Method for producing transformed plant, and method for transforming plant

Abstract

The present invention addresses the problem of providing: a novel method for more efficiently producing a transformed plant; and a novel method for more efficiently transforming a plant.　A method for producing a transformed plant, according to the present invention, includes: an explant acquisition step for acquiring an explant of a plant body; a gene introduction step for introducing an exogenous gene into the explant and culturing the explant in a medium; a callus induction step for culturing the explant after the gene introduction step in a callus induction medium to induce callus formation; and a redifferentiation step for culturing the culture after the callus induction step in a redifferentiation medium, the method further including a division step for dividing the explant after the gene introduction step and before the redifferentiation step.

Description

Method for producing transformed plants, method for transforming plants

[0001] This invention relates to a method for producing transformed plants. Furthermore, this invention relates to a method for transforming plants.

[0002] Introducing exogenous genes into plants to transform them and confer desired traits is a technique that has been used for a long time. Known methods for plant transformation include the Agrobacterium method, particle gun method, electroporation method, microinjection method, and polyethylene glycol method, with the Agrobacterium method being the most widely used.

[0003] Various studies have been conducted to date to achieve highly efficient plant transformation. For example, Craze et al. 2018 (Non-Patent Literature 1) discloses a method for efficiently transforming potatoes using the Agrobacterium method.

[0004] Furthermore, the transformation efficiency has been improved by adding silver nitrate to the culture medium used to cultivate tissue inoculated with Agrobacterium bacteria. Adly et al. 2022 (Non-Patent Literature 2) discloses the use of silver nitrate to promote potato transformation.

[0005] In addition, Hiei et al. 1994 (Non-Patent Literature 3) reported the use of a super-binary vector containing a portion of the pathogenicity genes of highly pathogenic Agrobacterium in transformation using the Agrobacterium method, and it has been shown that stable and highly efficient transformation can be achieved by performing plant transformation using such a super-binary vector.

[0006] The technique of conferring improved traits to plants through transformation is crucial for obtaining plants with desirable characteristics. To achieve improvements in plant traits, such as producing plants with resistance to diseases and pests, plants with resistance to environmental stress, and edible plants with increased yields, there is a need for more efficient methods for producing transformed plants.

[0007] Furthermore, transforming difficult-to-cultivate plant varieties is challenging, and from the perspective of achieving transformation in such plants, new technologies for producing transformed plants are needed.

[0008] Craze et al. , Current Protocols in Plant Pathology, (2018) 3(1) 33-41 Adly et al. , Horticulturae, (2022) 8(2) 113 1-13 Hiei et al. , The Plant Journal (1994) 8(2) 271-282

[0009] The object of the present invention is to provide a new method for more efficiently producing transformed plants. Furthermore, the object of the present invention is to provide a new method for more efficiently transforming plants.

[0010] To solve the above problems, the inventors conducted diligent research and found that transformed plants can be produced with high efficiency by dividing explant fragments collected from plant bodies. Furthermore, the inventors found that the efficiency of plant transformation can be further increased by adding a silver compound to the culture medium in addition to dividing the explant fragments as described above.

[0011] Accordingly, the present invention has the following features (1) to (17). (1) A method for producing a transformed plant, the method comprising: an explant acquisition step of obtaining an explant from a plant body; a gene introduction step of introducing an exogenous gene into the explant and culturing it in a culture medium; a callus induction step of culturing the explant after the gene introduction step in a callus induction medium to induce callus formation; and a redifferentiation step of culturing the culture after the callus induction step in a redifferentiation medium, wherein the method further comprises a division step of dividing the explant after the gene introduction step and before the redifferentiation step. (2) The method for producing the plant according to (1), wherein a silver compound is added to a culture medium in one or more steps selected from the gene introduction step, the callus induction step, and the redifferentiation step. (3) The method for producing the plant according to (1) or (2), wherein in one or more steps selected from the gene introduction step, the callus induction step, and the redifferentiation step, the explant into which the exogenous gene has been introduced is selected based on the expression of the exogenous gene. (4) The manufacturing method according to any one of (1) to (3), wherein the division step is performed after the callus induction step. (5) In the division step, the explant is 6 mm ２ ~25mm ２A method for producing a plant according to any one of (1) to (4), wherein the plant is divided into pieces of a certain size. (6) A method for producing a plant according to any one of (1) to (5), further comprising a plant culture step of culturing the redifferentiated tissue obtained after the redifferentiation step on a plant body. (7) A method for producing a plant according to any one of (1) to (6), wherein in the gene introduction step, the exogenous gene is introduced by the Agrobacterium method or the particle gun method. (8) A method for producing a plant according to any one of (1) to (7), wherein the plant is a dicotyledonous plant. (9) A method for producing a plant according to (8), wherein the dicotyledonous plant is a Solanaceae plant. (10) A method for producing a plant according to any one of (1) to (9), wherein the explant is derived from the leaves of a plant. (11) A method for producing a plant according to any one of (2) to (10), wherein the silver compound is silver nitrate. (12) A method for producing a plant according to any one of (1) to (11), wherein the exogenous gene contains the target gene. (13) The method for producing a product according to any one of (1) to (12), wherein the gene transfer step is performed in a culture for 1 to 6 days. (14) The method for producing a product according to any one of (1) to (13), wherein the callus induction step is performed in a culture for 2 to 10 days. (15) The method for producing a product according to any one of (1) to (14), wherein the redifferentiation step is performed in a culture for 2 to 12 weeks. (16) A method for transforming a plant, the method comprising: an explant acquisition step of obtaining an explant from a plant body; a gene introduction step of introducing an exogenous gene into the explant and culturing it in a culture medium; a callus induction step of culturing the explant after the gene introduction step in a callus induction medium to induce callus formation; and a redifferentiation step of culturing the culture after the callus induction step in a redifferentiation medium, the method further comprising a division step of dividing the explant after the gene introduction step and before the redifferentiation step, and adding a silver compound to the culture medium in one or more steps selected from the gene introduction step, the callus induction step and the redifferentiation step. (17) The method according to (16), comprising selecting the explant into which the exogenous gene has been introduced based on the expression of the exogenous gene in one or more steps selected from the gene introduction step, the callus induction step and the redifferentiation step. This specification contains the disclosures of Japanese Patent Application No. 2025-005963, which forms the basis of the priority of this application.

[0012] This invention makes it possible to efficiently produce transformed plants by dividing explant fragments collected from plant bodies. As a result, transformed plants can be obtained stably, reproducibly, and at a lower cost.

[0013] 1. Method for producing transformed plants 1-1. Overview The first aspect of the present invention is a method for producing transformed plants. The production method of the present invention includes the steps of obtaining an explant from a plant body, introducing an exogenous gene into the explant, culturing the gene-introduced explant to induce callus formation, and redifferentiating the callus-introduced explant, and further including the step of dividing the explant after the step of introducing the exogenous gene and before the step of redifferentiation.

[0014] In this invention, the efficiency of plant transformation can be further enhanced by combining the division of the explant fragments with the addition of a silver compound to the culture medium.

[0015] In the present invention, various dicotyledonous and monocotyledonous plants can be used as the target plants for transformation. Examples of dicotyledonous plants include Solanaceae plants (potatoes, tomatoes, tobacco, etc.); Convolvulaceae plants, which include Ipomoea genus plants (sweet potato); Myricaceae plants; Brassicaceae plants (Arabidopsis thaliana, cabbage, radish, wasabi, wild mustard, broccoli, etc.); Fabaceae plants (soybeans, etc.); Linaceae plants; Zygophyllaceae plants; Apiaceae plants; and Asteraceae plants (garland chrysanthemum, lettuce, burdock, butterbur, etc.). It is preferable that the target plant for transformation is a Solanaceae plant, and among Solanaceae plants, potatoes are particularly preferred.

[0016] Examples of monocotyledonous plants that can be transformed in this invention include grasses (rice, wheat, barley, oats, corn, sorghum, rye, adlay, sugarcane, etc.).

[0017] 1-2. Structure The steps of the plant manufacturing method of the present invention will now be described. The manufacturing method of the present invention includes, as essential steps, an explant acquisition step, a gene introduction step, a callus induction step, a redifferentiation step, and a division step. The most important feature of the present invention is that the division step, which divides the explant after the gene introduction step and before the redifferentiation step, is performed.

[0018] In the manufacturing method of the present invention, a silver compound may be added to the culture medium in one or more of the gene transfer step, callus induction step, and redifferentiation step, if necessary. Furthermore, the manufacturing method of the present invention may include a plant culture step after the redifferentiation step in which the redifferentiated tissue is cultured in a plant body.

[0019] The explant acquisition step, gene introduction step, callus induction step, redifferentiation step, and plant culture step of the present invention can be carried out in accordance with known plant transformation methods, unless otherwise specified.

[0020] 1-2-1. Explant Acquisition Process In the explant acquisition process of the present invention, explants are obtained by cutting out a portion of a plant body that has been cultivated in a sterile state with the intention of transformation. Alternatively, a portion of a plant body cultivated in a greenhouse or field can be sterilized, and the tissue washed with sterile water can be used as an explant.

[0021] 1-2-1-1. Collection of Explants In this specification, "plant body" means a complete individual, not a section or anything like that. In this specification, "explant" means a tissue piece cut out from a plant body for the purpose of transformation. Explants can be, but are not limited to, sections of leaves, stems, roots, etc., of a plant body, or the hypocotyl, cotyledons, etc., of a young plant body. In this invention, it is particularly preferable to use leaves of a plant body as explants. Such explants can be prepared by various means, such as tearing them off the plant body with tweezers or cutting them out with scissors, a scalpel, or other bladed tools.

[0022] 1-2-1-2. Size of Explants The size of the explants collected here is not particularly limited; any size that allows for efficient gene transfer, callus induction, and redifferentiation processes described below is acceptable. If the explant is a leaf, it should be 10-100 mm.２ The size of the range, preferably 20 to 80 mm ２ The size of the range, particularly preferably 30 to 60 mm ２ Leaf fragments within this size range can be obtained, but are not limited to this range. In the example below, 7 mm square, i.e., 49 mm ２ Potato leaf fragments are being obtained and used in experiments.

[0023] 1-2-2. Gene Introduction Process In the gene introduction process of the present invention, an exogenous gene is introduced into the explant obtained in the explant acquisition process and cultured in a culture medium of an appropriate composition. As a result, the target exogenous gene is introduced into the explant.

[0024] 1-2-2-1. Exogenous Genes An exogenous gene is any gene other than an endogenous gene present in the explant into which the gene is introduced. Exogenous genes may be target genes related to the properties intended to be conferred to the transformed plant, such as genes that confer resistance to plant diseases and pests, genes that confer resistance to plant environmental stress, or genes that promote plant growth. Examples of such genes include, but are not limited to, Xa21, RPM1, and Cf2, which confer resistance to plant diseases; Cry, which confers resistance to plant insect pests; GPS, which increases plant yield; and bar, mutant EPSPS, and modified AHAS, which confer resistance to plant herbicides.

[0025] Furthermore, the exogenous gene may be a reporter gene or selection marker gene that serves as an indicator of gene introduction and is useful for selecting transformed plants. Examples of reporter genes include the β-glucuronidase (GUS) gene, the green fluorescent protein (GFP) gene, the red fluorescent protein (RFP) gene, and the isopentenyltransferase (IPT) gene. Examples of selection marker genes include, but are not limited to, the kanamycin resistance gene (NPTII) and the hygromycin resistance gene (hyg or hpt), which confer antibiotic resistance. Introducing such reporter genes and selection marker genes into explants in addition to the target gene for traits to be conferred to plants is a preferred embodiment of the present invention. It should be noted that introducing only reporter genes or selection marker genes as exogenous genes without introducing the target gene is also included in the embodiments of the present invention.

[0026] Furthermore, the exogenous gene may be a gene that codes for a genome editing system. Here, the term "genome editing system" is not particularly limited as long as it is an artificial restriction enzyme system that can cause modifications at any site on the genomic DNA. Examples of such artificial restriction enzyme systems include the CRISPR / Cas system, the TALEN system, the ZFN system, and the PPR system.

[0027] Furthermore, the present invention also includes transient expression of exogenous genes without inserting them into the plant genome. Details of reporter genes and selection marker genes are described in detail in section 1-2-7 below.

[0028] 1-2-2-2. Gene Introduction Method The method for introducing exogenous genes into plants is not particularly limited and can be carried out according to known methods in the field for introducing nucleic acid molecules into plant cells. Generally, methods such as the Agrobacterium method, particle gun method, protoplast method, electroporation method, polyethylene glycol method, liposome method, microinjection method, whisker method, plasma method, and laser injection method are known and can be used. In this gene introduction process, introducing exogenous genes by the Agrobacterium method or particle gun method described below is a preferred embodiment.

[0029] (1) Agrobacterium method When the Agrobacterium method is used in the gene introduction step of the present invention, the exogenous gene is introduced into the plant explant by bringing a plant explant into contact with Agrobacterium bacteria containing the exogenous gene, inoculating it, and culturing it.

[0030] Methods using Agrobacterium fungi include transformation methods using Agrobacterium species (e.g., Agrobacterium tumefaciens, Agrobacterium rhizogenes) as transformation factors, and Ti plasmids and Ri plasmids derived from them, which allow for the introduction of exogenous genes into the explants of the target plant.

[0031] For example, methods include adding plant explants to a liquid culture medium containing Agrobacterium bacteria, directly dropping the Agrobacterium suspension onto explants on a co-existence culture medium, injecting the Agrobacterium suspension into plant explants, and immersing plant explants in the Agrobacterium suspension under reduced pressure. However, the method of inoculating Agrobacterium bacteria in the present invention is not limited to these.

[0032] As described above, fungi of the genus Agrobacterium have the property of introducing genes inserted into the T-DNA of plasmids within the fungus into the plant genome. By inoculating plant tissue with Agrobacterium fungi containing such plasmids, plant transformation can be achieved, thereby conferring desirable traits to plant cells in the tissue. Examples of Agrobacterium plasmids usable in the present invention include, but are not limited to, pIG121Hm, pSB131, U0009B, U0017S, pSB134, pNB131, and pLC41.

[0033] Furthermore, in the present invention, a binary vector system can be used, and the binary vector system consists of two plasmids. The first is a T-DNA binary vector (hereinafter referred to as the binary vector), which has two T-DNA boundary repeat sequences positioned on either side of the DNA sequence to be inserted into the host plant, and the exogenous gene is cloned between the T-DNA boundary repeat sequences of the binary vector. The second plasmid is called a vir helper plasmid and has the components necessary to insert the sequence sandwiched between the T-DNA boundary repeat sequences into the plant cell genome.

[0034] As a more preferred embodiment, a super-binary vector system (Hiei et al., 1994; Ishida et al., 1996) can be used. This system consists of a disarmed Ti plasmid having a vir region (virA, virB, virC, virD, virE and virG, hereinafter sometimes referred to as "vir fragment regions" respectively) and a plasmid having T-DNA, and is thus a kind of binary vector system. However, it is different in that a super-binary vector in which a fragment of the vir region obtained by substantially removing at least one vir fragment region among the vir fragment regions (preferably a fragment containing at least virB or virG, more preferably a fragment containing virB and virG) is incorporated into the plasmid having T-DNA, i.e., the binary vector, is used. In addition, in order to introduce a T-DNA region incorporating a desired gene into Agrobacterium having a super-binary vector, homologous recombination via a three-way crossing method can be used as an easy technique. This super-binary vector system has been found to provide a very high transformation efficiency in many plant species as compared with the various vector systems described above.

[0035] When inoculating Agrobacterium, in order to improve the transformation efficiency by Agrobacterium, for example, various additives such as acetosyringone, a surfactant, porous ceramics, etc. can be included in the suspension of Agrobacterium.

[0036] The Agrobacterium that can be used in the present invention is not particularly limited, and any known Agrobacterium that can be used in the transformation method by Agrobacterium may be used. In a preferred embodiment of the present invention, the Agrobacterium is, for example, LBA4404, EHA101, EHA105, AGL1, C58C1, etc., but is not limited thereto.

[0037] When a super binary vector is not used as the vector, from the viewpoint of transformation efficiency, it is preferable to use a strain containing the Ti plasmid pTiBo542 possessed by Agrobacterium A281.

[0038] In the following examples, the super binary vector pTOK233 (Hiei et al., Plant Journal (1994) 6(2) 271-82) was introduced into Agrobacterium tumefaciens LBA4404, and an exogenous gene was introduced into potato leaf segments.

[0039] (2) Gene introduction method using the particle gun method In the particle gun method, fine particles such as gold particles or tungsten particles are coated with DNA, RNA, and / or protein, and are injected into the target cells by the pressure of a high-pressure gas such as helium gas for introduction, which is a gene introduction method.

[0040] (3) Other gene introduction methods The protoplast method is a method of introducing a target nucleic acid, protein, etc. into plant cells using plant cells (mainly protoplasts) whose cell walls have been removed by enzymatic treatment with cellulase, etc. This method can be further classified into the electroporation method, microinjection method, polyethylene glycol method, etc. according to the gene introduction method. The electroporation method is a method of applying an electric pulse to a mixture of plant cells and a target nucleic acid, protein, etc. to introduce the nucleic acid, protein, etc. into the plant cells. The microinjection method is a method of directly introducing a target nucleic acid, protein, etc. into plant cells under a microscope using a fine needle. The polyethylene glycol method is a method of introducing a target nucleic acid, protein, etc. into plant cells by the action of polyethylene glycol.

[0041] All of the above methods are known methods in the art, and for details, an appropriate protocol for plant genetic manipulation may be referred to.

[0042] 1-2-2-3. Regarding Coexistence Mediums In this specification, among the media used in this process, the media used in the Agrobacterium method is referred to as "coexistence medium." Coexistence mediums may be those commonly used for culturing plant cells, and examples include, but are not limited to, media based on MS inorganic salts, media based on N6 inorganic salts, and media based on B5 inorganic salts. Specific examples of media include MS medium (Murashige and Skoog's medium), LS medium (Linsmailer and Skoog's medium), Gumborg B5 medium, White medium, Niche medium, KNUDSON C medium, SB medium, R2 medium, N6 medium, Tuleek medium, and basic media such as PCM (potato co-cultivation medium). The media may also contain sugars, vitamins, etc. The culture medium may be a solid medium or a liquid medium.

[0043] In this process, "cultivation" refers to placing plant tissue on a solidified culture medium or in a liquid culture medium and growing it under appropriate temperature, light / dark conditions, and duration. In the present invention, the form of the culture medium is not particularly limited as long as the culture medium components are sufficiently supplied to the plant tissue. The culture medium can be solidified by adding a solidifying agent known in the art, such as agarose and gellan gum. In the present invention, such a solidified culture medium can be suitably used.

[0044] The culture temperature in this process can be selected as appropriate, preferably 20°C to 35°C, and more preferably 25°C. The culture period in this process can also be selected as appropriate, preferably 1 to 6 days, preferably 2 to 4 days, and more preferably 2 days, but is not limited to this range.

[0045] 1-2-3. Callus Induction Process In the callus induction process of the present invention, the explant after the gene transfer process is cultured on a callus induction medium to induce callus formation. The means for inducing callus formation may be those commonly used in the art.

[0046] In this specification, the term "callus" refers to an amorphous, undifferentiated mass of cells that does not possess the original morphology of (differentiated) plant tissue, and is obtained by culturing differentiated explants in a medium containing plant hormones such as auxin and cytokinin.

[0047] Plants possess totipotency, the ability to regenerate the entire plant from a single cell. When a part of a plant is placed in a culture medium containing adjusted plant hormones, differentiated cells dedifferentiate and undergo repeated cell division, forming a mass of cells, or callus. In this specification, the process of inducing callus formation by placing an explant in a culture medium is referred to as "callus induction."

[0048] In this specification, the culture medium used in this process is referred to as "callus induction medium," and in this process, the explants after the gene transfer process are cultured in such a medium. This process involves removing Agrobacterium bacteria from plant cells after the gene transfer process and promoting the proliferation of dedifferentiated plant cells, i.e., inducing callus formation.

[0049] The callus induction medium used in this process may be any medium commonly used for culturing plant cells, such as MS inorganic salt-based medium, N6 inorganic salt-based medium, B5 inorganic salt-based medium, etc., but is not limited to these. Specific examples of media include MS medium (Murashige and Skoog's medium), LS medium (Linsmailer and Skoog's medium), Gamborg B5 medium, White medium, Niche medium, KNUDSON C medium, SB medium, R2 medium, N6 medium, Tuleek medium, and basic media such as PCM (potato co-cultivation medium). The medium may also contain sugars, vitamins, etc. The medium may be a solid medium or a liquid medium.

[0050] The callus induction medium in this process preferably contains plant hormones. Examples of such plant hormones include auxins and cytokinins. Examples of "auxins" include 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). Examples of "cytokinins" include, but are not limited to, zeatin, benzyladenine, and thidiazurone.

[0051] Furthermore, the callus induction medium in this process may contain drugs effective for selection using selection marker genes. For example, if the exogenous gene includes an antibiotic resistance gene as a selection marker gene, transformed calluses can be selected by culturing them in a callus induction medium containing that antibiotic.

[0052] In this process, "cultivation" refers to placing explants on a solidified callus induction medium containing plant hormones, or in a liquid callus induction medium containing plant hormones, and growing them under appropriate temperature, light / dark conditions, and duration. In this invention, the form of the culture medium is not particularly limited as long as the medium components are sufficiently supplied to the plant tissue. The callus induction medium can be solidified by adding a solidifying agent known in the art, such as agarose. The culture temperature in this process can be appropriately selected, preferably 20°C to 35°C, and more preferably 25°C. Furthermore, the culture in this process is preferably carried out in the dark, but is not limited thereto.

[0053] In the callus induction step, the gene-transformed explants are cultured on a callus induction medium. The culture period in this step is not particularly limited, but for example, it is 1 to 10 days, preferably 2 to 10 days, preferably 3 to 8 days, and more preferably 5 days.

[0054] 1-2-4. Redifferentiation Process In the present invention, a redifferentiation process is performed in which the culture after the callus induction process is redifferentiated by culturing it in a culture medium for redifferentiation. In this specification, the culture medium used in this process is referred to as the "redifferentiation medium".

[0055] In this invention, "redifferentiation" refers to the process by which plant tissue (callus), which has been dedifferentiated in whole or in part, regains the properties of the original plant tissue or plant body. That is, by culturing in a redifferentiation medium during the redifferentiation process, it is possible to form a unit of plant tissue consisting of a stem and leaves that grow on it, such as a "shoot." Unlike callus induction medium, which consists of a hormonal composition that dedifferentiates plant tissue, the redifferentiation medium consists of a composition that includes plant hormones that regenerate stem and leaf tissue, thereby enabling the redifferentiation of dedifferentiated tissue.

[0056] The culture medium used in this process is referred to herein as "redifferentiation medium." Examples of redifferentiation mediums include, but are not limited to, media based on MS inorganic salts, media based on N6 inorganic salts, and media based on B5 inorganic salts. Examples of specific media include MS medium (Murashige and Skoog's medium), LS medium (Linsmailer and Skoog's medium), Gumborg B5 medium, White medium, Niche medium, KNUDSON C medium, SB medium, R2 medium, N6 medium, Tuleek medium, and basic media such as PCM (potato co-cultivation medium). The medium may contain sugars, vitamins, etc. The medium may be a solid medium or a liquid medium.

[0057] Furthermore, the redifferentiation medium in this process may contain drugs effective for selection using selection marker genes. For example, if the exogenous gene includes an antibiotic resistance gene as a selection marker gene, it is possible to select transformed plant tissue, such as shoots, by culturing them in a redifferentiation medium containing that antibiotic.

[0058] In this process, "cultivation" refers to placing plant tissue on a solidified redifferentiation medium or in a liquid redifferentiation medium and growing it under appropriate temperature, light / dark conditions, and duration. In this invention, the form of the medium is not particularly limited as long as the medium components are sufficiently supplied to the plant tissue. The redifferentiation medium can be solidified using, for example, agarose, as described above. The cultivation temperature in this process can be appropriately selected, preferably 20°C to 35°C, and more preferably 25°C. The cultivation period in this process is 1 week to 16 weeks, preferably 2 weeks to 12 weeks, more preferably 2 weeks to 8 weeks, and even more preferably 6 weeks, but is not limited to this range.

[0059] 1-2-5. Dividing Process As already described, in the manufacturing method of the present invention, in addition to the explant acquisition process, gene introduction process, callus induction process, and redifferentiation process, a dividing process is performed to divide the explant after the gene introduction process and before the redifferentiation process. This dividing process makes it possible to increase the efficiency of callus induction, the efficiency of redifferentiation, and the efficiency of plant transformation, which is the most important feature of the present invention.

[0060] This division process can be performed at either the gene transfer step or the redifferentiation step. The explant can also be divided by cutting it with scissors or a scalpel. However, any method that allows for the division of the explant into desired sizes can be used.

[0061] The following three embodiments can be cited as examples of the division process. All of these embodiments are included in the present invention. 1) An embodiment in which the explant is divided after gene introduction but before culturing to induce callus formation. In this embodiment, the explant is divided after the introduction of an exogenous gene but before callus formation has been induced. 2) An embodiment in which the explant is divided during culturing to induce callus formation. In this embodiment, the explant is divided at an appropriate time during callus induction culture. 3) An embodiment in which the explant is divided after culturing to induce callus formation but before culturing in a redifferentiation medium. In this embodiment, the explant in which callus has formed is divided, and then culturing in a redifferentiation medium.

[0062] The embodiment of 3) above is preferred in the present invention. In this specification, performing the division step "after the callus culture step" in the present invention means this embodiment.

[0063] In this division step, the explant is ２ from 3 mm ２ to 40 mm ２ in size, preferably from 6 mm ２ to 25 mm ２ in size, more preferably from 4 mm ２ to 10 mm ２ in size, and most preferably from 3 mm ２ to 8 mm

[0064] and divided. The size of the divided explant here can be appropriately selected in consideration of various factors such as the part of the plant body from which the explant is derived (such as leaves, stems, roots, etc.) and the size of the formed callus. ２ ), when the leaf piece is preferably divided into 2 to 8 pieces. For example, when a 7 mm square leaf piece (49 mm ２ ) is divided into 2 to 8 pieces, the size of the divided leaf piece will be from 6 mm ２ to 25 mm.

[0065] In the following examples, a 7 mm square leaf piece was divided into 4 pieces of the leaf piece after callus culture was performed after gene introduction and cultured in a regeneration medium. It has been shown that the efficiency of regeneration increased compared to the case of culturing in a regeneration medium without performing leaf piece division.

[0066] 1-2-6. Addition of silver compound to the medium In the present invention, a silver compound may be added to the medium in one or more steps selected from the gene introduction step, callus induction step, and regeneration step. By adding a silver compound to the medium in combination with the division step described in 1-2-5 above, the efficiency of gene introduction and the efficiency of plant transformation can be further increased.

[0067] A silver compound may be added to the culture medium in any one of the following steps: the gene transfer step, the callus induction step, or the redifferentiation step. Alternatively, the silver compound may be added to the culture medium in both the callus induction step and the redifferentiation step, or in both the gene transfer step and the callus induction step, or in both the gene transfer step and the redifferentiation step. Furthermore, the silver compound may be added to the culture medium in all three steps: the gene transfer step, the callus induction step, and the redifferentiation step.

[0068] Examples of silver compounds include, but are not limited to, silver nitrate and silver sulfide (e.g., silver thiosulfate complex (STS)). Preferred silver compounds are silver nitrate and silver sulfate, with silver nitrate being particularly preferred.

[0069] The amount of silver compound to be added is not particularly limited and can be appropriately set depending on various factors such as the type of plant explant, the process of adding the silver compound, and the culture medium to which the compound is added. For example, when the silver compound is silver nitrate and the explant is a leaf, silver nitrate can be added to one or more of the coexistence medium, callus induction medium, and redifferentiation medium at a concentration of 0.1 mg / L to 10 mg / L, preferably 0.3 mg / L to 3 mg / L, and more preferably 0.5 mg / L to 1 mg / L. In the following examples, silver nitrate was added to the culture medium at a concentration of 0.85 mg / L to increase the efficiency of gene transfer and plant transformation.

[0070] Furthermore, the following examples demonstrate that adding silver nitrate to the culture medium improves the efficiency of redifferentiation in the callus induction step, the redifferentiation step, the two steps of the callus induction step and the redifferentiation step, and the three steps of the gene introduction step, the callus induction step, and the redifferentiation step.

[0071] 1-2-7. Selection of Explants into which Exogenous Genes Have Been Introduced The manufacturing method of the present invention may include selecting the explants into which the exogenous genes have been introduced (transformed) based on the expression of the exogenous genes.

[0072] In order to select transformants based on the presence or absence of gene transfer, selecting transformants based on the expression of exogenous genes is a commonly practiced method in this art. While this selection is not essential in the method for producing transformed plants of the present invention, it is useful for efficiently selecting transformed individuals. The following description regarding selection is illustrative, and the present invention is not limited thereto.

[0073] In order to perform this selection, the exogenous gene must include a reporter gene or selection marker gene for selection purposes. Reporter genes or selection marker genes that can be used for this purpose are known in the art.

[0074] Such reporter genes are genes whose expression allows us to confirm whether or not a gene has been introduced. Examples include, but are not limited to, the GUS gene, GFP gene, RFP gene, and IPT gene.

[0075] Furthermore, the exogenous gene may be a selection marker gene that enables the selection of explants into which the gene has been introduced, based on resistance to antibiotics or herbicide components present in the culture medium. Such a selection marker gene is used to select explants into which the gene has been introduced. For example, if an antibiotic resistance gene is introduced as a selection marker gene, explants of non-transformed plants will have difficulty surviving when cultured in a medium containing antibiotics, thereby enabling the selection of transformed explants. The selection marker gene may also be an antibiotic resistance gene, such as a hygromycin resistance gene or a kanamycin resistance gene. Alternatively, the selection marker gene may also be a herbicide component resistance gene, such as a bar gene, a mutated EPSPS gene, or a modified AHAS gene. Furthermore, the selection marker gene may also be a pmi gene or a cah gene, which are genes that convert substances that are normally unusable by plant cells into usable compounds through the enzyme they encode, thereby allowing only transformed cells to selectively survive. The selection marker genes are not limited to those mentioned above; any gene that can be used as an indicator of gene introduction is acceptable.

[0076] Selection can be repeated multiple times by changing the composition of the culture medium. For example, performing selection multiple times increases the certainty of selection and increases the possibility of obtaining explants, callus, or plants (transformed explants, callus, or plants) into which exogenous genes have been introduced. Therefore, this selection is carried out in at least one step, preferably two steps, and more preferably three steps.

[0077] Specifically, selection can be performed in the gene transfer step, in the callus induction step, in the redifferentiation step, in the gene transfer step and the redifferentiation step, in the callus induction step and the redifferentiation step, and in all steps of the gene transfer step, callus induction step and redifferentiation step.

[0078] 1-2-8. Preparation of Plants After the redifferentiation step in 1-2-4, transformed plants can be obtained by further culturing the shoots in a redifferentiation medium or by culturing them in a rooting medium with a different plant hormone composition.

[0079] 2. Method for transforming plants 2-1. Overview A second aspect of the present invention is a method for transforming plants. The method for transforming plants of the present invention includes the steps of obtaining an explant from a plant body, introducing an exogenous gene into the explant, culturing the gene-introduced explant to induce callus formation, and redifferentiating the callus-induced explant, and further including the step of dividing the explant after the step of introducing the exogenous gene and before the step of redifferentiation.

[0080] In the method of the present invention, the efficiency of plant transformation can be further enhanced by combining the step of dividing the explant fragments with the addition of a silver compound to the culture medium.

[0081] 2-2. Structure The plant transformation method of the present invention includes, as essential steps, an explant acquisition step, a gene introduction step, a callus induction step, a redifferentiation step, and a division step. In the manufacturing method of the present invention, in addition to the explant acquisition step, gene introduction step, callus induction step, and redifferentiation step, the most important feature is that a division step is performed to divide the explant after the gene introduction step and before the redifferentiation step.

[0082] Each of the above steps is the same as described in 1-2-1 to 1-2-5 in the method for producing the transformed plant described above. The addition of the silver compound is also the same as described in 1-2-6. Furthermore, in the plant transformation method of the present invention, the same selection as described in 1-2-7 may be performed.

[0083] The present invention will be described in more detail with reference to the following embodiments, but the technical scope of the present invention is not limited by these embodiments.

[0084] <Example 1: Effect of adding silver nitrate to culture medium on redifferentiation efficiency> Example 1-1: Improvement of redifferentiation rate by adding silver nitrate (Variety: Russet Burbank) Potato transformation was performed by modifying the method of Craze et al. (2018). Specifically, the super binary vector pTOK233 (Hiei et al. 1994), which has a gene cassette in which a hygromycin resistance gene (hpt gene) driven by the cauliflower mosaic virus 35S promoter and a GUS gene bound to the intron of castor catalase are driven by the cauliflower mosaic virus 35S promoter in the T-DNA region, was introduced into the Agrobacterium tumefaciens LBA4404 strain. LBA4404 (pTOK233) bacteria were cultured on YP agar medium (powdered yeast extract: 5 g / L, peptone: 10 g / L, sodium chloride: 5 g / L, agar: 15 g / L, pH 6.8) at 28°C for 1 day. The grown bacteria were scraped off and suspended in PCM liquid medium (MS inorganic salts, MS vitamins, 2,4-D: 2 mg / L, zeatin: 0.5 mg / L, MES: 0.5 g / L, saccharose: 30 g / L, pH 5.7) containing 100 μM acetosyringone at a concentration where OD600 was 0.5, and used as an inoculum.

[0085] Leaves were taken from potatoes (variety: Russet Burbank) that were being subcultured in MS20 medium (MS inorganic salts, MS vitamins, MES: 0.5 g / L, saccharose: 20 g / L, agar: 5 g / L, pH 5.8), and the leaves were cut into approximately 7 mm squares on filter paper containing the inoculant. The leaf pieces were transferred to a petri dish containing the inoculant and left to stand at room temperature for 5 to 10 minutes. The leaf pieces were placed on the filter paper, the inoculant was removed, and the leaf surface was placed facing upwards on PCM-coexisting medium (PCM liquid medium supplemented with agarose 5 g / L and silver nitrate: 0.85 mg / L). The petri dish was sealed with surgical tape and incubated at 25°C for 2 days under dim lighting.

[0086] Leaf fragments were placed with the upper surface facing upwards in a PCM callus induction medium (MS inorganic salts, MS vitamins, 2,4-D: 2 mg / L, zeatin: 0.5 mg / L, MES: 0.5 g / L, silver nitrate: 0.85 mg / L, saccharose: 20 g / L, agarose: 5 g / L, pH 5.7) containing 20 mg / L hygromycin and 150 mg / L thimentin, and cultured under illumination at 25°C for 5 days. Since the PCM callus induction medium contains 2,4-D, callus formation was induced. Furthermore, since this medium contains hygromycin, transformed calluses were selected.

[0087] Leaf fragments were placed with the upper surface facing upwards in PSM redifferentiation medium containing 20 mg / L hygromycin and 150 mg / L thimentin (MS inorganic salts, MS vitamins, zeatin: 0.5 mg / L, gibberellic acid: 2 mg / L, MES: 0.5 g / L, silver nitrate: 0.85 mg / L, saccharose: 20 g / L, agarose: 5 g / L, pH 5.7), and cultured under light at 25°C for two weeks.

[0088] Leaf fragments were transferred to a culture medium of the same composition and cultured under the same conditions for two weeks. This procedure was repeated twice, resulting in a total of six weeks of culture in PSM redifferentiation medium. After culturing, the number of redifferentiated shoots (number of redifferentiated individuals) from each leaf fragment was counted. Since the PSM redifferentiation medium does not contain 2,4-D but contains zeatin and gibberellic acid, redifferentiation from callus and shoot elongation were observed. Furthermore, because this medium contains hygromycin, it was possible to select redifferentiated individuals that had undergone transformation.

[0089] As a result, no redifferentiated individuals were obtained in the untreated silver nitrate group, while redifferentiated individuals (redifferentiation rate 0.79) were obtained in the silver nitrate treated group (Table 1). Therefore, it was suggested that adding silver nitrate to the culture medium improved the redifferentiation rate.

[0090]

[0091] Example 1-2: Improvement of redifferentiation rate by adding silver nitrate (Variety: Konahime) Transformation was carried out in a different potato variety, Konahime, using the same method as in Example 1-1. In this example, the effect of the presence or absence of silver nitrate on the redifferentiation rate was investigated in the PCM co-existing medium, PCM callus induction medium, and PSM redifferentiation medium used in each step.

[0092] As a result, treating the callus induction medium or redifferentiation medium with silver nitrate during leaf tissue culture improved the redifferentiation efficiency (Table 2). Treating both the callus induction medium and the redifferentiation medium with silver nitrate further improved the redifferentiation rate. Furthermore, treating all stages—coexistence medium, callus induction medium, and redifferentiation medium—with silver nitrate further improved the redifferentiation rate. Therefore, it was suggested that treatment with silver nitrate in the callus induction stage, the redifferentiation stage, or all stages significantly improves the redifferentiation rate.

[0093]

[0094] <Example 2: Effect of leaf segmentation on redifferentiation efficiency> Example 2-1: Improvement of redifferentiation rate by leaf segmentation alone and improvement of redifferentiation rate by a combination of leaf segmentation and silver nitrate addition (Variety: Konahime) The effect of leaf segmentation on redifferentiation efficiency was investigated using the same method as in Example 1 (Variety: Konahime). For the segmentation group, the leaf segments were divided into four using a scalpel when placed on the PSM redifferentiation medium. In the silver nitrate treatment group, silver nitrate (0.85 mg / L) was added to all of the coexistence medium, callus induction medium, and redifferentiation medium. The culture period in the PSM redifferentiation medium was 6 weeks, and the number of leaf segments that formed shoots (number of redifferentiated leaf segments) was counted (Table 3).

[0095] As a result, in the silver nitrate-free group, redifferentiated leaf fragments were observed after leaf fragment division. The redifferentiated leaf fragment ratio, expressed as the number of redifferentiated leaf fragments divided by the number of inoculated leaf fragments, was 0.091. Furthermore, in the silver nitrate-treated group, the redifferentiated leaf fragment ratio was 0.45 when leaf fragment division was not performed, while it was 0.91 when leaf fragment division was performed, indicating a significant improvement in redifferentiation efficiency. In other words, it was suggested that combining leaf fragment division and silver nitrate treatment, compared to treating each treatment individually, leads to a greater improvement in redifferentiation efficiency.

[0096]

[0097] Example 2-2: Improvement of redifferentiation rate by combination of leaf division and silver nitrate addition (Variety: Russet Burbank) The effect of leaf division on the redifferentiation rate was investigated using the same method as in Example 2-1 (Variety: Russet Burbank). In all test plots, silver nitrate (0.85 mg / L) was added to the culture medium in all stages of the process: coexistence, callus induction, and redifferentiation. Five or eight weeks after inoculation with Agrobacterium, the number of leaf fragments that formed shoots (redifferentiated leaf fragments) and the number of shoots redifferentiated from each leaf fragment (redifferentiated individuals) were counted. As shown in Table 4, on all survey days, leaf division increased the number of redifferentiated leaf fragments and the number of redifferentiated individuals. As a result, it was confirmed that even when using Russet Burbank, leaf division improved the redifferentiation rate (number of redifferentiated individuals / number of inoculated leaf fragments).

[0098]

[0099] Example 2-3: Investigation of changes in redifferentiation rate depending on the timing of silver nitrate addition using a combination of leaf segmentation and silver nitrate addition (Variety: Konayutaka) We investigated whether the method of combining silver nitrate and leaf segmentation is effective even for Konayutaka, a potato variety that is difficult to cultivate. The experimental method was the same as in the example described above. For the segmented leaves, the leaves were divided into four sections with a scalpel when placed on the PSM redifferentiation medium. In addition, the effect of the presence or absence of silver nitrate on the redifferentiation rate was investigated in each process using PCM co-existing medium, PCM callus induction medium, and PSM redifferentiation medium.

[0100] As a result, redifferentiated individuals were obtained only in the test plots that combined leaf division and silver nitrate addition (Table 5). Therefore, it was demonstrated that the present invention, which combines silver nitrate and leaf division, is effective even for difficult-to-cultivate varieties.

[0101]

[0102] The present invention relates to a method for obtaining explants from a plant body, performing transformation by introducing exogenous genes, inducing callus formation from the transformed explants, and redifferentiating the resulting callus, wherein the explants are divided after gene introduction but before redifferentiation culture. The present invention makes it possible to provide a new method for efficiently producing transformed plants and a new, highly efficient method for transforming plants. Because the present invention relates to a method of transformation with high efficiency, it can be used to improve plant traits, for example, to produce plants with resistance to diseases and pests, plants with resistance to environmental stress, and edible plants with increased yields. All publications, patents and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A method for producing a transformed plant, the method comprising: an explant acquisition step of obtaining an explant from a plant body; a gene introduction step of introducing an exogenous gene into the explant and culturing it in a culture medium; a callus induction step of culturing the explant after the gene introduction step in a callus induction medium to induce callus formation; and a redifferentiation step of culturing the culture after the callus induction step in a redifferentiation medium, the method further comprising a division step of dividing the explant after the gene introduction step and before the redifferentiation step.

2. The manufacturing method according to claim 1, wherein a silver compound is added to the culture medium in one or more steps selected from the gene introduction step, the callus induction step, and the redifferentiation step.

3. The manufacturing method according to claim 1 or 2, comprising selecting explants into which the exogenous gene has been introduced based on the expression of the exogenous gene in one or more steps selected from the gene introduction step, callus induction step, and redifferentiation step.

4. The manufacturing method according to any one of claims 1 to 3, wherein the division step is performed after the callus induction step.

5. In the division step, the explant is cut to 6 mm ２ ~25mm ２ A manufacturing method according to any one of claims 1 to 4, which divides the material into pieces of a certain size.

6. The manufacturing method according to any one of claims 1 to 5, further comprising a plant culture step of culturing the redifferentiated tissue obtained after the redifferentiation step into a plant body.

7. The manufacturing method according to any one of claims 1 to 6, wherein the exogenous gene is introduced in the gene introduction step by the Agrobacterium method or the particle gun method.

8. The manufacturing method according to any one of claims 1 to 7, wherein the plant is a dicotyledonous plant.

9. The manufacturing method according to claim 8, wherein the dicotyledonous plant is a plant of the Solanaceae family.

10. The manufacturing method according to any one of claims 1 to 9, wherein the explant is derived from a plant leaf.

11. The manufacturing method according to any one of claims 2 to 10, wherein the silver compound is silver nitrate.

12. The manufacturing method according to any one of claims 1 to 11, wherein the exogenous gene includes the target gene.

13. The manufacturing method according to any one of claims 1 to 12, wherein the gene introduction step is performed in a culture for 1 to 6 days.

14. The manufacturing method according to any one of claims 1 to 13, wherein the callus induction step is performed for 2 to 10 days.

15. The manufacturing method according to any one of claims 1 to 14, wherein the redifferentiation step is performed by culturing for 2 to 12 weeks.

16. A method for transforming a plant, the method comprising: an explant acquisition step of obtaining an explant from a plant body; a gene introduction step of introducing an exogenous gene into the explant and culturing it in a culture medium; a callus induction step of culturing the explant after the gene introduction step in a callus induction medium to induce callus formation; and a redifferentiation step of culturing the culture after the callus induction step in a redifferentiation medium, the method further comprising a division step of dividing the explant after the gene introduction step and before the redifferentiation step, and adding a silver compound to the culture medium in one or more steps selected from the gene introduction step, the callus induction step, and the redifferentiation step.

17. The method according to claim 16, comprising selecting explants into which the exogenous gene has been introduced based on the expression of the exogenous gene in one or more steps selected from the gene introduction step, callus induction step, and redifferentiation step.

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