Fusion cells, method for producing fusion cells, cell mass, plant body, and method for producing plant body

Abstract

To provide a fused cell that can be grown to obtain a plant, a fused cell production method, a cell aggregation, a plant, and a plant production method.SOLUTION: Provided is a method for producing a fused cell of a plant. The method comprises a step for obtaining a fused cell by fusing a plant egg cell or a cell derived therefrom, and a plant sperm cell or a cell derived therefrom.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a fused cell, a method for producing a fused cell, a cell mass, a plant, and a method for producing a plant. [Background technology]

[0002] In the agricultural and horticultural markets, there is demand for plants that produce high seed or fruit yields, plants that produce large flowers, plants that have improved resistance to environmental stresses, etc. It is known that plants with such traits can be obtained, for example, by crossbreeding.

[0003] However, fertilization between different species is often difficult, and efficient interspecies hybridization techniques are desired. In response to this, the inventors have developed a technique for fusing egg cells and sperm cells of different plant species by electrofusion (e.g., Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6436701 Summary of the Invention [Problem to be solved by the invention]

[0005] However, fertilized eggs obtained from egg cells and sperm cells of different plant species may exhibit phenotypes such as embryonic lethality, and may not be able to be cultivated to obtain plants. Therefore, an object of the present invention is to provide fused cells, a method for producing fused cells, a cell mass, a plant, and a method for producing a plant, which can be grown to give a plant. [Means for solving the problem]

[0006] The present invention includes the following aspects. [1] A method for producing a fused cell, comprising a step of fusing a plant egg cell or a cell derived therefrom with a plant sperm cell or a cell derived therefrom to obtain a fused cell. [2] The method of producing the present invention described in [1], wherein the egg cells include egg cells of two or more different plant species. [3] A manufacturing method described in [1] or [2], wherein the taxonomic group to which the plant species from which the egg cells are derived belongs and the taxonomic group to which the plant species from which the sperm cells are derived belong belong to the same family or lower taxonomic group. [4] The method according to any one of [1] to [3], wherein the egg cell is an egg cell of a plant species belonging to the family Poaceae. [5] The method according to any one of [1] to [4], wherein the egg cell is an egg cell of a plant species belonging to the subfamily Poaceae, the subfamily Ehrhartineae, or the subfamily Panniculidae. [6] A method for producing a cell cluster, comprising the step of culturing the fused cells obtained by the production method according to any one of [1] to [5] to obtain a cell cluster. [7] A method for producing a plant, comprising the step of producing a plant from the fused cell obtained by the production method according to any one of [1] to [5]. [8] A fused cell obtained by the production method described in any one of [1] to [5]. [9] A plant grown from the fused cells obtained by the production method described in any one of [1] to [5].

[10] A fusion cell obtained by fusing a plant egg cell or a cell derived therefrom with a plant sperm cell or a cell derived therefrom, wherein the nuclear genomic DNA of the fusion cell contains all or part of the nuclear genomic DNA of a first plant species, the nuclear genomic DNA of the fusion cell contains 30% or less of the nuclear genomic DNA of a second plant species, and the first plant species and the second plant species are different.

[11] The fusion cell described in

[10] , wherein the mitochondrial genomic DNA of the fusion cell has part or all of the mitochondrial genomic DNA of a first plant species and part or all of the mitochondrial genomic DNA of a second plant species.

[12] The fusion cell described in

[10] or

[11] , wherein the plastid genomic DNA of the fusion cell has part or all of the plastid genomic DNA of the first plant species and part or all of the plastid genomic DNA of the second plant species.

[13] The fused cell according to any one of

[10] to

[12] , which is a fused cell between a plant egg cell or a cell derived therefrom and a plant sperm cell or a cell derived therefrom.

[14] The fused cell according to any one of

[10] to

[13] , wherein the first plant species and / or the second plant species is a plant species belonging to the family Poaceae.

[15] The fusion cell described in any of

[10] to

[14] , wherein the first plant species and / or the second plant species is a plant species belonging to the subfamily Poaceae of the Poaceae family, the subfamily Ehrhartineae of the Poaceae family, or the subfamily Panniculidae of the Poaceae family.

[16] The first plant species is a plant species belonging to the Poaceae family, subfamily Poaceae, The fused cell according to any one of

[10] to

[15] , wherein the second plant species is a plant species belonging to the subfamily Ehrharta family of the family Poaceae.

[17] A plant grown from the fused cell according to any one of

[10] to

[16] . Effect of the Invention

[0007] According to the present invention, it is possible to provide fused cells, a method for producing fused cells, a cell mass, a plant, and a method for producing a plant, which can be grown to give a plant. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic diagram illustrating a method for producing fusion cells according to one embodiment of the present invention. [Diagram 2] FIG. 1 is a schematic diagram illustrating a method for producing fusion cells according to one embodiment of the present invention. [Diagram 3] FIG. 1 is a schematic diagram illustrating a method for producing fusion cells according to one embodiment of the present invention. [Figure 4] FIG. 1 is a schematic diagram illustrating a method for producing fusion cells according to one embodiment of the present invention. [Diagram 5] FIG. 1 is a schematic diagram illustrating a method for producing fusion cells according to one embodiment of the present invention. [Figure 6] FIG. 1 is a schematic diagram illustrating a method for producing fusion cells according to one embodiment of the present invention. [Figure 7] This is a photograph of a plant body grown from a fused cell obtained by fusing a rice egg cell, a wheat egg cell, and a wheat sperm cell. [Figure 8] 1 is a photograph of a plant produced by cultivating a fused cell obtained by fusing a rice egg cell, a rice sperm cell, a wheat egg cell, and a wheat sperm cell. [Figure 9] This is a photograph of a plant grown from a hybrid cell obtained by fusing a rice sperm cell isolated from a tetraploid rice with a wheat egg cell. [Figure 10] Photographs of embryoid bodies, cell masses, and plants grown from fused cells obtained by fusing maize sperm cells and wheat egg cells. [Figure 11A] FIG. 1 shows the results of mapping the nuclear genomic DNA, mitochondrial genomic DNA, and plastid genomic DNA of a plant grown from a fused cell obtained by fusing a rice egg cell, a wheat egg cell, and a wheat sperm cell. [Figure 11B] FIG. 1 shows the results of mapping the nuclear genomic DNA, mitochondrial genomic DNA, and plastid genomic DNA of a plant grown from a fused cell obtained by fusing a rice egg cell, a wheat egg cell, and a wheat sperm cell. [Figure 11C] FIG. 1 shows the results of mapping the nuclear genomic DNA, mitochondrial genomic DNA, and plastid genomic DNA of a plant grown from a fused cell obtained by fusing a rice egg cell, a wheat egg cell, and a wheat sperm cell. [Figure 12] FIG. 1 shows the compositions of mitochondrial genomic DNA and plastid genomic DNA of fused cells obtained by fusing a rice egg cell, a wheat egg cell, and a wheat sperm cell, and of plants grown from fused cells obtained by fusing a wheat egg cell, a rice egg cell, a wheat sperm cell, and a rice sperm cell. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] <Method for producing fused cells> In one embodiment, the present invention provides a method for producing a fused cell, comprising a step of fusing a plant egg cell or a cell derived therefrom with a plant sperm cell or a cell derived therefrom to obtain a fused cell.

[0010] As used herein, the term "egg cell" refers to a female gamete formed in the pistil by meiosis of an embryo sac mother cell. The method for isolating egg cells is not limited, but for example, the ovary is cut in a solution with an appropriate osmotic pressure, and the egg cells emerging from the cut surface can be isolated using a glass capillary under a microscope.

[0011] As used herein, the term "sperm cells" refers to male gametes formed in the anthers of the stamens by meiosis of pollen mother cells. There are no limitations on the method for isolating sperm cells. For example, when pollen collected from an anther is immersed in a solution of appropriate osmotic pressure, the pollen contents including sperm cells are released from the pollen into the solution after a few minutes, and the sperm cells can be isolated using a glass capillary under a microscope.

[0012] In this embodiment, the "cell derived from a plant egg cell" means a cell obtained by fusing a plant egg cell with any cell, and may be, for example, a cell obtained by fusing a plurality of egg cells, or a cell obtained by fusing an egg cell with a sperm cell. The origin of a cell can be determined by analyzing the genomic information possessed by the cell. The ploidy of the cell obtained by fusing multiple egg cells is not particularly limited, and may be, for example, diploid, triploid, tetraploid, pentaploid, hexaploid, octaploid, etc. Among these, the cell obtained by fusing multiple egg cells is preferably diploid, triploid, or tetraploid, and more preferably diploid.

[0013] In this embodiment, the plant species of the egg cells is not particularly limited and may be one species or two or more species. It is preferable that the egg cells include egg cells of two or more different plant species. When the plant species of the egg cells are two or more different plant species, it is possible to produce fused cells having new, superior traits derived from, for example, the genetic information of different cytoplasms.

[0014] Two or more different plant species may mean that the plant species belong to different species or higher taxonomic groups, different genera or higher taxonomic groups, different subfamilies or higher taxonomic groups, or different families or higher taxonomic groups.

[0015] In this embodiment, the "cell derived from a plant sperm cell" means a cell obtained by fusing a plant sperm cell with any cell, and may be, for example, a cell obtained by fusing a plurality of sperm cells, or a cell obtained by fusing a sperm cell with an egg cell. The origin of a cell can be determined by analyzing the genomic information possessed by the cell.

[0016] In this embodiment, assuming that the number of haploid egg cells that serve as material for the fused cells is m and the number of haploid sperm cells that serve as material for the fused cells is n, it is preferable that n is equal to or less than m. For example, when two haploid egg cells are fused in the production of fused cells, it is preferable to fuse two haploid egg cells with one or two haploid sperm cells.

[0017] Examples of plant species families to which egg cells and / or sperm cells belong include Malvaceae, Sterculiaceae, Chenopodiaceae, Rubiaceae, Cannabaceae, Brassicaceae, Linaceae, Poaceae, Cucurbitaceae, Anacardiaceae, Ebenaceae, Bilberry family, Asteraceae, Asparagaceae, Mulberry family, Juglandaceae, Pedaceae, Piperaceae, Araceae, Lamiaceae, Zingiberaceae, Apiaceae, Polygonaceae, Ericaceae, Theaceae, Solanaceae, Bromeliaceae, Musaceae, Nelumbaceae, Papaya family, Rosaceae, Convolvulaceae, Fagaceae, Actinidiaceae, Fabaceae, Rutaceae, Oleaceae, Dioscoreaceae, Liliaceae, and the like.

[0018] Furthermore, examples of the genera of plant species to which the egg cells and / or sperm cells belong include the genera shown below. Examples of genera in the Poaceae family include Phyllostachys, Hordeum, Triticum, Oryza, Bucconata, Turfgrass, Saccharum, Panicum, Barnyard Millet, Sorghum, and Maize. Examples of genera in the Brassicaceae family include Brassica, Arabidopsis, Wasabi, Horseradish, Shepherd's Purse, Euonymus purse, Radish, and Shepherd's Purse. Examples of genera in the Chenopodiaceae family include the genus Spinacea and the genus Beet. Examples of genera in the Fabaceae family include Phaseolus, Pisum, Vicia, Sword bean, Glycine, Kudzu, Vigna, Coryo, Labia, Pigeon pea, Arachis, Chickpea, Rosewood, Buttercup, Astragalus, Licorice, Broom, Quercus, Lotus, Lupinus, and Wisteria. Examples of genera in the Solanaceae family include Solanum, Capsicum, Nicotiana, Datura, Physalis, and Petunia.

[0019] In this embodiment, the taxonomic group of the plant species to which the egg cell belongs is preferably the Poaceae or Leguminosae family. Egg cells of plant species belonging to the family Poaceae are larger than egg cells of other plant species, and the egg cells are easier to handle and fused cells can be produced, making it easier to produce fused cells, and the resulting fused cells can be cultivated to produce plants. Since legume species are capable of nitrogen fixation in symbiosis with nitrogen-fixing bacteria, plants useful in the fields of horticulture and agriculture can be produced from the resulting fused cells.

[0020] The egg cell is preferably an egg cell of a plant species belonging to the subfamily Poaceae, the subfamily Ehrhartineae, or the subfamily Panniculidae of the family Poaceae. In this case, the obtained fused cell and a plant body obtained by cultivating the fused plant can be cultivated as food and are useful in agriculture. The taxonomic group of plant species to which the egg cells belong is preferably the genus Hordeum, the genus Triticum, or the genus Sesquieoideae in the family Poaceae, subfamily Poaceae. Alternatively, the taxonomic group of plant species to which the egg cells belong is preferably the genus Oryza in the subfamily Ehrhartidae of the family Poaceae. Alternatively, the taxonomic group of plant species to which the egg cells belong is preferably the genus Saccharum, the genus Sorghum, the genus Maize, the genus Millet, or the genus Barnyard Millet in the subfamily Panicum of the family Poaceae. More specifically, plant species to which the egg cells belong include Asian rice (O. sativa L.), Oryza rufipogon (O. rufipogon sensu lato), African rice (O. glaberrima Steud.), wild rice (O. rufipogon, O. barthii, O. longistaminata, O. meridionalis, etc.), bread wheat (Triticum aestivum), durum wheat (Triticum durum), corn (Zea mays), etc.

[0021] In the production method according to the present embodiment, the taxonomic group of the plant species to which the egg cell belongs and the taxonomic group of the plant species to which the sperm cell belongs, for at least one pair of egg cell and sperm cell, are preferably taxonomic groups belonging to the same family or lower, more preferably taxonomic groups belonging to the same genus or lower, and even more preferably taxonomic groups belonging to the same species or lower. Examples of taxonomic groups below the species include cultivars, lineages, subspecies, varieties, etc. In the manufacturing method of this embodiment, it is preferable that the taxonomic group of the plant species to which the egg cells belong and the taxonomic group of the plant species to which the sperm cells belong are from the same family, more preferably from the same genus, and even more preferably from the same species. If the taxonomic group of the plant species to which the egg cells belong and the taxonomic group of the plant species to which the sperm cells belong belong to the same subfamily or lower taxonomic group, development will be favorable, and the resulting fused cells can be cultivated to more easily produce a plant body.

[0022] In this embodiment, the plant to which the egg cell belongs is, for example, C 3 It may be a plant, C 4 It may be a plant. Said C 3 Examples of plant taxonomic groups include the genus Oryza and the genus Triticum. Said C4 Examples of plant taxonomic groups include the genus Saccharum, Sorghum, Maize, Millet, Barnyard Millet, and Setaria. C 3 Since the plant is a staple crop, the resulting fused cells can be cultivated to produce plants useful in horticulture and agriculture. From the point of view of producing useful agricultural crops, the plant to which the egg cells belong is C. 4 Preferably, it is a plant. 4 Plants are capable of fixing carbon dioxide even in high temperature, dry environments, etc., and the resulting fused cells can be cultivated to produce plants that are useful in horticulture and agriculture and have high stress resistance.

[0023] Among the above examples, the taxonomic group of plant species to which the egg cells belong is preferably the subfamily Poaceae of the family Poaceae, and more preferably the genus Triticum. Furthermore, the taxonomic group of the plant species to which at least one pair of egg cells and sperm cells belongs is preferably the subfamily Poaceae of the family Poaceae, and more preferably the genus Triticum. When the egg cells include egg cells of two or more different plant species, the taxonomic group of the plant species to which the egg cells belong preferably belongs to the family Poaceae and not to the subfamily Poaceae, more preferably to the subfamily Ehrhartineae of the Poaceae family or the subfamily Panniculinae of the Poaceae family, and even more preferably to the subfamily Ehrhartineae of the Poaceae family.

[0024] The egg cells and sperm cells may be obtained from a genetically modified plant, where the genetically modified plant may be, for example, a plant whose genomic DNA has been modified or a plant that harbors a vector. The egg cells, sperm cells, and fused cells may be those into which nucleic acids, proteins, peptides, etc. have been introduced. Examples of the method for introducing the nucleic acids include the methods described in JP-A-2019-129705 and JP-A-2020-72645.

[0025] In this embodiment, the plant species to which the sperm cells belong can be, for example, the same species as the plant species to which the egg cells belong.

[0026] More specifically, for example, the following first to fourth embodiments can be mentioned: In each embodiment, the plant species of the egg cell and the plant species of the sperm cell can be exemplified by the above-mentioned plant species.

[0027] The first to fourth embodiments will be described with reference to the drawings shown in FIGS.

[0028] First Embodiment The production method according to this embodiment, as exemplified in FIG. 1, is a method for producing a fused cell by fusing a first egg cell with a first sperm cell. The plant species of the egg cell is different from the plant species of the sperm cell. Examples of the taxonomic groups of plant species to which egg cells and sperm cells belong include the taxonomic groups mentioned above. The taxonomic group of the plant species of the egg cells and the plant species of the sperm cells is preferably the Poaceae family, more preferably the subfamily Pooideae, the subfamily Ehrhartineae, or the subfamily Panniculinae. The sperm cells may be, for example, diploid sperm cells, and more specifically, may be diploid sperm cells obtained from a tetraploid plant of a plant species belonging to the subfamily Ehrhartidae of the family Poaceae. Alternatively, the plant species of the sperm cells may be a plant species belonging to the family Poaceae, subfamily Panniculidae.

[0029] <Second embodiment> The method for producing a fused cell according to this embodiment is a method for producing a fused cell by fusing a first egg cell, a second egg cell, and a first sperm cell.

[0030] FIG. 2 is a schematic diagram illustrating a method for producing fusion cells according to one embodiment of the present invention. In FIG. 2, nuclei of cells having genome information that is more closely related to one another are shown using the same hatching, and this is also true in the following FIGS. 3 to 6.

[0031] The manufacturing method of this embodiment may include a step of fusing a first egg cell with a first sperm cell to obtain a first fusion cell, and a step of fusing the first fusion cell with a second egg cell, as illustrated in Figure 2. In addition, the manufacturing method of this embodiment may include a step of fusing a second egg cell with a first sperm cell to obtain a first fusion cell, and a step of fusing the first fusion cell with the first egg cell. In addition, the manufacturing method of this embodiment may include a step of fusing a first egg cell with a second egg cell to obtain a first fusion cell, and a step of fusing the first fusion cell with a first sperm cell, as illustrated in Figure 3.

[0032] In the production method of this embodiment, at least one of the first egg cell and the second egg cell is preferably from a plant species different from that of the sperm cell. It is preferable that the family or lower taxonomic group of the plant species to which the first sperm cell belongs is the same as the subfamily or lower taxonomic group of the plant species to which the first egg cell belongs, or the subfamily or lower taxonomic group of the plant species to which the second egg cell belongs. In this case, the initial development of the fused cells proceeds stably, and the fused cells can be more reliably cultivated to obtain a plant body. It is preferable that the taxonomic group of the plant species to which the first egg cell belongs is different from the taxonomic group of the plant species to which the second egg cell belongs. In this case, the genetic information of the obtained fused cell has both the genetic information of the first egg cell and the genetic information of the second egg cell, and fused cells with diverse traits can be obtained. Examples of the taxonomic groups of plant species to which egg cells and sperm cells belong include the taxonomic groups mentioned above. The taxonomic group of the plant species to which the first sperm cells belong is preferably the Poaceae family, and more preferably the Poaceae subfamily. When the taxonomic group of the plant species to which the first sperm cell belongs and the taxonomic group of the plant species to which the first egg cell belongs is the subfamily Poaceae, Poaceae, the taxonomic group of the plant species to which the second egg cell belongs is not particularly limited, but is preferably the family Poaceae, more preferably the subfamily Ehrhartineae, Poaceae or the subfamily Panniculinae, and even more preferably the subfamily Ehrhartineae, Poaceae.

[0033] <Third embodiment> The production method according to this embodiment is a method of producing a fused cell by fusing a first egg cell, a second egg cell, a first sperm cell, and a second sperm cell. In the production method of this embodiment, at least one egg cell of the two egg cells is from a different plant species from at least one sperm cell of the two sperm cells.

[0034] The manufacturing method of this embodiment may include, as illustrated in Figure 4, a step of fusing a first egg cell with a first sperm cell to obtain a first fusion cell, a step of fusing the first fusion cell with a second sperm cell to obtain a second fusion cell, and a step of fusing the second fusion cell with a second egg cell. Furthermore, the manufacturing method of this embodiment may include, as illustrated in FIG. 5, a step of fusing a first egg cell with a first sperm cell to obtain a first fusion cell, a step of fusing a second egg cell with a second sperm cell to obtain a second fusion cell, and a step of fusing the first fusion cell with the second fusion cell. In addition, the manufacturing method of this embodiment may include, as illustrated in Figure 6, a step of fusing a first egg cell with a second egg cell to obtain a first fusion cell, a step of fusing the first fusion cell with a first sperm cell to obtain a second fusion cell, and a step of fusing the second fusion cell with the second sperm cell.

[0035] It is preferable that the taxonomic group of the plant species to which at least one of the two egg cells belongs is the same as the taxonomic group of the plant species to which at least one of the two sperm cells belongs. In this case, the initial development of the fused cells proceeds stably, and the fused cells can be more reliably cultivated to obtain a plant body. It is preferable that the taxonomic group of the plant species to which the first egg cell belongs is different from the taxonomic group of the plant species to which the second egg cell belongs. In this case, the genetic information of the obtained fused cell has both the genetic information of the first egg cell and the genetic information of the second egg cell, and fused cells with diverse traits can be obtained. Examples of the taxonomic groups of plant species to which egg cells and sperm cells belong include the taxonomic groups mentioned above. More preferably, the taxonomic group of the plant species to which at least one of the egg cells belongs is the family Poaceae, subfamily Poaceae. More preferably, the plant species to which at least one of the sperm cells belongs is the family Poaceae, subfamily Poaceae. It is further preferable that the taxonomic group of the plant species to which the egg cells and sperm cells belong is the subfamily Poaceae, the subfamily Ehrhartineae, or the subfamily Panniculidae.

[0036] <Fourth embodiment> The production method according to the present embodiment is a method of fusing egg cells or cells derived therefrom of two or more plant species with sperm cells of one or more plant species. In the present embodiment, the total number of haploid egg cells and haploid sperm cells used as materials is 5 or more, and the number of haploid egg cells is 3 or more.

[0037] Examples of the taxonomic groups of plant species to which egg cells and sperm cells belong include the taxonomic groups mentioned above. Preferably, the plant species to which at least one of the egg cells belongs is the family Poaceae. More preferably, the taxonomic group of the plant species to which at least one of the egg cells belongs is the family Poaceae, subfamily Poaceae. The taxonomic group of the plant species to which at least one of the sperm cells belongs is preferably the Poaceae family, subfamily Poaceae.

[0038] <Fusion> As used herein, "cell fusion" refers to fusing the cytoplasm of two or more cells of the same or different species, and preferably artificial fusion. An example of artificial fusion is in vitro fusion. As used herein, "fused cells" refers to cells formed by fusing two or more cells of the same or different species, and preferably refers to cells formed by fusing two or more cells having the same or different cytoplasm. The method of fusing cells is not particularly limited, but is preferably performed by electric fusion.

[0039] When cell fusion is carried out by electrofusion, conditions such as voltage and osmotic pressure of the solution can be appropriately set by those skilled in the art. The conditions for electric fusion are not particularly limited, but examples thereof include the following conditions (see, for example, Japanese Patent No. 6436701). The DC voltage used when electrofusion of plant cells is preferably 5 to 30 kv / cm, more preferably 8 to 20 kv / cm, and even more preferably 12 to 15 kv / cm. The lower limit of the osmotic pressure of the solution for electrofusion of plant cells is 380 mosmol / kg H 2 O or higher, and 390 mosmol / kg H 2 More preferably, it should be 400 mosmol / kg H or more. 2 It is more preferable to set the upper limit at 470 mosmol / kg H. 2 It is preferable to keep the blood glucose level at 460 mosmol / kg H or less. 2 It is more preferable to keep it at 450 mosmol / kg H or less. 2 It is more preferable that the content is 0 or less. The upper and lower limits can be appropriately selected by those skilled in the art.

[0040] <Method for producing cell aggregates> In one embodiment, the present invention provides a method for producing a cell cluster, comprising the step of culturing the fused cells obtained by the above-mentioned production method to obtain a cell cluster. In this embodiment, examples of the cell cluster include a globular embryo, an embryoid body, a callus, etc.

[0041] The method for culturing the above-mentioned fused cells to obtain cell clusters is not particularly limited and may be a method known to those skilled in the art. For example, the following method for obtaining cell clusters includes forming callus from the fused cells to obtain cell clusters. First, the fused cells are placed in a medium and cultured with shaking. Examples of the medium include liquid MS medium (T. Murashige et al., Physiol. Plant., 15, 473 (1962)), B5 medium (OL Gamborg et al., Experimental Cell Research, 50, 151-158 (1968)), and N6 medium (Chu et al., Sci. Sinica, 18, 659-668 (1975)) to which auxins such as 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid have been added. The shaking speed may be 30 to 50 rpm, and the culture temperature may be 24 to 28°C. The culture is preferably carried out in the dark. The concentration of auxin added to the medium may be 0.1 to 0.3 mg / L. It is preferable to add feeder cells to the medium. The culture period may be 4 to 7 days. The globular embryos with a diameter of about 50 to 200 μm obtained by shaking culture are transferred to the above-mentioned medium to which no feeder cells have been added, and further cultured for about 10 to 20 days to obtain cell colonies. Thereafter, the callus is formed by culturing the callus in any medium containing auxin and / or kinetin, for example, N6 medium. In this case, the culturing is preferably performed under light irradiation, and the light may be, for example, 50 to 400 μmol photons m -2 sec -1 The medium for callus formation preferably contains a support, and examples of the support that can be used include agar, gellan gum, and gellite.

[0042] <Production method of plant body> In one embodiment, the present invention provides a method for producing a plant, comprising the step of producing a plant from the fused cell obtained by the above-mentioned production method. The plant obtained by this embodiment is fertile and capable of producing seeds, and therefore, by cultivating the seeds, it is possible to easily grow a large number of progeny of the plant.

[0043] As used herein, the term "plant" refers to the first generation plant line cultivated from the fused cells and its descendants. In addition, as used herein, the term "plant" refers to the plant itself, as well as parts thereof such as plant organs and plant tissues, such as leaves, stems, and roots, and seeds.

[0044] A progeny plant line of the first generation plant line cultivated from the fused cells can be obtained, for example, by obtaining seeds from the first generation plant body, and the obtained seeds can be germinated and cultivated. The cultivation method can be appropriately selected by those skilled in the art. Alternatively, a progeny plant line of a first-generation plant line may be obtained by dedifferentiating the first-generation plant line, redifferentiating the dedifferentiated cells, and cultivating the redifferentiated cells to obtain a progeny plant line. The method of dedifferentiation and redifferentiation can be appropriately selected by one skilled in the art.

[0045] ≪Fused cells≫ In one embodiment, the present invention provides a fusion cell obtained by the method for producing a fusion cell according to the above embodiment. In this embodiment, the fused cells also include cells cultured from the fused cells obtained by the method for producing fused cells of the above-mentioned embodiment, and cells that constitute a plant body grown from the fused cells obtained by the method for producing fused cells of the above-mentioned embodiment. Methods for culturing and growing the fused cells include, for example, the methods described above.

[0046] In one embodiment, the present invention provides a fusion cell, wherein the nuclear genomic DNA of the fusion cell comprises all or a portion of the nuclear genomic DNA of a first plant species and 30% or less of the nuclear genomic DNA of a second plant species, and the first plant species and the second plant species are different.

[0047] Examples of the fused cells include those exemplified in the above <<Method for producing fused cells>>. When the taxonomic group of the plant species to which the egg cell belongs and the taxonomic group of the plant species to which the sperm cell belongs are taxonomic groups belonging to the same subfamily or lower (preferably the same genus or lower, more preferably the same species or lower), the plant species of the egg cell and / or sperm cell can be exemplified as the first plant species.

[0048] When the egg cells include egg cells of two or more different plant species, the second plant species may be different from the first plant species. The second plant species may belong to a different species or higher taxonomic group from the first plant species, may belong to a different genus or higher taxonomic group from the first plant species, may belong to a different subfamily or higher taxonomic group from the first plant species, or may belong to a different family or higher taxonomic group from the first plant species.

[0049] More specifically, as shown in the Examples below, for example, a case can be exemplified in which the sperm cell and egg cell of the first plant species are from wheat, and the egg cell of the second plant species is from rice.

[0050] As will be shown in the Examples below, it is presumed that nuclear genomic DNA derived from egg cells and / or sperm cells used in a combination that has the same or a close phylogenetic relationship to each other tends to be contained in a larger amount in the nuclear genome of the fused cell.

[0051] As used herein, "the nuclear genomic DNA of the fused cell contains X% of the nuclear genomic DNA of the first plant species" means that "the total number of sequences derived from the nuclear genomic DNA of the first plant species contained in the nuclear genomic DNA of the fused cell is X% of the total number of sequences of the entire nuclear genomic DNA of the first plant species." The same applies to the second plant species. Here, the "sequence derived from the nuclear genomic DNA of the first plant species" used as a reference is a sequence possessed by the first plant species used in fusion as a raw material for producing the fusion cell. The reference sequence may refer to the wild type of the plant species or the genomic DNA sequence possessed by a registered variety. If duplication of the reference sequence is found in the fusion cell, the duplicated sequence is not included in the ratio. In addition, with regard to the proportions relating to the mitochondrial genome and plastid genome described below, the above nuclear genome can be read as the mitochondrial genome or the plastid genome, respectively.

[0052] The fused cell of this embodiment preferably contains 70% or more and 100% or less of the nuclear genomic DNA of the first plant species, more preferably 80% or more and 100% or less, and even more preferably 90% or more and 100% or less. The fusion cell of this embodiment loses more than half of the nuclear genomic DNA of the second plant species. The nuclear genomic DNA of the fusion cell of this embodiment may contain more than 0% to 20% of the nuclear genomic DNA of the second plant species, more than 0% to 15% of the nuclear genomic DNA of the second plant species, or more than 0% to 10% of the nuclear genomic DNA of the second plant species. In order to diversify the characteristics of the fused cell, the nuclear genomic DNA of the fused cell preferably contains 1% or more of the nuclear genomic DNA of the second plant species, more preferably 3% or more, even more preferably 5% or more, and particularly preferably 10% or more.

[0053] The fused cell according to this embodiment may be a fused cell between a plant egg cell or a cell derived therefrom and a plant sperm cell or a cell derived therefrom. The fused cells according to this embodiment may be obtained by the above-mentioned method for producing plant fused cells. In the fused cell according to the present embodiment, the plant species of the egg cell is not particularly limited, and may be one type or two or more types. The plant species of the sperm cell may be one type or two or more types. Examples of the plant species of the egg cell and the sperm cell include the plant species mentioned above in the method for producing the fused cell.

[0054] In the nuclear genomic DNA of the fused cell of this embodiment, a portion of the nuclear genomic DNA derived from the first plant species and a portion of the nuclear genomic DNA derived from the second plant species may be deleted. In the nuclear genomic DNA of the fused cell of this embodiment, a portion of the nuclear genomic DNA derived from the first plant species and a portion of the nuclear genomic DNA derived from the second plant species may overlap.

[0055] The sequence of the nuclear genomic DNA of the fused cell according to this embodiment can be determined by isolating the genomic DNA of the fused cell and analyzing the obtained genomic DNA by next-generation sequencing or the like. Furthermore, by using known base sequence analysis software, the nuclear genomic DNA sequence of the first plant species and the nuclear genomic DNA sequence of the second plant species can be mapped to the nuclear genomic DNA sequence of the fusion cell of this embodiment.

[0056] The mitochondrial genomic DNA of the fused cell of this embodiment may have all or a portion of the mitochondrial genomic DNA of a first plant species and all or a portion of the mitochondrial genomic DNA of a second plant species.

[0057] In the mitochondrial genomic DNA of the fused cell of this embodiment, a portion of the mitochondrial genomic DNA derived from the first plant species and a portion of the mitochondrial genomic DNA derived from the second plant species may be deleted. In the mitochondrial genomic DNA of the fused cell of this embodiment, a portion of the mitochondrial genomic DNA derived from the first plant species and a portion of the mitochondrial genomic DNA derived from the second plant species may overlap.

[0058] The mitochondrial genomic DNA of the fused cell preferably contains 80% or more and 100% or less of the mitochondrial genomic DNA of the first plant species, more preferably 90% or more and 100% or less, and even more preferably 95% or more and 100% or less. The mitochondrial genomic DNA of the fused cell preferably contains 5% or more and 100% or less of the mitochondrial genomic DNA of the second plant species, more preferably 20% or more and 100% or less, and even more preferably 40% or more and 100% or less.

[0059] This can also be rephrased as follows: Of the sequences of the mitochondrial genomic DNA of the fused cell (100%), the sequences derived from the mitochondrial genomic DNA of the first plant species are preferably 50% or more and 95% or less, and more preferably 50% or more and 80% or less. Of the sequences (100%) of the mitochondrial genomic DNA of the fused cell, the sequences derived from the mitochondrial genomic DNA of the second plant species are preferably 5% to 50%, more preferably 10% to 50%.

[0060] The sequence of the mitochondrial genomic DNA of the fused cell according to this embodiment can be determined by isolating the genomic DNA of the fused cell and analyzing the obtained genomic DNA by next-generation sequencing or the like. Furthermore, by using known base sequence analysis software, the sequence of the mitochondrial genomic DNA of the first plant species can be mapped to the sequence of the mitochondrial genomic DNA of the fused cell of this embodiment, and the sequence of the mitochondrial genomic DNA of the first plant species can be mapped to the sequence of the mitochondrial genomic DNA of the first plant species.

[0061] The plastid genomic DNA of the fused cell according to this embodiment may have all or a part of the plastid genomic DNA of a first plant species, and all or a part of the plastid genomic DNA of a second plant species.

[0062] In the plastid genomic DNA of the fused cell of this embodiment, a portion of the plastid genomic DNA derived from the first plant species and a portion of the plastid genomic DNA derived from the second plant species may be deleted. In the plastid genomic DNA of the fused cell of this embodiment, a portion of the plastid genomic DNA derived from the first plant species and a portion of the plastid genomic DNA derived from the second plant species may overlap.

[0063] The plastid genomic DNA of the fused cell preferably contains 80% or more and 100% or less of the plastid genomic DNA of the first plant species, more preferably 90% or more and 100% or less, and even more preferably 95% or more and 100% or less. The plastid genomic DNA of the fused cell preferably contains 3% or more and 100% or less of the plastid genomic DNA of the second plant species, preferably 5% or more and 100% or less, more preferably 10% or more and 100% or less, and even more preferably 20% or more and 100% or less.

[0064] This can also be rephrased as follows: Of the sequences (100%) of the plastid genomic DNA of the fused cell, the sequences derived from the plastid genomic DNA of the first plant species are preferably 50% or more and 98% or less, and more preferably 50% or more and 95% or less. Of the sequences (100%) of the plastid genomic DNA of the fused cell, the sequences derived from the plastid genomic DNA of the second plant species are preferably 2% to 50%, more preferably 5% to 50%.

[0065] The sequence of the plastid genomic DNA of the fused cell according to this embodiment can be determined by isolating the genomic DNA of the fused cell and analyzing the obtained genomic DNA by next-generation sequencing or the like. Furthermore, by using known base sequence analysis software, the sequence of the plastid genomic DNA of the first plant species can be mapped to the sequence of the plastid genomic DNA of the fusion cell of this embodiment, and the sequence of the plastid genomic DNA of the first plant species can be mapped to the sequence of the plastid genomic DNA of the first plant species.

[0066] In this embodiment, the fused cell may be a fused cell between a plant egg cell or a cell derived therefrom and a plant sperm cell or a cell derived therefrom. Examples of the taxonomic group of the first plant species and / or the second plant species include the taxonomic groups described above in the method for producing fused cells. The first plant species and / or the second plant species are preferably plant species belonging to the family Poaceae. It is more preferable that the first plant species and / or the second plant species are plant species belonging to the subfamily Poaceae, the subfamily Ehrhartineae, or the subfamily Panniculidae of the family Poaceae.

[0067] More specifically, the first plant species may be a plant species belonging to the subfamily Poaceae of the family Poaceae, and the second plant species may be a plant species belonging to the subfamily Ehrhartidae of the family Poaceae. In this case, the fused cell is preferably a fused cell of an egg cell of a plant species belonging to the subfamily Poaceae of the family Poaceae, an egg cell of a plant species belonging to the subfamily Ehrhartafamily of the family Poaceae, and a sperm cell of a plant species belonging to the subfamily Poaceae of the family Poaceae. In this case, the nuclear genomic DNA of the fused cell preferably contains 70% or more, more preferably 80% or more, and even more preferably 90% or more of the nuclear genomic DNA of the first plant species, and the nuclear genomic DNA of the plant body grown from the fused cell preferably contains 3% or more, more preferably 5% or more, and even more preferably 10% or more of the nuclear genomic DNA of the second plant species.

[0068] In this specification, the content (%) of each genomic DNA can be obtained by mapping the DNA base sequence by next-generation sequencing, etc., with the sequence information of each genomic DNA (each genome, mitochondrial genome, plastid genome) of the original cell of the fused cell being taken as 100%. Note that, if duplication occurs in the sequence information as a result of fusion in the fused cell, the duplication is excluded.

[0069] ≪Plant body≫ In one embodiment, the present invention provides a plant grown from the fused cells obtained by the method for producing fused cells of the above-mentioned embodiment. The method for cultivating a plant from the fused cells is not particularly limited, and may be any of the above-mentioned plant cultivating methods.

[0070] According to one embodiment of the method for producing fused cells, hybrid cells of different plant species can be stably obtained. By culturing and cultivating these hybrid cells, first-generation and subsequent generations of plants can be obtained. According to the above-mentioned embodiment of the method for producing fused cells, by containing cytoplasmic genomic information such as mitochondrial genomic DNA and plastid genomic DNA contained in egg cells of two or more different plant species, hybrid plants with excellent traits such as improved environmental stress resistance and improved yield can be easily obtained. The fused cells and plants produced by fusing non-genetically modified cells of existing plant species using the above-mentioned embodiment of the method for producing fused cells are not genetically modified plants, and therefore can be easily cultivated without undergoing safety evaluation tests, etc. Conventional methods for producing hybrid cells by fusing protoplasts of different plant species were applicable only to limited plant species such as Brassicaceae, Solanaceae, etc. In contrast, the method for producing plant fusion cells according to one embodiment of the present invention is applicable to many plant species. By using the technique of cell fusion by electrofusion, egg cells and sperm cells of different plant species can be easily fertilized to obtain hybrid cells. EXAMPLES

[0071] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0072] Materials and Methods (Isolation of rice egg cells and rice sperm cells) Isolation of rice egg cells and rice sperm cells was performed according to the literature (Toda E et al. (2016) Electro-fusion of Gametes and Subsequent Culture of Zygotes in Rice. Bio-protocol. Vol 6, Iss 24, December 20, 2016.). The rice variety used in the experiment was Oryza sativa L.cv Nipponbare. 0.3 mL of mineral oil was dropped onto a siliconized cover glass, and several 1-2 μL drops of mannitol solution (370 mosmol / kg H) were then dispensed into the mineral oil using a micro glass capillary. 2 Droplets of O) were prepared. Mannitol solution (370 mosmol / kg H 2 Ovary was removed from unopened flowers and incubated in a mannitol solution (370 mosmol / kg H 2 The ovary was cut using a razor in a 30-well plate (O), and then the rice egg cells were released from the ovary using a glass needle. The obtained rice egg cells were transferred into the droplet on the cover glass using a micro glass capillary. The egg cells were used for each experiment within 6 hours after isolation. If the egg cells were not used on the day, they were stored at 4°C overnight and used for the experiment the next day. Pollen was taken from unopened flowers and soaked in a mannitol solution (370 mosmol / kg H 2 O) to obtain sperm cells.

[0073] (Isolation of wheat egg cells and wheat sperm cells) The wheat species used in the experiment was Triticum aestivum L. cv. Fielders. Wheat egg cells and wheat sperm cells were isolated in the same manner as in the above-mentioned method for isolating rice egg cells and rice sperm cells.

[0074] (Isolation of corn sperm cells) The corn varieties used in the experiments were corn (B73 or A188). Maize sperm cells were isolated in the same manner as in the above-mentioned isolation methods for rice egg cells and rice sperm cells.

[0075] (Cell electrofusion) The egg cell and sperm cell were placed in a droplet on the cover glass described above, and the egg cell was attached to an electrode in an AC current field (1 MHz, 5 Vrms), and then the sperm cell was attached to the egg cell. 0.5-1 μL of mannitol solution (520 mosmol / kg H 2 O) was added to the droplets, and then a direct current pulse (50 μs, 12 to 15 kV / cm) was applied to induce cell fusion, and the resulting fused cells were collected.

[0076] (Culture of fused cells) The fused cells obtained by electrofusion were soaked in a mannitol solution (450 mosmol / kg H 2 O). Next, 0.2 mL of modified N6Z medium for fertilized egg culture and the fused cells were placed into the Millicell-CM insert, and the Millicell containing the fused cells was cultured in medium containing 40 to 60 μL of wheat cultured cells (feeder cells) at 26°C overnight in the dark. The culture was then continued for an additional 7 days with shaking at 30 rpm. After removing the feeder cells, the Millicell containing the cultured fused cells was placed in 2 mL of fertilized egg culture medium and cultured for 20 days.

[0077] The modified N6Z medium for culturing fertilized eggs was composed of 2 g / L CHU(N6) basal salt mixture (Sigma-Aldrich), 0.025 mg / L Na 2 MoO 4 2H2 O, 0.025 mg / L CoCl 2 6H 2 O, 0.025 mg / L CuSO 4 5H 2 O, 0.01mg / L retinol, 0.01mg / L calciferol, 0.01mg / L biotin, 1mg / L thiamine H 2 The supplements were: 1mg / L nicotinic acid, 1mg / L pyridoxine·HCl, 1mg / L choline chloride, 1mg / L Ca-pantothenic acid, 0.2mg / L riboflavin, 0.2mg / L 2,4-D, 0.02mg / L cobalamin, 0.02mg / L p-aminobenzoic acid, 0.4mg / L folic acid, 2mg / L ascorbic acid, 40mg / L malic acid, 40mg / L citric acid, 40mg / L fumaric acid, 20mg / L Na-pyruvic acid, 1,000mg / L glutamine, 250mg / L casein hydrolysate, and 100mg / L myo-inositol. The osmolarity was controlled by glucose at 450mosmol / kg H 2 The solution was adjusted to 0, pH 5.7, and sterilized by filter.

[0078] Next, the cell colonies in the Millicell were transferred to callus induction medium (a solid medium of modified N6Z medium, which is a medium in which the glucose in the modified N6Z medium for fertilized egg culture described above has been replaced with maltose (2.7%) and solidified with 0.4% Gelrite) and cultured at 30°C under continuous light for 12 to 30 days to form callus. The resulting calli were cultured on regeneration and rooting medium (Ishida, Y., Tsunashima, M., Hiei, Y., Komari, T. (2015) Wheat (Triticum aestivum L.) transformation using immature embryos. Methods Mol. Biol. 1223: 189-98.) under a 13-h / 11-h light / dark cycle at 28°C for 11 to 20 days to obtain plantlets.

[0079] (Genomic DNA analysis) Genomic DNA was isolated from the leaves of plants obtained by regenerating the callus of the fused cells. A sequence library was prepared from the obtained genomic DNA using the Nextera DNA Flex Library Prep Kit (Illumina). The obtained library was sequenced using Illumina HiSeqX_Ten; Paired End 150bp. The reads obtained by sequencing were mapped using HomeoRoq (Akama et al., Genome-wide quantification of homeolog expression ratio revealed nonstochastic gene regulation in synthetic allopolyploid Arabidopsis. Nucleic Acids Research 42: e46 (2014)).

[0080] [Experimental Example 1] The isolated wheat sperm cells and rice egg cells were electrofused to generate the first fused cells. Subsequently, the obtained first fusion cell was electrofused with a wheat egg cell to produce a second fusion cell. The resulting second fusion cells were cultured to obtain callus. The resulting callus was cultured and cultivated to obtain a plant body. Of the five second fusion cells, three formed plants. Figure 7 shows an example of the obtained plant body. Figure 7 shows a plant body 119 days after the second fusion cells were produced. Seeds were obtained from this plant, which were then germinated and cultivated to yield more seeds.

[0081] [Experimental Example 2] The isolated rice sperm cells and rice egg cells were electrofused to produce the first fused cells. The isolated wheat sperm cells and wheat egg cells were electrofused to generate secondary fusion cells. Subsequently, the first fusion cell obtained and the second fusion cell obtained were electrofused to produce a third fusion cell. The obtained third fusion cells were cultured to obtain callus. The obtained callus was cultured and cultivated to obtain plant bodies. Of the seven third fusion cells, five formed plants. Figure 8 shows an example of the obtained plant body. Figure 8 shows a plant body 121 days (left in Figure 8), a plant body 120 days (center in Figure 8), and a plant body 117 days (right in Figure 8) after the production of the third fusion cells. Seeds were obtained from these plants, which were then germinated and cultivated to yield more seeds.

[0082] [Experimental Example 3] Hybrid cells were produced by electrofusion of rice sperm cells isolated from tetraploid rice with wheat egg cells. The resulting fused cells were cultured to obtain callus. The resulting callus was cultured and cultivated to obtain a plant body. Of the 10 fused cells, 3 formed a plant body. Figure 9 shows an example of the obtained plant body. Seeds were obtained from this plant.

[0083] [Experimental Example 4] Maize sperm cells isolated from wheat were electrofused with wheat egg cells to produce fused cells. The resulting fused cells were cultured to obtain callus. The resulting callus was cultured and cultivated to obtain plant bodies. Of the seven fused cells, five formed plants. Figure 10 shows the growth process of the resulting plants. In Figure 10, DAF indicates the number of days elapsed since the production of the fused cells.

[0084] [Experimental Example 5] The fertilized cells obtained by fusing wheat egg cells, rice egg cells, and wheat sperm cells were cultured to obtain plants. Genomic DNA was isolated from the leaves of the obtained plants, and the sequence of the genomic DNA was analyzed and mapped.

[0085] The mapping results are shown in FIG. It was revealed that the nuclear genomic DNA of the above-mentioned leaves contains more than 95% of the nuclear genomic DNA of hexaploid wheat and 3-10% of the nuclear genomic DNA of diploid rice. The mitochondrial genomic DNA of the leaves mentioned above contained almost all of the wheat mitochondrial genomic DNA and about half of the rice mitochondrial genomic DNA. The plastid genomic DNA of the leaves mentioned above contained almost all of the wheat plastid genomic DNA and approximately 10% of the rice plastid genomic DNA.

[0086] This can also be rephrased as follows: The size of the nuclear genomic DNA of hexaploid wheat is approximately 40 times that of the nuclear genomic DNA of diploid rice. The size of wheat mitochondrial genome DNA is approximately the same as that of rice mitochondrial genome DNA. The size of the wheat plastid genomic DNA is almost the same as that of the rice plastid genomic DNA. Approximately 99.8% of the nuclear genomic DNA in the leaves mentioned above was derived from wheat nuclear genomic DNA, and approximately 0.2% of the nuclear genomic DNA in the leaves was derived from rice nuclear genomic DNA. Approximately 65% ​​of the mitochondrial genomic DNA in the leaves mentioned above was derived from wheat mitochondrial genomic DNA, and approximately 35% of the mitochondrial genomic DNA in the leaves was derived from rice mitochondrial genomic DNA. Approximately 95% of the plastid genomic DNA in the leaves mentioned above was derived from wheat plastid genomic DNA, and approximately 5% of the plastid genomic DNA in the leaves was derived from rice plastid genomic DNA.

[0087] Similarly, we fused a wheat egg cell, a rice egg cell, and a wheat sperm cell to obtain fertilized cells, and cultured them to obtain a plant body (Cybrid-1). Furthermore, we fused a wheat egg cell, a rice egg cell, a wheat sperm cell, and a rice sperm cell to obtain fertilized cells, and cultured them to obtain a plant body (Cybrid-2 and -3). We analyzed the mitochondrial genome and plastid genome of these plants.

[0088] The results are shown in Figure 12. 12, the graph for mitochondrial genomic DNA shows the ratio of the number of sequences of rice-derived mitochondrial genomic DNA and the number of sequences of wheat-derived mitochondrial genomic DNA to the total number of sequences of mitochondrial genomic DNA in leaves, while the graph for plastid genomic DNA shows the ratio of the number of sequences of rice-derived plastid genomic DNA and the number of sequences of wheat-derived plastid genomic DNA to the total number of sequences of plastid genomic DNA in leaves.

[0089] As shown in FIG. 12, 60 to 90% of the mitochondrial genomic DNA in the above-mentioned leaves was derived from wheat mitochondrial genomic DNA, and approximately 10 to 40% of the mitochondrial genomic DNA in the leaves was derived from rice mitochondrial genomic DNA. As shown in FIG. 12, about 75 to 95% of the plastid genomic DNA in the above-mentioned leaves was derived from wheat plastid genomic DNA, and about 5 to 25% of the plastid genomic DNA in the leaves was derived from rice plastid genomic DNA.

[0090] This can also be rephrased as follows: In the cybrid plants (Cybrid-1 to Cybrid-3), the mitochondrial genomic DNA contained almost all of the wheat mitochondrial genomic DNA and 12 to 65% of the rice mitochondrial genomic DNA. The size of wheat mitochondrial genome DNA is almost the same as that of rice mitochondrial genome DNA.

[0091] In the cybrid plants (Cybrid-1 to Cybrid-3), the plastid genomic DNA contained almost all of the wheat plastid genomic DNA and 5.5 to 35% of the rice plastid genomic DNA. The size of wheat plastid genomic DNA is almost the same as that of rice plastid genomic DNA. [Industrial Applicability]

[0092] According to the present invention, it is possible to provide fused cells, a method for producing fused cells, a cell mass, a plant, and a method for producing a plant, which can be grown to give a plant. The fused cells, the method for producing the fused cells, the cell mass, the plant, and the method for producing the plant are useful in agriculture and horticulture.

Claims

1. A step of electrofusing a first sperm cell and a second egg cell to obtain a first fused cell, and a step of electrofusing the first fused cell and a first egg cell, wherein the taxonomic groups of the plant species to which the first sperm cell belongs and the plant species to which the first egg cell belongs are the Poaceae Fragariinae, and the taxonomic group of the plant species to which the second egg cell belongs is the Poaceae Ehrharta subfamily or the Poaceae Panicoideae. A method for producing a fused cell.

2. A step of electrofusing a first egg cell and a first sperm cell to obtain a first fused cell, a step of electrofusing a second egg cell and a second sperm cell to obtain a second fused cell, and a step of electrofusing the first fused cell and the second fused cell, wherein one of the taxonomic groups of the plant species to which the first egg cell and the second egg cell belong is the Poaceae Fragariinae, and the other of the taxonomic groups of the plant species to which the first egg cell and the second egg cell belong is the Poaceae Ehrharta subfamily or the Poaceae Panicoideae, and one of the taxonomic groups of the plant species to which the first sperm cell and the second sperm cell belong is the Poaceae Fragariinae, and the other of the taxonomic groups of the plant species to which the first sperm cell and the second sperm cell belong is the Poaceae Ehrharta subfamily or the Poaceae Panicoideae. A method for producing a fused cell.

3. A step of electrofusing a first egg cell and a first sperm cell to obtain a first fused cell, a step of electrofusing the first fused cell and a second sperm cell to obtain a second fused cell, and a step of electrofusing the second fused cell and a second egg cell, wherein one of the taxonomic groups of the plant species to which the first egg cell and the second egg cell belong is the Poaceae Fragariinae, and the other of the taxonomic groups of the plant species to which the first egg cell and the second egg cell belong is the Poaceae Ehrharta subfamily or the Poaceae Panicoideae, and one of the taxonomic groups of the plant species to which the first sperm cell and the second sperm cell belong is the Poaceae Fragariinae, and the other of the taxonomic groups of the plant species to which the first sperm cell and the second sperm cell belong is the Poaceae Ehrharta subfamily or the Poaceae Panicoideae. A method for producing a fused cell.

4. A step of obtaining a fused cell by the production method according to any one of Claims 1 to 3, and a step of culturing the fused cell to obtain a cell mass. A method for producing a cell mass.

5. A step of obtaining a fused cell by the production method according to any one of Claims 1 to 3, A method for producing a plant, comprising the step of producing a plant from the fusion cell.

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