Transformant selection method for plant

Abstract

The purpose of the present invention is to develop and provide a transformation method that improves the selection rate of a transformant of a plant and reduces the appearance rate of escape individuals and chimeric individuals in regenerated individuals.　According to the present invention, a transformant can be efficiently selected by using a step for introducing a gene into a plant cell and subsequently immersing a tissue, generated in a selection medium to which a selection pressure is applied, in a solution containing the same agent as a selection agent used in the medium.

Description

Method for selecting plant transformants ,

[0005]

[0001] The present invention relates to a method for selecting plant transformants.

[0002] Leguminous plants are extremely important plants in agriculture that are closely related to human life as agricultural crops, green manure plants, ornamental plants, nectar source plants, and even timber. In particular, soybean (Glycine max) is used as a raw material for processed foods such as tofu and natto, seasonings such as soy sauce and miso, as well as a raw material for edible oil and livestock feed, and is one of the extremely important agricultural crops in the world.

[0003] By the way, in order to obtain leguminous plants having useful traits such as high yield and disease resistance, generally, variety improvement by crossing based on breeding methods is carried out. However, since this method requires about 10 years per variety until the target variety is obtained, in recent years, it has the problem that it cannot respond immediately to abnormal weather and the spread of infectious diseases that have become apparent. Therefore, variety improvement of plants by gene recombination technology and genome editing technology is expected to become even more important in the future.

[0004] In both gene recombination technology and genome editing technology, the transformation technology of the target plant is important. In leguminous plants, a soybean transformation method was developed in 1988 and has contributed to the field of molecular biology for the purpose of variety improvement and plant physiological research (Non-Patent Documents 1 and 2). However, after that, despite being studied for more than 35 years and various research and developments being carried out, the transformation rate of soybeans has not improved since the initial development and remains at 5-10% to this day. This is in contrast to the fact that the transformation methods of rice, corn, and wheat were developed around the same time as soybeans, and their efficiency has improved dramatically through subsequent development (Non-Patent Documents 3-5).

[0005] Furthermore, in the case of soybeans, transformation is not possible for the vast majority of varieties, and the period from nucleic acid inoculation using Agrobacterium, etc., to rooting of regenerated individuals takes more than four months, compared to two months for potatoes, corn, and wheat. In addition, there are many escape individuals and chimeric individuals among the regenerated individuals produced by the transformation method, among other issues that make up the current transformation method (Non-Patent Literature 6). In addition to these problems, although various methods have been used to encourage rooting from transformed soybean shoots, there is no method that consistently produces rooting with high efficiency, which is a problem in cultivating transformed soybeans.

[0006] Hinchee et al., 1988, Bio / Technology, 6; 915-922Mccabe et al., 1988, Bio / Technology, 6; 923-926Hiei and Komari, 2008, Nature Protocols, 3; 824-834Ishida et al., 2007, Nature Protocols, 2; 1614-1621Ishida et al., 2015, Methods in Molecular Biology, 1223; 189-198Xu et al., 2022, Front Plant Sci., 13:900318; 1-33

[0007] The problem to be solved by the present invention is to develop and provide a new transformation method for improving the selection rate of plant transformants, including soybeans, and for reducing the occurrence rate of escape individuals and chimeric individuals in the selected individuals.

[0008] To solve the above problems, the inventors focused on the selection process of transformants in transformation methods and diligently conducted research. As a result, they succeeded in isolating only transformed individuals with extremely high efficiency by immersing tissues such as shoots or buds that have grown in a selection medium under selection pressure after gene introduction into a solution containing the same selection agent used in the medium. This method also demonstrated that the appearance of escape individuals and chimeric individuals, which have been a problem in conventional soybean transformation methods, can be efficiently suppressed. The present invention is based on these findings and includes the following.

[0009] (1) A method for selecting plant transformants, comprising: an introduction step of co-introducing a selection marker gene and a target gene into target plant cells; a shoot induction step of culturing the target plant cells after the introduction step in a shoot-forming medium to induce shoot tissue; an immersion step of immersing the target plant cells after the shoot induction step in a solution that enables selection based on the expression of the selection marker gene; and a transformant selection step of selecting transformants from the shoot tissue after the immersion step based on the expression of the selection marker gene. (2) A method for selecting plant transformants, comprising: an introduction step of co-introducing a selection marker gene and a target gene into target plant cells; a shoot induction step of culturing the target plant cells after the introduction step in a shoot-forming medium to induce shoot tissue; a selection step of culturing the target plant cells after the shoot induction step in a selection medium containing a selection agent that enables selection based on the expression of the selection marker gene; an immersion step of immersing the shoot tissue generated after the selection step in a solution containing the selection agent; and a transformant selection step of selecting transformants from the shoot tissue after the immersion step based on the expression of the selection marker gene. (3) The method according to (1), wherein the immersion step is carried out under reduced pressure. (4) The method according to (1) or (2), wherein the selection marker gene is a drug resistance gene to the selection agent. (5) The method according to any one of (1) to (3), wherein the co-introduction of the gene in the introduction step is carried out by any method selected from the group consisting of the Agrobacterium method, the particle gun method, the electroporation method, and the protoplast method. (6) The method according to any one of (1) to (4), wherein the shoot tissue is a shoot or a sprout. (7) The method according to any one of (1) to (5), wherein the plant is a legume. (8) A method for producing a transformed regenerated plant, comprising a regeneration step of culturing a transformed plant obtained after the transformant selection step in the plant transformant selection method according to any one of (1) to (7) to regenerate the plant body. This specification includes the disclosures of Japanese Patent Application No. 2025-020143, which forms the basis of the priority of this application.

[0010] According to the transformant selection method of the present invention, the appearance of escape individuals and chimeric individuals can be suppressed, and at the same time, the desired transformants can be efficiently selected.

[0011] According to the transformation method of the present invention, the selection rate of plant transformants can be dramatically improved compared to conventional methods.

[0012] This figure shows the tissue on solid culture media after each step in Example 1. A shows half-cotyledons of soybean cotyledons cultured on co-existence medium after inoculation with Agrobacterium into which the bar gene was introduced in the introduction step. B shows soybean tissue in which multiple shoots were induced by selection using SI medium (shoot induction medium) containing phosphinolysin. C shows soybean tissue cultured on shoot elongation medium containing phosphinolysin after immersion treatment of soybean tissue induced on the selection medium in B with an immersion solution consisting of 200-fold diluted Basta® solution. In the figure, arrows that appear green indicate shoots whose survival was confirmed. D shows shoots obtained in C, indicated by black arrows, that were collected and stained with X-gluc. In the figure, the shoots stained black, indicated by arrowheads, are the target transformants. Figure E shows soybean tissue induced on selection medium B, immersed in a immersion solution without Basta® solution, and then cultured on shoot growth medium containing phosphinolysin. In the figure, the arrows indicate shoots that are green and whose survival has been confirmed. Figure F shows shoots obtained in E, indicated by the black arrows, that have been collected and stained with X-gluc. In the figure, the shoots stained black, indicated by the arrowheads, are the target transformants. Most are not stained, indicating that most of the shoots whose survival was confirmed in E were escaped individuals.

[0013] 1. Method for Selecting Plant Transformants 1-1. Overview The first aspect of the present invention is a method for selecting plant transformants. The method of the present invention is characterized by the steps of introducing a gene, inducing shoot tissue, and then immersing the obtained shoot tissue, or shoot tissue cultured in a selection medium with further selection pressure, in a solution containing a selection agent that enables selection based on the expression of the selection marker gene. According to the method of the present invention, the appearance of escape individuals and chimeric individuals can be suppressed and the desired transformants can be efficiently selected.

[0014] 1-2. Definitions of Terms The following terms used herein are defined. In this specification, “plant” refers to mosses, ferns, angiosperms, and gymnosperms. Angiosperms include both dicotyledonous and monocotyledonous plants. They also include both herbaceous and woody plants. Plants particularly preferred in the present invention include leguminous plants.

[0015] In this specification, "legume plants" refers to plants belonging to the family Fabaceae, order Fabales, of the dicotyledonous angiosperms. The family Fabaceae includes subfamilies such as the subfamilies Faboideae, Mimosoideae, and Caesalpinioideae, but the legume plants referred to herein may be species belonging to any of these subfamilies. Preferably, they are species belonging to the subfamilies Faboideae. For example, this includes species belonging to the genera Glycine, Phaseolus, Vigna, Vicia, Pisum, Lens, Arachis, Canavalia, Cicer, Glycyrrhiza, Medicago, Lupinus, Lotus, Cytisus, and Lespedeza.

[0016] In this specification, "plant body" refers to the entire plant that forms a single individual.

[0017] In this specification, "plant cell" refers to a cell that makes up a plant body. It may be a cell that makes up any part of the plant body. For example, it includes cells that make up the whole plant, leaves, stems, flowers, roots, buds, and seeds, as well as cells that make up shoot tissue, tissue fragments (leaf fragments, shoot apex, terminal bud fragments), and cell fragments (differentiated cell fragments, callus fragments).

[0018] In this specification, "shoot tissue" means a shoot or a bud.

[0019] In this specification, "shoot" refers to the tissue consisting of the apical tissue formed in an explant and the stem and leaves derived therefrom.

[0020] In this specification, "multiple shoot" is also referred to as "multiple shoot" and means a mass of cells having multiple shoots.

[0021] In this specification, "explant" refers to the plant parts or plant tissues used in cultivation.

[0022] In this specification, "cotyledonous node" refers to the part of a plant between the cotyledons and primary leaves.

[0023] In this specification, "cotyledon" refers to the first leaf formed in the embryo within a seed.

[0024] In this specification, "primordial leaf" refers to the first leaf that appears after the cotyledons unfold but before the true leaves emerge, mainly in leguminous plants.

[0025] In this specification, "target plant" or "target plant cell" means a plant or plant cell subjected to transformation.

[0026] In this specification, "transformed plant" means a plant or plant cell that has been transformed by introducing a target gene into the cell.

[0027] In this specification, "target gene" refers to a gene introduced into plant cells in order to confer a desired trait to the target plant being transformed. Examples include genes related to disease resistance, insect pest resistance, yield increase, herbicide resistance, etc.

[0028] In this specification, a "selection marker gene" is a gene that can confer a new trait to a target plant that the target plant does not possess, and encodes a selection marker protein used for selecting transformants. Based on the activity of the selection marker protein produced by the expression of the gene, it is possible to identify transformants that possess the introduced selection marker gene. The selection marker gene can be any known gene and is not particularly limited. Examples include drug resistance genes, reporter genes, or nutrient genes. Although not limited, drug resistance genes are suitable as selection marker genes because they can efficiently select the desired transformants when used in combination with the selection agents described later.

[0029] In this specification, "drug resistance gene" refers to a gene that confers resistance to a specific drug (selected drugs as described later) to a target plant. While not limited to these, specific examples of drug resistance genes include the hygromycin B resistance gene (hpt gene) to hygromycin B, the chloramphenicol resistance gene to chloramphenicol, the neomycin resistance gene to neomycin, and the bar gene for phosphinothricin (PPT) (glufosinate).

[0030] In this specification, "reporter gene" refers to a gene that can be used to confirm whether or not it has been introduced into a target plant. While not limited to reporter genes, examples include genes encoding β-glucuronidase (GUS), fluorescent proteins, or luminescent enzymes. Specific examples of fluorescent proteins include GFP (including derivatives such as EGFP), CFP, RFP, DsRed, YFP, and PE. An example of a luminescence-labeling gene is the luciferase gene that catalyzes the chemiluminescent reaction of luciferin.

[0031] A "vector" is a nucleic acid molecule used in genetic engineering to amplify, maintain, and introduce recombinant nucleic acids. Examples include plasmids, viral vectors, cosmids, bacmids, and YACs. The vector is not particularly limited as long as it can be used to achieve the desired effect in plant cells, and for example, pBI vectors, pBluescript vectors, and pUC vectors can be used. Examples of pBI vectors include pBI121, pBI101, pBI101.2, pBI101.3, and pBI221. Binary vectors such as pBI vectors are preferred because they can introduce the target nucleic acid into plant cells via Agrobacterium, which will be discussed later. Examples of pBluescript vectors include pBluescript SK(+), pBluescript SK(-), pBluescript II KS(+), pBluescript II KS(-), pBluescript II SK(+), and pBluescript II SK(-). Examples of pUC-based vectors include pUC19 and pUC119. pBluescript-based vectors and pUC-based vectors are preferred because they allow for the direct introduction of nucleic acids into plants. Furthermore, binary vectors such as the pGreen series, pCAMBIA series, and pLC series (WO2007 / 148819), as well as super-binary vectors such as pSB11 (Komari et al., 1996, Plant J, 10:165-174) and pSB200 (Komori et al., 2004, Plant J, 37:315-325) are also suitable. Moreover, vectors created by combining parts of these vectors can also be preferably used.

[0032] An "expression vector" refers to a vector that contains a nucleic acid molecule encoding a protein or functional nucleic acid in an operable state and can control the expression of that nucleic acid molecule. In this specification, "operable state" means that the target nucleic acid molecule is placed under the control of a promoter within the expression vector. This results in a state where the expression of the target nucleic acid molecule is initiated by the activation of the promoter. In this specification, the expression vector contains the aforementioned selection marker gene and target gene in an operable state. The expression vector also further includes regulatory regions such as the promoter, enhancer, and terminator. In this expression vector, these regulatory regions only need to operate within plant cells. For example, if the target plant is a leguminous plant, a promoter, enhancer, and terminator that operate within the cells of that leguminous plant are preferred. The expression vector may also include other elements such as multi-cloning sites and replication initiation sites.

[0033] A "promoter" refers to a regulatory region on a nucleic acid sequence that controls the expression of a nucleic acid molecule located under a downstream control range. In this specification, a promoter may be any of the following, depending on the desired expression pattern: an overexpression promoter, a constitutive promoter, a site-specific promoter, a growth stage-specific promoter, and / or an inducible promoter. Specific examples of overexpression constitutive promoters include, for example, the 35S promoter (P35S) derived from cauliflower mosaic virus (CaMV), the Pnos promoter for the nopaline synthase gene derived from the Ti plasmid, the ubiquitin promoter derived from maize, the actin promoter derived from rice, and the PR protein promoter derived from tobacco. In addition, the small subunit (Rubisco ssu) promoter of ribulose diphosphate carboxylase, histone promoters, ubiquitin promoters, cysteine ​​protease promoters, metallothionein (MTT) 1 promoter, and MTT2 promoter can also be used.

[0034] An "enhancer" is a regulatory region that controls the activity of a promoter. Typically, it is located upstream or downstream of the promoter in the nucleotide sequence, or within the 5'-UTR, 3'-UTR, or introns within a gene, and has the function of enhancing promoter activity. For example, the hemacatalase intron functions as an enhancer for the upstream CaMV P35S.

[0035] "Terminator" refers to a regulatory region in the exogenous gene expression vector of this embodiment that consists of a nucleotide sequence capable of terminating the transcription of the target gene or the like when it is expressed.

[0036] In this specification, "selection agent" refers to a chemical used in combination with a specific selection marker gene to select transformants. Examples include low-molecular-weight compounds and polypeptides. Generally, it refers to chemicals that may negatively affect the development and growth of the target plant. Specific examples of selection agents include antibiotics such as hygromycin B, chloramphenicol, or neomycin, and herbicidal active ingredients such as phosphinolycin.

[0037] In this specification, when "culture medium" is used, unless otherwise specified, it refers to a basic culture medium commonly used in plant tissue culture. Examples include MS (Murashige & Skoog) medium, Gamborg's B5 medium, N6 medium, etc. These basic culture media may contain sugars, vitamins, etc. The culture medium may be a solid medium or a liquid medium. Although not limited, a solid medium is preferred in this invention.

[0038] In this specification, "shoot-forming medium" refers to a medium that induces the formation of shoots or buds in plant tissue or plant cells. While not limited, a medium containing cytokinin as a base medium is typically used. The cytokinin concentration may be 0.1 mg / L to 5 mg / L, 0.5 mg / L to 4 mg / L, 0.8 mg / L to 3 mg / L, or 1 mg / L to 2 mg / L. The shoot-forming medium may also contain auxin as needed. If auxin is included, there is no lower limit to the auxin concentration, but the upper limit may be 1 mg / L or less, 0.8 mg / L or less, 0.5 mg / L or less, 0.3 mg / L or less, or 0.1 mg / L.

[0039] In this specification, "selection medium" refers to a culture medium containing a selection agent. It is primarily used for the selection or maintenance of transformants. While not limited to these, a selection medium is usually a basic culture medium containing a selection agent capable of selecting or maintaining the desired transformants.

[0040] In this specification, "coexistence medium" refers to a medium used to inoculate plant tissue with Agrobacterium bacteria and culture it for a certain period of time in order to infect plant cells with the bacteria.

[0041] Auxin is a plant hormone that is biosynthesized in the apical bud of a plant and suppresses the growth of axillary buds by moving basophilically through the stem via the auxin transporter protein PIN. Specifically, this includes indoleacetic acid (IAA), indolebutyric acid (IBA), naphthaleneacetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).

[0042] Cytokinins are plant hormones that promote the growth of axillary buds in plants. When axillary buds are released from dormancy and begin to elongate, they are biosynthesized locally and transiently in the stem and supplied to the axillary buds to promote elongation. Specifically, these include zeatin, benzyladenine, and thidiazurone.

[0043] In this specification, "immersion" means immersing all or part of an object in a liquid.

[0044] As used herein, the "immersion solution" refers to a solution containing a selection agent.

[0045] As used herein, the "transformation rate" refers to the ratio of the number of cells or tissues transformed after gene introduction to the total number of cells or tissues used for gene introduction.

[0046] As used herein, the "selection rate" refers to the ratio of the number of actually transformed cells or tissues to the total number of apparently transformed cells or tissues selected in the selection process based on the introduced selection marker gene after gene introduction. In this specification, for example, it corresponds to the ratio of the number of target transformed shoot tissues to the total number of tissues including the target transformed shoot tissues, escape tissues, and / or chimeric tissues generated and elongated in the selection step described below.

[0047] 1-3. Method The method for selecting a transformant of a plant according to the present invention is characterized by including a step of immersing, after gene introduction, the tissue generated in a selection solution to which a selection pressure is applied or in a solution containing the same agent as the selection agent used in the selection medium to which a selection pressure is applied, in the medium. The method for selecting a transformant of a plant according to the present invention includes an introduction step, a shoot induction step, an immersion step, and a transformant selection step as essential steps, and includes a selection step as an optional step. Hereinafter, each step will be specifically described.

[0048] (1) Introduction step The "introduction step" is a step of co-introducing a selection marker gene and a target gene into target plant cells.

[0049] The method of introducing a selection marker gene and a target gene into a target plant may be carried out according to a method known in the art for introducing a nucleic acid molecule into a plant cell, and is not particularly limited. The plant part into which the gene is introduced may be any part containing dividing cells or cells having redifferentiation ability, and examples include introduction into cells such as cotyledon nodes, adventitious embryos, leaf pieces, shoot apices, and apical bud pieces. Hereinafter, taking the gene introduction into cotyledon nodes as an example, the method of introducing a nucleic acid molecule into plant cells will be specifically described.

[0050] To prepare the cotyledon nodes, if using mature seeds, first allow the mature seeds of the target plant (beans in the case of legumes) to absorb water. At this time, vernalization treatment may be performed before water absorption if necessary. Vernalization treatment is a process that simulates winter by exposing the plant to low temperatures for a certain period of time. Water absorption can be achieved by soaking the seeds in water and incubating them at a predetermined absorption temperature. For example, a water absorption temperature of 15-25°C is preferable for soybeans. The water may be changed as needed during the water absorption period.

[0051] When the selection marker gene and the target gene are contained in an expression vector consisting of a plasmid, known genetic engineering techniques can be used. Generally, methods such as the Agrobacterium method, protoplast method, particle gun method, electroporation method, PEG-calcium phosphate method, liposome method, microinjection method, whisker method, plasma method, and laser injection method can be used.

[0052] The Agrobacterium method is a transformation method that uses Agrobacterium species (e.g., Agrobacterium tumefaciens, Agrobacterium rhizogenes, etc.) and derived tumor induction (Ti) plasmids as binary vectors. Specifically, for example, a binary vector linking the target gene and a selection marker gene is used with Agrobacterium introduced by electroporation or the like. Agrobacterium containing the binary vector is cultured in a bacterial culture medium such as YP medium, and the resulting cells are suspended in Agrobacterium suspension medium. This suspension is used as an inoculant, and the target plant tissue is inoculated by immersion in the inoculant, thereby introducing the target gene into the target plant's genomic DNA. After inoculation, it is preferable to co-culture the target plant tissue on a co-culture medium.

[0053] The protoplast method is a method of introducing target nucleic acids into plant cells using plant cells (protoplasts) from which the cell wall has been removed by enzymatic treatment such as cellulase. This method can be further classified into electroporation, microinjection, and polyethylene glycol methods depending on the method of DNA introduction. The electroporation method is a method of introducing nucleic acids into protoplasts by applying electrical pulses to a mixture of protoplasts and the target nucleic acid. Like the Agrobacterium method mentioned above, it can be used not only for introduction into plant cells but also into bacteria. The microinjection method is a method of directly introducing target nucleic acids, proteins, etc. into protoplasts using a microneedle under a microscope. The polyethylene glycol method is a method of introducing target nucleic acids, proteins, etc. into protoplasts by reacting them with polyethylene glycol.

[0054] The particle gun method involves attaching the target nucleic acid to fine particles such as gold or tungsten, and then injecting these particles into the cells of a target plant using high-pressure gas to introduce the target nucleic acid into the cells. This method allows for the production of transformed cells in which the target gene has been incorporated into the genomic DNA of the target plant.

[0055] Furthermore, if the expression vector is a viral vector (e.g., CaMV, BGMV, TMV, etc.), transformants can be obtained by infecting the target plant with the expression vector. For details on gene transfer methods using such viral expression vectors, one should follow methods known in the field. For example, one can refer to the method of Hohn et al. (Molecular Biology of Plant Tumors, Academic Press, New York, 1982, pp. 549).

[0056] (2) Shoot induction process The "shoot induction process" is a process in which the target plant cells after the introduction process are cultured in a shoot-forming medium to induce shoot tissue.

[0057] Shoot tissue induction is achieved by suspending, burying, or placing plant tissue or plant cells in a shoot-forming medium and maintaining it at a temperature suitable for shoot induction. The temperature is not limited, but is usually 15-30°C, 20-28°C, or 25°C.

[0058] Furthermore, a selection process may be carried out simultaneously with this process by adding a selection agent that enables selection based on the expression of a selection marker gene to the shoot-forming medium during this process.

[0059] The culture conditions can be any of the usual conditions used for shoot induction in this field, and are not particularly limited. For example, the plants may be cultured under illumination and at the aforementioned temperature for 1 to 3 weeks, 10 to 18 days, or 14 days.

[0060] (3) Selection Process The "selection process" is a process in which the target plant cells after the shoot induction process are cultured in a selection medium containing a selection agent that enables selection based on the expression of selection marker genes. This process is a selection process and may be performed as needed, but it is preferable to perform it in order to improve the selection rate of transformants.

[0061] In this process, the target plant cells or target plant tissue obtained after the shoot induction process are selected by applying a selection pressure using a selection agent.

[0062] The selection medium used in this process is, in principle, a selection medium containing a selection agent corresponding to the selection marker gene introduced into the target plant cells in the introduction process. Since the selection agent corresponding to the selection marker gene is predetermined, the selection agent to be included in the selection medium in this process should be appropriately determined according to the selection marker gene introduced in the introduction process. For example, if the introduced selection marker gene is a drug resistance gene, the selection agent paired with that gene will be included in the selection medium. Through transformation, only the transformants can grow in the presence of the selection agent due to the drug resistance performance conferred by the drug resistance gene to the target plant cells.

[0063] More specifically, for example, if the bar gene is introduced as a selection marker gene in the introduction process, the selection medium used in this process only needs to contain phosphinolysin (glufosinate) as a selection agent.

[0064] The "bar gene" is a gene isolated from the soil microorganism Streptomyces hygroscopicus and encodes phosphinothricin N-acetyltransferase (PAT). Phosphinothricin acetyltransferase inactivates phosphinolysin, the active ingredient in herbicides. Therefore, if the bar gene is introduced as a selection marker gene during the introduction process, transformed tissue incorporating the bar gene can be obtained by culturing it in a selection medium containing phosphinolysin.

[0065] The concentration of the selection agent in the selection medium should be determined appropriately depending on the type of selection agent used. For example, for phosphinolysin, concentrations of 0.5 mg / L to 100 mg / L, 1 mg / L to 50 mg / L, 3 mg / L to 25 mg / L, or 5 mg / L to 10 mg / L are acceptable. For kanamycin, concentrations of 1 mg / L to 200 mg / L, 3 mg / L to 150 mg / L, 5 mg / L to 100 mg / L, or 10 mg / L to 50 mg / L are acceptable. For hygromycin B, concentrations of 1 mg / L to 200 mg / L, 3 mg / L to 150 mg / L, 5 mg / L to 100 mg / L, or 10 mg / L to 50 mg / L are acceptable.

[0066] The selection medium used in this process may be a basic medium containing a selection agent, or a shoot-forming medium to which a selection agent has been added. By using the latter medium, transformed target plant cells or target plant tissues can be selected while maintaining shoot induction.

[0067] The culture conditions in this process can be any conditions that are normal in the field, and are not particularly limited. For example, the culture can be performed under illumination, at 15-30°C, 20-28°C, or 25°C for 1 to 3 weeks, 10 to 18 days, or 14 days.

[0068] If necessary, the proliferated target plant cells or target plant tissues may be isolated and obtained, and then subjected to selective culture again in the selection medium under the same or similar conditions.

[0069] (4) Immersion Step The "immersion step" is a step in which shoot tissue after the shoot induction step, or shoot tissue generated after the selection step, is immersed in a solution containing a selection agent based on the expression of a selection marker gene. This step is the most important step in the plant transformant selection method of the present invention. After the shoot induction step or selection step, the shoot tissue contains not only transformed shoot tissue but also untransformed shoot tissue as escape tissue and chimeric tissue. These untransformed tissues have been a major cause of the selection rate of transformants in plants such as soybeans.

[0070] Therefore, in this process, the objective is to improve the selection efficiency in the shoot tissue by immersing the shoot tissue obtained after the shoot induction process or the selection process in a solution containing a selection agent, thereby ensuring sufficient contact between the shoot tissue and the selection agent.

[0071] In this process, shoot tissue obtained after the shoot induction process or selection process is used. The shoot tissue may consist only of tissues such as shoots or buds, or it may be bud tissue including cotyledons that was subjected to the selection process.

[0072] The immersion solution contains a selection agent based on the expression of the introduced selection marker gene. If the selection process is performed, the selection agent may have the same or equivalent activity as the selection agent contained in the selection medium used in the selection process. In this specification, "agent with equivalent activity" refers to an agent having the same or comparable activity as a specific agent, for example, a derivative of a specific agent.

[0073] The concentration of the selection agent in the immersion solution can be appropriately increased or decreased depending on the selection pressure in this process. For example, if the selection agent is phosphinolysin, the concentration may be 20 mg / L to 2000 mg / L, 50 mg / L to 1800 mg / L, 100 mg / L to 1500 mg / L, 500 mg / mL to 1200 mg / L, or 800 mg / L to 1000 mg / L. If a selection process is performed, the concentration should be the same as or greater than the concentration in the selection medium, preferably higher.

[0074] The solvent in the immersion solution is not particularly limited, as long as it does not inhibit contact of the included selection agent with the shoot tissue or reduce or destroy the activity of the selection agent. Examples include water, buffer solution, and liquid culture medium.

[0075] The immersion solution may be an existing solution, provided it contains the specified selection agent. For example, if the selection agent is phosphinolysin, a commercially available herbicide, Basta® liquid (BASF), can be used. Basta® liquid contains 18.5% glufosinate ammonium as the active ingredient (Basta® liquid product safety data sheet: BASF). Glufosinate ammonium is a chemically synthesized product and is a racemic mixture containing equimolar ratios of the L and D isomers of phosphinolysin, the selection agent. Of these, only the L isomer exhibits herbicidal activity. Therefore, Basta® liquid may be diluted with a suitable solvent and used as the immersion solution in this process. The dilution ratio should be such that the L-form phosphinolysin is within the aforementioned concentration range. Specifically, dilutions of 20 to 2000 times, 50 to 1000 times, 80 to 500 times, or 100 to 300 times are acceptable.

[0076] In this step, the shoot tissue is immersed in the immersion solution, but the immersion rate of the shoot tissue in the immersion solution is not limited. For example, it may be 10% or more, 20% or more, 40% or more, 60% or more, 80% or more, or 100%. However, considering that the purpose of this step is to allow the selection agent to come into sufficient contact with the shoot tissue obtained in the shoot tissue induction step or selection step, it is preferable to immerse the entire shoot tissue in the immersion solution, i.e., an immersion rate of 100%.

[0077] This process may be carried out under reduced pressure after immersion to more efficiently penetrate the chute tissue with the selection agent. The reduced pressure conditions are not particularly limited as long as they do not affect the survival of the chute tissue. For example, -0.01 MPa to -0.1 MPa, -0.02 MPa to -0.08 MPa, or -0.04 MPa to -0.06 MPa relative to atmospheric pressure (e.g., 0.1 MPa) would be acceptable.

[0078] The immersion time is not limited. It should be determined appropriately, taking into account the type and concentration of the selection agent contained in the immersion solution. For example, it could be 1 minute to 6 hours, 5 minutes to 4 hours, 10 minutes to 2 hours, or 15 minutes to 1 hour.

[0079] (5) Transformant Selection Process The "transformant selection process" is a process of selecting transformants from the shoot tissue after the immersion process based on the expression of the selection marker gene. By performing selection on the shoot tissue after the immersion process, this process eliminates escape tissue and chimeric tissue that were mixed in with the shoot tissue obtained after the shoot tissue induction process or the selection process, making it possible to efficiently obtain the desired transformed shoot tissue (often referred to as "transformed shoot tissue" in this specification).

[0080] This step involves culturing the shoot tissue after the immersion step on a selection medium. The selection agent contained in the selection medium used in this step may be the same as the selection agent used in the selection step and / or immersion step. For example, if phosphinolysin was used as the selection agent in the selection step and / or immersion step, then phosphinolysin may also be used as the selection agent in the selection medium of this step. The concentration of the selection medium contained in the culture medium is not limited, but it should be the same as or greater than the concentration of the selection agent used in the selection step and / or immersion step.

[0081] The culture conditions (temperature, culture period, etc.) for this process are not limited, but are basically the same as those for the selection process described above.

[0082] In this process, escape tissue and chimeric tissue are killed by the selection agent, but transformed shoot tissue can survive based on the expression of selection marker genes. Therefore, shoot tissue that has grown without dying in this process can be selectively obtained as the desired transformant.

[0083] The plant transformant selection method according to the above embodiment of the present invention makes it possible to efficiently select and obtain transformed shoot tissue, which is the target transformant, by eliminating escape tissue and chimeric tissue.

[0084] 2. Method for Producing Transgenic Regenerated Plants 2-1. Overview The second aspect of the present invention is a method for producing transgenic regenerated plants. In the method of the present invention, the transformed shoot tissue obtained using the plant transformant selection method of the first aspect is cultured to regenerate the plant body. According to the method of the present invention, it is possible to efficiently produce the desired transgenic plants (transgenic plants) without the presence of escaped regenerated plants or chimeric regenerated plants.

[0085] 2-2. Method The method for producing transformed and regenerated plants of the present invention includes the following essential steps: (1) introduction step, (2) shoot induction step, (3) selection step, (4) immersion step, (5) transformant selection step, and (6) regeneration step. Of these, the introduction step, shoot induction step, selection step, immersion step, and transformant selection step may be the same as the corresponding steps (1) to (5) described in the plant transformant selection method described in the first embodiment. In other words, the present invention is a method for regenerating a transformant obtained after carrying out the plant transformant selection method described in the first embodiment into a plant in the regeneration step. Therefore, the explanation of steps (1) to (5) described in the first embodiment, which are redundant, will be omitted here, and the regeneration step unique to the present invention will be explained in detail below.

[0086] (6) Regeneration process The "regeneration process" is a process in which the transformed organisms obtained after the transformed organism selection process are cultured and the plant body is regenerated.

[0087] In the transformant selection process, escape tissues and chimeric tissues that are mixed in during the shoot induction process or after the selection process are removed through an immersion process, and only the transformed shoot tissue, which is the target transformant, is selected and isolated.

[0088] In this process, rooting is induced in isolated transformed shoot tissue, and then the tissue is cultured until it grows into a plant. Rooting from the elongated shoots is achieved by culturing in a medium containing the plant hormone indole-3-butyric acid (IBA). However, for individuals that are difficult to root, grafting using a non-transformed soybean plant as rootstock is an extremely effective method for obtaining a plant.

[0089] The method of culturing the transformants in this process is not particularly limited, but the culture medium used can be appropriately changed according to the growth stage of the transformants. For example, if the transformed tissue obtained after the selection process is at the shoot or multi-bud stage, it can be cultured in the selection medium. The selection medium can be the same medium used in the selection process, or a medium containing the same selection agent as that medium. Once the shoots or multi-buds have elongated and entered the rooting stage, or once plants have been grown by grafting, they can be transplanted into seedling soil or liquid culture medium and cultivated.

[0090] After transplanting into potting soil, if cultivated under appropriate temperature and light / dark conditions, the plants will flower, bear fruit, and produce offspring seeds.

[0091] <Example 1> (Objective) To select transformants using the plant transformant selection method of the present invention and to verify the concentration of the selection agent in the immersion solution and the selection rate.

[0092] (Method) Fully matured soybean seeds (variety: Fayette) sterilized with ethanol and antiformin were washed with sterile distilled water and soaked in sterile distilled water overnight. After removing the seed coat, the hypocotyl was removed from the seeds, leaving the base 1-2 mm, and the seeds were cut in half lengthwise. Subsequently, the primary leaves at the cotyledon nodes were removed, and the cotyledons were washed with sterile distilled water.

[0093] A binary vector, pLC41 (GenBank accession No. LC215698), containing a GUS gene expression cassette (P35S-Icat-GUS-Tnos) and a bar gene expression cassette (P35S-bar-T35S) on T-DNA, was introduced into the Agrobacterium tumefaciens EHA105 strain to create EHA105 (pLC41 P35S-Icat-GUS P35S-Bar). The GUS gene expression cassette drives the GUS gene (Ohta et al., 1990, Plant Cell Physiol. 31 (6); 805-813), which has an intron in the castor flower (Ricinus communis) catalase gene, with the cauliflower mosaic virus 35S promoter (CaMV-p35S). The bar gene expression cassette drives the bar gene with CaMV-p35S.

[0094] Agrobacterium EHA105 (pLC41 P35S-Icat-GUS P35S-Bar), pre-cultured for 1 day on YP agar medium (Non-patent Literature 4: Ishida et al., 2007, Nature Protocols, 2; 1614-1621), was suspended in Agrobacterium suspension medium (Paz et al., 2006, Plant Cell Reports, 25(3): 206-213) and used as an inoculant.

[0095] The cotyledons were immersed in the inoculant and stored at room temperature for 15 minutes. The inoculated cotyledons were placed on a co-existence medium lined with filter paper (Paz et al. 2006) and cultured under illumination at 25°C for 3 days. Subsequently, the cotyledons were placed on SI medium (shoot induction medium) (Paz et al. 2006) so that they were embedded in the medium, and cultured under illumination at 25°C for 2 weeks. The grown stems and leaves were removed, and the cotyledons were placed on SI medium containing 5 mg / L phosphinolysin. After culturing under illumination at 25°C for 2 weeks, the proliferated tissue was placed on SI medium containing the same concentration of phosphinolysin and cultured under the same conditions for a further 2 weeks.

[0096] Basta® solution (BASF), filtered and sterilized with sterile distilled water containing 150 mg / L of cimentine, was diluted 40,000-fold, 2,000-fold, 200-fold, and 100-fold. Basta® solution is a commercially available herbicide containing 18.5% glufosinate ammonium. As mentioned above, glufosinate ammonium is a racemic mixture containing L- and D-isomers of phosphinothlysin in equimolar ratios. Since only the L-isomer is the active ingredient exhibiting herbicidal activity, the concentration of the L-isomer in the Basta® solution is half the calculated glufosinate concentration. As a control group, an immersion solution containing only cimentine and without Basta® solution was used.

[0097] Soybean sprout tissue was immersed in each dilution of Basta® solution and allowed to stand under reduced pressure (-0.06 MPa) for 15 minutes. After removing excess Basta® solution on filter paper, the soybean sprout tissue was placed on SI medium containing 5 mg / L phosphinolysin and cultured under illumination at 25°C for 2 weeks. Green stems and leaves were collected from the cultured tissue and stained with X-gluc.

[0098] (Results) The results are shown in Table 1 and Figure 1.

[0099]

[0100] In the control group (glufosinate concentration: 0 mg / L) which did not contain Basta® solution in the immersion solution, only 15.4% showed GUS positivity, which corresponds to the selection rate. In other words, it became clear that even though the selection process involved using a selection medium containing 5 mg / L phosphinolysin and culturing for 6 weeks under selection pressure, more than 80% of the viable tissues that retained their green color were untransformed escape tissues.

[0101] On the other hand, only 11.8% of the tissue treated with an immersion solution consisting of a 40,000-fold diluted Basta® solution (glufosinate concentration: 4.6 mg / L) showed GUS positivity, and, as with the control group, most of these were escapes.

[0102] However, when tissue was treated with an immersion solution consisting of a 2000-fold diluted Basta® solution (glufosinate concentration 93 mg / L), only 28.6% showed GUS positivity, indicating that approximately 30% were transformed. Furthermore, when tissue was treated with an immersion solution consisting of a 200-fold diluted Basta® solution (glufosinate concentration 930 mg / L) and an immersion solution consisting of a 100-fold diluted Basta® solution (glufosinate concentration 1850 mg / L), the GUS positivity rates were 92.3% and 66.7%, respectively, confirming that a large proportion of these tissues were transformed.

[0103] From these results, it was demonstrated that when the Basta® liquid solution, diluted 2000 times or less, was used as the immersion solution for immersion treatment, the selection rate was clearly improved compared to the control group treated with an immersion solution that did not contain the active ingredient.

[0104] <Example 2> (Objective) In Example 1, when immersion treatment was performed using an immersion solution consisting of Basta® liquid solution diluted 200 times, the selection efficiency of transformants was 90% or more. Therefore, the plant transformant selection method of the present invention was carried out again using an immersion solution consisting of Basta® liquid solution diluted 200 times, and transformants were selected.

[0105] (Method) The basic method was the same as that described in Example 1. Soybean cotyledons (variety: Fayette) inoculated with EHA105 (pLC41 P35S-Icat-GUS P35S-Bar) were cultured for 4 weeks in a medium containing 5 mg / L of phosphinolysin, and then immersed in a 200-fold diluted solution of Basta.

[0106] After culturing again in a medium containing 5 mg / L phosphinolysin for 2 weeks, the green-colored tissue was collected and placed on a shoot growth medium containing 5 mg / L phosphinolysin (MS inorganic salts, MS vitamins, 20 g / L sucrose, 0.5 g / L MES, 0.5 mg / L zeatin, 2 mg / L gibberellic acid, 150 mg / L thimentin, 5 mg / L phosphinolysin, 5 g / L agarose, pH 5.7), and cultured under illumination at 25°C for 2 weeks. Green-colored stems and leaves were collected from the cultured tissue and stained with X-gluc.

[0107] (Results) 98% of the stained shoots or leaf tissues showed GUS positivity. This means that the selection rate was 98%. From these results, it became clear that using a 200-fold dilution of Basta® solution as the immersion solution is effective in efficiently obtaining transformants.

[0108] <Example 3> (Objective) To confirm that the plant transformant selection method of the present invention is applicable to transformations using selection marker genes other than the bar gene used in Example 1, and to confirm whether a surfactant is essential in the immersion solution.

[0109] (Method) The basic procedure was the same as in Example 1. A binary vector containing the GUS gene expression cassette (P35S-Icat-GUS-Tnos) and the ht gene expression cassette (P35S-hpt-T35S) on T-DNA was introduced into the Agrobacterium tumefaciens EHA105 strain to produce EHA105 (pLC41 P35S-Icat-GUS P35S-hpt). The ht gene expression cassette drives the ht gene with CaMV-p35S.

[0110] Soybean seed cotyledons (variety: Fayette) prepared in the same manner as in Example 1 were inoculated with an inoculum containing EHA105 (pLC41 P35S-Icat-GUS P35S-hpt) suspended in Agrobacterium suspension medium. Similar to Example 1, the inoculated cotyledons were cultured in co-existence medium and shoot induction medium (SI medium), then placed on SI medium containing 10 mg / L hygromycin B and cultured under illumination at 25°C for 2 weeks. The proliferated tissues were then placed on SI medium containing the same concentration of hygromycin B and cultured under the same conditions for a further 2 weeks.

[0111] A hygromycin B solution was prepared by adding filtration-sterilized hygromycin B to sterile distilled water containing 150 mg / L of cimentin to a concentration of 933 mg / L. The immersion solution was a hygromycin B solution. The Basta® solution used as the immersion solution in Example 1 contains a surfactant (Basta solution product label) in addition to the active ingredient glufosinate. However, the immersion solution in this example does not contain a surfactant. Soybean sprout tissue was immersed in the immersion solution and left to stand under reduced pressure (-0.06 MPa) for 15 minutes. After removing excess immersion solution on filter paper, the soybean sprout tissue was placed on SI medium containing 10 mg / L of hygromycin B and cultured under illumination at 25°C for 2 weeks. Green stems and leaves were collected from the cultured tissue and stained with X-gluc.

[0112] (Results) The results are shown in Table 2.

[0113]

[0114] Green tissue randomly collected from multi-buds that were not immersed in the hygromycin B solution was all GUS-negative. In other words, the selection rate was 0%, and it contained no transformants; all were escape tissues.

[0115] On the other hand, in the case of multiple buds subjected to immersion treatment, 35% of the randomly collected green tissue showed positive GUS, indicating that transformed tissue was selected. These results confirm that the plant transformant selection method of the present invention is effective regardless of the type of selection marker gene.

[0116] Furthermore, as mentioned above, although the immersion solution in this embodiment does not contain surfactants, the same selection process was carried out as when using the Basta® liquid agent used as the immersion solution in Example 1. This demonstrates that surfactants are not essential for the selection of transformed tissues by immersion treatment.

[0117] <Example 4> (Objective) From the results of Examples 1 to 3, it was clear that the selection rate improves by immersion treatment. On the other hand, we will confirm whether the transformation rate also improves by immersion treatment.

[0118] (Method) Forty-two soybean cotyledons (Fayette variety) inoculated with EHA105 (pLC41 P35S-Icat-GUS P35S-Bar) were cultured for four weeks in a medium containing 5 mg / L of phosphinolysin, and then half of the cotyledons (21 in total) were immersed in a 200-fold diluted solution of Basta®. After culturing in the same medium for two weeks, they were cultured for a total of six weeks in shoot growth medium using the same method as in Example 2.

[0119] The remaining 21 cotyledonous segments were not treated with immersion in Basta® solution, while the others were treated in the same manner as described above.

[0120] The number of shoots formed in each case was counted, and the shoots or leaf fragments were stained with X-gluc. The transformation rate was calculated from the number of transformed cotyledonous nodes relative to the number of inoculated cotyledonous nodes.

[0121] (Results) The results are shown in Table 3.

[0122]

[0123] Of the 21 cotyledonous nodes that were subcultured in a medium containing phosphinolysin without immersion in the Basta® solution, two shoots formed from two cotyledonous nodes, one from each node. When the expression of the GUS gene in the two resulting shoots was examined, the tissue of one shoot was stained dark blue, while the base of the other shoot was only lightly stained. In other words, the transformation rate was 4.8%.

[0124] On the other hand, in the cotyledonous nodes that underwent immersion treatment, a total of 11 shoots were formed from 6 out of 21 cotyledonous nodes. When the expression of the GUS gene was examined in the 11 resulting shoots, all 11 shoots showed dark blue staining. In other words, the transformation rate was 28.6%.

[0125] From these results, it was confirmed that immersion treatment in the Basta® liquid solution also improves the transformation rate.

[0126] <Example 5> (Objective) To verify that the plant transformation selection method of the present invention can efficiently obtain shoots regardless of the transformation method or target plant.

[0127] (Method) The basic method was the same as in Example 1. A binary vector pLC41, which has a GFP mutant gene BFP (sGFPS65TY66H) expression cassette (PcUbi-BFP-Tnos) and the bar gene expression cassette (P35S-bar-T35S) on T-DNA, was introduced into the Agrobacterium tumefaciens EHA105 strain to produce EHA105 (pLC41 PcUbi-BFP P35S-Bar). The BFP expression cassette is driven by a parsley ubiquitin promoter.

[0128] EHA105 (pLC41 PcUbi-BFP P35S-Bar) suspension medium and EHA105 (pLC41 P35S-Icat-GUS P35S-Bar) suspension medium prepared in Example 1 were inoculated into soybean cotyledons (varieties: Jack, Williams82, Fayette, Maple Arrow, and Essex) prepared in the same manner as in Example 1, respectively. Shoot induction culture, immersion treatment with a 200-fold diluted Basta® solution, and shoot elongation culture were then performed in the same manner as in Example 2, and the number of phosphinolysin-resistant shoots formed was counted.

[0129] Jack, Fayette, and Williams82 are varieties that have been used in many trials and studies on soybean transformation (Non-Patent Document 6). However, Maple Arrow and Essex are common cultivated varieties for which no transformations have been reported to date.

[0130] (Results) The results are shown in Table 4.

[0131]

[0132] In the table, "Shoot formation rate" is the ratio of the number of shoots observed to the number of cotyledonous nodes inoculated.

[0133] In all soybean varieties, the tissues immersed in the Basta® solution showed elongation of many phosphinolysin-resistant shoots, with shoot formation rates of 20% or higher in all cases. In particular, Jack and Fayette showed high shoot formation rates of 30% to 40% or higher in most of the test plots.

[0134] Based on these results, the plant transformant selection method of the present invention enabled the formation of drug-resistant shoots with high efficiency, regardless of the type of promoter or soybean variety.

[0135] <Example 6> (Objective) To confirm that soybean shoots obtained by the plant transformation selection method of the present invention are uniformly transformed.

[0136] (Method) Thirty-two Fayette phosphinolysin-resistant shoots obtained in Example 5 were used as scions, and Fayette seedlings were used as rootstock for grafting. Of the 32 grafted plants, approximately half (15 plants) showed scion growth, and these 15 plants were transplanted into pots with soil and cultivated in a greenhouse. Generally, it is considered difficult for soybeans to root from shoots. However, this method, using grafting, made it possible to cultivate them in a greenhouse at a high frequency.

[0137] Leaves that had developed during cultivation were coated with a 1000-fold dilution of Basta® solution using a cotton swab. As a negative control, plants derived from untransformed Fayette seeds were used. Two weeks after application of the Basta® solution, the application sites on each leaf were examined.

[0138] (Results) The results are shown in Table 5.

[0139] S: Dead (Susceptible, Non-transformed) R: Healthy (Resistant, Transformed) Position: Location of test leaves (from top to bottom)

[0140] In the negative control group, all leaves treated with the diluted Basta® solution withered at the application site. In contrast, none of the 15 plant lines derived from Fayette phosphinolysin-resistant shoots obtained in Example 5 showed any leaf death, and all leaves demonstrated resistance to the Basta® solution.

[0141] The results above indicate that all parts of the tested plants were transformed, and that no escaped or chimeric individuals were included. This demonstrates that immersion treatment in Basta® solution is an extremely effective method for obtaining transformed plants.

[0142] <Example 7> (Objective) To confirm that the transformed soybeans obtained by the plant transformation selection method of the present invention are not chimeras, and that the introduced genes are inherited by the subsequent generations of plants.

[0143] (Method) Seeds were collected from two lines of Fayette-transformed plants obtained in test plots No. 6 and 7 of Example 5, and from one line of Essex-transformed plants obtained in test plot No. 9 of Example 5, through self-pollination.

[0144] Two strains of Fayette (No. 6 and 7) and one strain of Essex (No. 9) were subjected to GUS assays after their immature seeds were extracted from ripe pods.

[0145] One of Fayette's strains (No. 7) was cultivated until the pods withered, and the fully matured seeds were extracted and sown in pots filled with soil.

[0146] Parts of leaves from germinated and grown plants were cut off and subjected to a GUS assay. Additionally, a 1000-fold diluted solution of Basta® was applied to a portion of the leaves at each location using a cotton swab.

[0147] (Results) Of the seven immature seeds obtained from one Essex strain (No. 9), five were GUS-positive and the remaining two were negative, demonstrating that the transformed GUS gene is inherited by the progeny according to Mendel's laws. We also confirmed that the introduced GUS gene is inherited by the progeny in two Fayette strains (No. 6 and No. 7).

[0148] Of the three plants derived from mature seeds of Fayette line No. 7, two were GUS-positive and showed resistance to Basta, while one was GUS-negative and showed sensitivity to Basta (registered trademark). This indicates that both types of introduced genes are similarly inherited by the progeny.

[0149] From these results, it was revealed that the transgenic plants obtained by the selection method involving immersion in Basta® liquid solution had the introduced gene stably incorporated and inherited by the progeny.

[0150] <Example 8> (Objective) To confirm that transformants can be obtained even when a immersion step is performed on cotyledonous nodes cultured in shoot-inducing medium for 2 weeks without going through a selection step, followed by a transformant selection step.

[0151] (Method) In the same manner as in Example 1, 26 soybean cotyledons (Fayette variety) inoculated with EHA105 (pLC41 P35S-Icat-GUS P35S-Bar) were immersed in a 200-fold diluted Basta® solution without being cultured in a medium containing 5 mg / L of phosphinolysin. After being cultured for 2 weeks in a medium containing 5 mg / L of phosphinolysin, they were cultured for a total of 6 weeks in shoot growth medium in the same manner as in Example 2.

[0152] The number of cotyledonous nodes that formed shoots was counted, and the formed shoots were stained with X-gluc. The transformation rate was calculated from the number of transformed cotyledonous nodes relative to the number of inoculated cotyledonous nodes.

[0153] (Results) The results are shown in Table 6.

[0154]

[0155] Of the 26 cotyledonous segments that were subcultured in a medium containing phosphinolysin without immersion in the Basta® solution, shoots formed from 5 cotyledonous segments. When the expression of the GUS gene in these shoots was examined, shoots derived from 4 cotyledonous segments were stained blue. In other words, the transformation rate was 15.4%.

[0156] From the above results, it was confirmed that transformed plants can be obtained with high efficiency even when the immersion process is performed after the shoot induction process without going through the selection process. All publications, patents and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A method for selecting transformants of plants, comprising: an introduction step of co-introducing a selection marker gene and a target gene into target plant cells; a shoot induction step of culturing the target plant cells after the introduction step in a shoot-forming medium to induce shoot tissue; an immersion step of immersing the target plant cells after the shoot induction step in a solution containing a selection agent that enables selection based on the expression of the selection marker gene; and a transformant selection step of selecting transformants from the shoot tissue after the immersion step based on the expression of the selection marker gene.

2. A method for selecting transformants of plants, comprising: an introduction step of co-introducing a selection marker gene and a target gene into target plant cells; a shoot induction step of culturing the target plant cells after the introduction step in a shoot-forming medium to induce shoot tissue; a selection step of culturing the target plant cells after the shoot induction step in a selection medium containing a selection agent that enables selection based on the expression of the selection marker gene; an immersion step of immersing the shoot tissue generated after the selection step in a solution containing the selection agent; and a transformant selection step of selecting transformants from the shoot tissue after the immersion step based on the expression of the selection marker gene.

3. The method according to claim 1 or 2, wherein the immersion step is performed under reduced pressure.

4. The method according to claim 1 or 2, wherein the selection marker gene is a drug resistance gene to the selected drug.

5. The method according to claim 1 or 2, wherein the co-introduction of the gene in the introduction step is carried out by any method selected from the group consisting of the Agrobacterium method, the particle gun method, the electroporation method, and the protoplast method.

6. The method according to claim 1, wherein the shoot tissue is a shoot or a bud.

7. The method according to claim 1 or 2, wherein the plant is a legume.

8. A method for producing a transformed regenerated plant, comprising a regeneration step of culturing a transformant obtained after the transformant selection step in the plant transformant selection method described in claim 1 or 2, and regenerating the plant body.

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