placed on callus-forming medium CIM3 to induce the formation of embryogenic callus. Specifically, in order to remove Agrobacterium from the surface of leaf slices, repeat the leaf slices 4 to 5 times, immerse them in sterile distilled water and stir to wash away Agrobacterium, and immerse them in a 300 mg/L aqueous agmentin sulfate solution at room temperature. The mixture was stirred for 4 hr so that antibiotics were absorbed into the leaf slices. Thereafter, the leaf slices were brought into contact with a paper towel to remove moisture, and placed on a callus-forming medium CIM3 to which a concentration of 3 mg/l Basta was added, and cultured under weak light conditions. After about 2-3 weeks, it was transferred to a new medium. When Agrobacterium grew, Agrobacterium was removed and transferred to a new medium according to the above method. Callus was formed on most leaf slices after about 4-6 weeks.

(4) Induction of somatic embryo development and acquisition of transgenic plants

The leaf slices with callus formed or the isolated callus mass were transferred to the somatic embryo development medium EID3 to which 5 mg/L of Basta was added and cultured. As a result, about 2 weeks after cultivation in the somatic embryo development medium EID3, the development of somatic embryos was observed, and after 6 to 8 weeks, the development of secondary embryos progressed to form clusters or clusters. Thereafter, the primary somatic embryos or the clusters or clusters of the secondary somatic embryos were transferred to the plant development medium SDM3 containing 5 mg/l of Basta, and cultured, and as a result, a complete plant was developed. It stems and leaves are fully developed and planted to grow the plant roots have developed a planter soil Medicare cargo agent reonka Tula (Medicago truncatula ) A17 transgenic plants were obtained.

(5) Verification of transformation of plants

Medicago Trunkatula obtained earlier ( Medicago truncatula ) A17 transgenic plant was verified by whether or not green fluorescent protein (GFP) contained in the transformation vector was introduced into the plant. Specifically, the obtained Medicago Trunkatula ( Medicago truncatula ) A17 transgenic plants were examined with a fluorescence microscope to observe whether or not green fluorescence appeared. In addition, Medicago Truncatula obtained using polymerase chain reaction (PCR) truncatula ) A17 transgenic plants were amplified and the GFP gene was measured in the amplification product. The Medicago Trunkatula ( Medicago truncatula ) The primers and nucleotide sequences used to amplify the GFP gene from the transgenic plant of A17 are as follows.

* Forward primer: 5'-TTCATCTGCACCACCGGCAAG-3'

* Reverse primer: 5'-GATCGCGCTTCTCGTTGGGGT-3'

(6) Experiment result

7 is a step-by-step view of the production process of a Medicago truncatula A17 plant transformed by a basta resistance gene. Contents of each step shown in FIG. 7 are as follows.

* Step A: Medicago Trunkatula truncatula ) A17 leaf slices of plant Agrobacterium tumefaciens ( Agrobacterium tumefaciens ), and after co-culture in the co-culture medium CCM3, the leaf sections are transferred to the callus formation medium CIM3 and cultured to induce callus formation.The top is the second week of culture and the bottom is the green fluorescent protein ( Green fluorescent protein, GFP) fluorescence was observed.

* Step B: When cultured in CIM3, a culture medium for callus formation, for 4 weeks, embryogenic callus is formed (indicated by an arrow).

* Step C: The appearance of somatic embryos when the leaf section with callus was transferred to EID3 for somatic embryo development and cultured for 4 weeks.

* Step D: When the callus in which the somatic embryo has occurred is transferred and cultured on the plant development medium SDM3, it is developing into a plant.

* Step E: Medicago Truncatula transformed by the Basta resistance gene grown by planting in a pot truncatula ) A17 plant.

FIG. 8 is a result of observing the fluorescence of a green fluorescent protein (GFP) of a Medicago truncatula A17 plant transformed by a Basta resistance gene. 8A is a view before fluorescence is irradiated, and FIG. 8B is a view observed with a fluorescence microscope. As shown in FIG. 8, the transgenic plant exhibited a distinct green fluorescence on the stem of the leaf in fluorescence microscopy.

9 is a Medicago Truncatula transformed by a Basta resistance gene using a chain polymerase reaction (PCR) ( Medicago truncatula ) This is the result of confirming the green fluorescent protein (GFP) gene in the A17 plant. As shown in FIG. 9, the amplified DNA of the same size as 514 bp of the vector plasmid pGK3304 used as a positive control sample was shown in all of the 23 transgenic plants to be investigated. Thus, it can be seen that transformation has occurred in all plants.

4. Conventional Somatic embryo Transformation efficiency comparison with the generation method

Somatic embryo development medium P4 10:4:1:5 (Nolan et al., 2014, PLOS ONE, Volume) of the method claimed to have high somatic embryo generation efficiency among the conventional methods and can be applied to the Medicago Truncatula A17 genotype. 9, Issue 6, e99908) was compared with EID3, the medium for somatic cell embryo development of the present invention. In Table 13 below, components and concentrations of the somatic cell embryo development medium P4 10:4:1:5 are shown.

Composition density B5 Basal Salt 1× Sucrose 30 g/ℓ B5 vitamins solution 1× MES 0.5 g/ℓ Casein hydrolysates 0.5 g/ℓ NAA 10 μM BAP 4 μM ABA 1 μM GA 5 μM Plant agar 8 g/ℓ

* NAA: 1-Naphthaleneacetic acid

* BAP: 6-Benzylaminopurine

* ABA: Abscisic Acid

* GA: Gibberellic Acid

The characteristic of somatic embryo development medium P4 10:4:1:5 is that 1 μM ABA and 5 μM GA are added to the hormone combination of 10 μM NAA and 4 μM BAP to promote somatic embryo development.Medicago Trunka It has been argued that the Tula 2HA genotype showed high somatic embryogenicity, and the A17 genotype also showed low somatic embryogenicity.

In order to compare the somatic embryo generation efficiency of the conventional somatic embryo development medium P4 10:4:1:5 and the somatic embryo development medium EID3 of the present invention with respect to Medicago truncatula A17, Nolan et al. Except for the use of P4 10:4 (10 μM NAA, 4 μM BAP) used in. And P4 10:4:1:5 used in Nolan et al. In the same way, the kanamycin (Kanamycin) resistance gene transformed by Medicago truncatula ( Medicago truncatula ) A17 plants and Medicago truncatula A17 plants transformed by the Basta resistance gene were produced. The somatic embryo generation efficiency of the conventional method and the present invention was evaluated by the percentage (%) of leaf slices with somatic embryos and the number of somatic embryos generated per leaf slice. Figure 10 is the present invention when producing a Medicago truncatula A17 plant transformed by a kanamycin resistance gene using Nolan et al.'s callus formation medium and somatic cell embryo development medium Medicago trunkatula transformed by kanamycin resistance gene using a medium for callus formation and a medium for somatic embryo development of truncatula ) shows the development of somatic embryos when A17 plants are produced. In FIG. 10, the left side is the result when the conventional method is used, and the right side is the result when the method of the present invention is used. Medicago truncatula transformed by a kanamycin resistance gene by a conventional method ( Medicago truncatula ) When A17 plants were produced, the percentage (%) of leaf slices with somatic embryos was about 8%, and the number of somatic embryos per leaf slice was about 0.1. On the other hand, when producing a Medicago truncatula A17 plant transformed by a kanamycin resistance gene by the method of the present invention, the proportion (%) of leaf fragments with somatic embryos is about 86% and leaves It was found that the number of somatic embryos per section was about 4.78. When producing a Medicago truncatula A17 plant transformed by a Basta resistance gene using Nolan et al.'s callus formation medium and somatic cell embryo development medium and the callus formation of the present invention Medicago Truncatula transformed by a Basta resistance gene using a medium for cultivation and somatic embryo development. truncatula ) A17 plants showed similar results. As a result, the medium EID3 for somatic cell embryo development of the present invention showed about 10 times higher somatic cell embryo generation efficiency than the conventional somatic cell embryo development medium P4 10:4:1:5.

As described above, the present invention has been described through the above embodiments, but the present invention is not necessarily limited thereto, and various modifications may be possible without departing from the scope and spirit of the present invention. Accordingly, the scope of protection of the present invention should be construed as including all embodiments falling within the scope of the claims appended to the present invention.

Claims (16)

delete delete delete delete delete delete Medicago truncatula ( Medicago truncatula ) the step of co-culturing the explant (Explant) of the plant with the transgene introduced Agrobacterium bacteria;
Culturing the co-cultured explants in a callus-forming medium composition to form a callus;
Culturing the callus in a medium composition for generating somatic embryos to generate somatic embryos; And
A method comprising the step of developing the somatic embryo into a plant,
The callus-forming medium composition includes 1-naphthaleneacetic acid (NAA) at a concentration of 4 to 15 μM and 6-benzylaminopurine at a concentration of 2 to 8 μM (6-Benzylaminopurine, BAP) based on the total volume of the medium composition. It is a medium composition comprising a,
The medium composition for somatic embryo development includes 1-naphthaleneacetic acid (NAA) at a concentration of 0.8 to 2 μM and Thidiazuron (TDZ) at a concentration of 1.5 to 3 μM based on the total volume of the medium composition. A method for transforming a plant of the genus Medica, characterized in that it is a medium composition.
8. The method of claim 7, wherein the explants are leaf slices.
The method of claim 7, wherein the transgene is at least one selected from antibiotic resistance gene, herbicide resistance gene, disease resistance gene, virus resistance gene, stress resistance gene, enzyme biosynthesis gene, recombinant protein expression gene, or plant growth promoting gene. The transformation method of a plant of the genus Medica, characterized in that consisting of.
8. The method of claim 7, wherein the Agrobacterium genus is Agrobacterium tumefaciens .
The method of claim 7, wherein the callus-forming medium is 1-naphthaleneacetic acid (NAA) at a concentration of 4 to 15 μM and 6-benzylaminopurine at a concentration of 2 to 8 μM based on the total volume of the medium composition. -Benzylaminopurine, BAP), (NH ₄ )H ₂ PO ₄ ·H ₂ O at a concentration of 200~400 mg/ℓ, KNO _{3 at a} concentration of 2000~3000 mg/ℓ, CaCl ₂ ·2H at a concentration of 75~275 mg/ℓ ₂ O, 300~500 mg/ℓ concentration of MgSO ₄ ·7H ₂ O, 10~50 mg/ℓ concentration of Fe-EDTA·3H ₂ O, 5~25 mg/ℓ concentration of MnSO ₄ ·H ₂ O, 1 H ₃ BO _{3 at a} concentration of ~15 mg/ℓ, KI at a concentration of 0.2 to 4 mg/ℓ, ZnSO ₄ 7H ₂ O at a concentration of 0.2 to 4 mg/ℓ, CuSO ₄ 5H _{2 at a} concentration of 0.05 to 1 mg/ℓ O, 0.01~0.25 mg/l concentration of Na ₂ MoO ₄ ·2H ₂ O, 0.01~0.25 mg/l concentration of CoCl ₂ ·6H ₂ O, 10~250 mg/l concentration of myo-inositol, Nicotinic acid at a concentration of 0.1 to 2.5 mg/l, Pyridoxine HCl at a concentration of 0.1 to 2.5 mg/l, Thiamine HCl at a concentration of 1 to 25 mg/l, 10 to 35 g/l Sucrose at concentration, Casein hydrolysate at concentration of 0.1 to 1.5 g/ℓ, MES [2-(N-morpholino)ethanesulfonic acid] at concentration of 0.1 to 1.5 g/ℓ, 0.1 to 1.5 g/ ℓ concentration of calcium gluconate, 0.2-2.5 μM concentration of AVG [L-α-(2-aminoethoxyvinyl) glycine], 400-800 mg/ℓ concentration of Augmentin (Augmentin; Amoxicillin clavulanate) and 1- A type of plant of the genus Medica, characterized in that it contains gellan gum at a concentration of 5 g/ℓ Vaginal conversion method.
The method of claim 7, wherein the medium composition for somatic cell embryo development is 1-Naphthaleneacetic acid (NAA) at a concentration of 0.8 to 2 μM, thidiazuron at a concentration of 1.5 to 3 μM, 200 based on the total volume of the medium composition. ~400 mg/ℓ concentration of (NH ₄ )H ₂ PO ₄ ·H ₂ O, 2000 ~ 3000 mg/ℓ concentration of KNO ₃ , 75 ~ 275 mg/ℓ concentration of CaCl ₂ ·2H ₂ O, 300 ~ 500 mg /ℓ concentration of MgSO ₄ ·7H ₂ O, 10 ~ 50 ㎎ / ℓ concentration of Fe-EDTA · 3H ₂ O, 5 ~ 25 ㎎ / ℓ concentration of MnSO ₄ ·H ₂ O, 1 ~ 15 ㎎ / ℓ concentration of H ₃ BO ₃ , KI at a concentration of 0.2~4 mg/ℓ, ZnSO ₄ ·7H ₂ O at a concentration of 0.2~4 mg/ℓ, CuSO ₄ ·5H ₂ O at a concentration of 0.05~1 mg/ℓ, 0.01~0.25 mg/ ℓ concentration of Na ₂ MoO ₄ ·2H ₂ O, 0.01~0.25 mg/ℓ concentration of CoCl ₂ ·6H ₂ O, 10~250 mg/ℓ concentration of myo-inositol, 0.1~2.5 mg/ℓ concentration Nicotinic acid, Pyridoxine HCl at a concentration of 0.1 to 2.5 mg/L, Thiamine HCl at a concentration of 1 to 25 mg/L, Sucrose at a concentration of 10 to 35 g/L , Casein hydrolysate at a concentration of 0.1~1.5 g/ℓ, MES[2-(N-morpholino)ethanesulfonic acid] at a concentration of 0.1~1.5 g/ℓ, calcium gluconate at a concentration of 0.1~1.5 g/ℓ ( Calcium gluconate), 0.2~2.5 μM AVG[L-α-(2-aminoethoxyvinyl)glycine], 200~900 mg/L augmentin (Augmentin; Amoxicillin clavulanate) and 1~5 g/L gellan A method for transforming a plant of the genus Medica, comprising a gum (Gellan gum).
Medicago truncatula ( Medicago truncatula ) step of culturing the explant (Explant) of the plant in a callus-forming medium composition to form a callus;
Co-culturing the callus with the Agrobacterium genus into which the transgene is introduced;
Culturing the co-cultured callus in a medium composition for generating somatic cell embryos to generate somatic cell embryos; And
A method comprising the step of developing the somatic embryo into a plant,
The callus-forming medium composition includes 1-naphthaleneacetic acid (NAA) at a concentration of 4 to 15 μM and 6-benzylaminopurine at a concentration of 2 to 8 μM (6-Benzylaminopurine, BAP) based on the total volume of the medium composition. It is a medium composition comprising a,
The medium composition for somatic embryo development includes 1-naphthaleneacetic acid (NAA) at a concentration of 0.8 to 2 μM and Thidiazuron (TDZ) at a concentration of 1.5 to 3 μM based on the total volume of the medium composition. A method for transforming a plant of the genus Medica, characterized in that it is a medium composition.
The method of claim 13, wherein the Agrobacterium genus is Agrobacterium tumefaciens .
The method of claim 13, wherein the callus-forming medium is 1-naphthaleneacetic acid (NAA) at a concentration of 4 to 15 μM and 6-benzylaminopurine at a concentration of 2 to 8 μM based on the total volume of the medium composition. -Benzylaminopurine, BAP), (NH ₄ )H ₂ PO ₄ ·H ₂ O at a concentration of 200~400 mg/ℓ, KNO _{3 at a} concentration of 2000~3000 mg/ℓ, CaCl ₂ ·2H at a concentration of 75~275 mg/ℓ ₂ O, 300~500 mg/ℓ concentration of MgSO ₄ ·7H ₂ O, 10~50 mg/ℓ concentration of Fe-EDTA·3H ₂ O, 5~25 mg/ℓ concentration of MnSO ₄ ·H ₂ O, 1 H ₃ BO _{3 at a} concentration of ~15 mg/ℓ, KI at a concentration of 0.2 to 4 mg/ℓ, ZnSO ₄ 7H ₂ O at a concentration of 0.2 to 4 mg/ℓ, CuSO ₄ 5H _{2 at a} concentration of 0.05 to 1 mg/ℓ O, 0.01~0.25 mg/l concentration of Na ₂ MoO ₄ ·2H ₂ O, 0.01~0.25 mg/l concentration of CoCl ₂ ·6H ₂ O, 10~250 mg/l concentration of myo-inositol, Nicotinic acid at a concentration of 0.1 to 2.5 mg/l, Pyridoxine HCl at a concentration of 0.1 to 2.5 mg/l, Thiamine HCl at a concentration of 1 to 25 mg/l, 10 to 35 g/l Sucrose at concentration, Casein hydrolysate at concentration of 0.1 to 1.5 g/ℓ, MES [2-(N-morpholino)ethanesulfonic acid] at concentration of 0.1 to 1.5 g/ℓ, 0.1 to 1.5 g/ ℓ concentration of calcium gluconate, 0.2-2.5 μM concentration of AVG [L-α-(2-aminoethoxyvinyl) glycine], 400-800 mg/ℓ concentration of Augmentin (Augmentin; Amoxicillin clavulanate) and 1- The plant of the genus Medica, characterized in that it contains gellan gum at a concentration of 5 g/ℓ Transformation method.
The method of claim 13, wherein the medium composition for developing somatic cell embryos is 1-Naphthaleneacetic acid (NAA) at a concentration of 0.8 to 2 μM, and thidiazuron at a concentration of 1.5 to 3 μM, based on the total volume of the medium composition. ~400 mg/ℓ concentration of (NH ₄ )H ₂ PO ₄ ·H ₂ O, 2000 ~ 3000 mg/ℓ concentration of KNO ₃ , 75 ~ 275 mg/ℓ concentration of CaCl ₂ ·2H ₂ O, 300 ~ 500 mg /ℓ concentration of MgSO ₄ ·7H ₂ O, 10 ~ 50 ㎎ / ℓ concentration of Fe-EDTA · 3H ₂ O, 5 ~ 25 ㎎ / ℓ concentration of MnSO ₄ ·H ₂ O, 1 ~ 15 ㎎ / ℓ concentration of H ₃ BO ₃ , KI at a concentration of 0.2~4 mg/ℓ, ZnSO ₄ ·7H ₂ O at a concentration of 0.2~4 mg/ℓ, CuSO ₄ ·5H ₂ O at a concentration of 0.05~1 mg/ℓ, 0.01~0.25 mg/ ℓ concentration of Na ₂ MoO ₄ ·2H ₂ O, 0.01~0.25 mg/ℓ concentration of CoCl ₂ ·6H ₂ O, 10~250 mg/ℓ concentration of myo-inositol, 0.1~2.5 mg/ℓ concentration Nicotinic acid, Pyridoxine HCl at a concentration of 0.1 to 2.5 mg/L, Thiamine HCl at a concentration of 1 to 25 mg/L, Sucrose at a concentration of 10 to 35 g/L , Casein hydrolysate at a concentration of 0.1~1.5 g/ℓ, MES[2-(N-morpholino)ethanesulfonic acid] at a concentration of 0.1~1.5 g/ℓ, calcium gluconate at a concentration of 0.1~1.5 g/ℓ ( Calcium gluconate), 0.2~2.5 μM AVG[L-α-(2-aminoethoxyvinyl)glycine], 200~900 mg/L augmentin (Augmentin; Amoxicillin clavulanate) and 1~5 g/L gellan A method for transforming a plant of the genus Medica, comprising a gum (Gellan gum).

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