WO2020203806A1 - Procédé de suppression de l'agrégation de cellules végétales isolées - Google Patents

Procédé de suppression de l'agrégation de cellules végétales isolées Download PDF

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WO2020203806A1
WO2020203806A1 PCT/JP2020/014111 JP2020014111W WO2020203806A1 WO 2020203806 A1 WO2020203806 A1 WO 2020203806A1 JP 2020014111 W JP2020014111 W JP 2020014111W WO 2020203806 A1 WO2020203806 A1 WO 2020203806A1
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Prior art keywords
plant
cells
cell
isolated
fertilized egg
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PCT/JP2020/014111
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English (en)
Japanese (ja)
Inventor
松井　南
龍史 岡本
加藤　紀夫
雅子 市川
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日本たばこ産業株式会社
国立研究開発法人 理化学研究所
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Publication of WO2020203806A1 publication Critical patent/WO2020203806A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues

Definitions

  • the present invention relates to a method of suppressing aggregation of isolated plant cells and a method of introducing a substance into a plant.
  • Non-Patent Document 1 describes a method of electrically fusing corn egg cells and sperm cells to produce fertilized egg cells (in vitro fertilized eggs) and culturing them into a plant.
  • Non-Patent Document 1 uses a high-concentration mixture of plant tissue-degrading enzymes for the separation of egg cells.
  • Non-Patent Document 2 describes a method of producing a fertilized egg by electric fusion of male and female gametes of rice and culturing it into a plant body.
  • Artificial insemination of plants involves isolation of gamete cells (egg cells and sperm cells) from pre-pollination flowers, fusion of isolated cells (fertilization), and culture of fused cells (fertilized eggs). It consists of three steps. Typical successful examples are corn (Non-Patent Document 1) and rice (Non-Patent Document 2), and there are several reports of wheat and tobacco.
  • egg cells are obtained by cutting the ovule before pollination or by degrading the ovule or ovule treated with a plant tissue degrading enzyme such as cellulase or pectinase under a microscope, and sperm cells are suitable.
  • Non-Patent Documents 1 and 2 Three types of fusion methods for gamete cells have been reported: electrical fusion (Non-Patent Documents 1 and 2), calcium ion fusion (Non-Patent Document 3), and polyethylene glycol fusion (Non-Patent Documents 4 and 5).
  • electrical fusion Non-Patent Documents 1 and 2
  • Non-Patent Document 3 calcium ion fusion
  • Non-Patent Documents 4 and 5 polyethylene glycol fusion
  • a fertilized egg grows into an embryo and regenerates into a plant only for a fertilized egg produced by electrical fusion
  • Non-Patent Documents 1 and 2 These prior literatures indicate that it is possible to induce plants from fertilized egg cells that are artificially fused with male and female gametes.
  • the points such as gene transfer and transformation into fertilized eggs are not described at all, and it is completely unknown whether transformation can be performed using in vitro fertilized eggs.
  • Non-Patent Document 6 Fertilized offspring in species such as corn (Non-Patent Document 6), rice (Non-Patent Document 7), wheat (Non-Patent Document 8), barley (Non-Patent Documents 9 and 11), tobacco (Non-Patent Document 10). It is also known that fertilized eggs are taken out from bunches and ovules and cultured to produce plants. Non-Patent Documents 6 and 11 relating to maize and barley describe that DNA was introduced into fertilized egg cells by a microinjection method. However, the fact that a plant transformation method by a microinjection method for fertilized eggs has been put into practical use has not been reported. In addition, there is no knowledge about gene transfer by other methods.
  • the microinjection method can also introduce a gene into a cell having a cell wall, and it is not particularly necessary to remove the cell wall of the plant cell to be introduced by plant tissue degrading enzyme treatment or the like.
  • it has the disadvantage that only one cell can be handled by one transfer operation, and is not suitable for medium to large-scale gene transfer experiments using a large number of plant cells.
  • Other methods for introducing a gene into cells include a polyethylene glycol method (polyethylene glycol: PEG method), a peptide method (Non-Patent Document 12), an electroporation method, an Agrobacterium method, and the like, in addition to the microinjection method.
  • the electroporation method, the peptide method and the PEG method, particularly the PEG method are simpler than the microinjection method and have the advantage of being able to handle a large number of cells at one time.
  • plant cells have a cell wall
  • the tissues and cells are treated with plant tissue-degrading enzymes such as cellulase, pectinase, protease, and hemicellulase, especially when performing the PEG method or electroporation method. It is common to lyse the cell wall and protoplast the cells. For this reason, the above methods have so far targeted materials such as leaves, cultured cells, and callus that can obtain a large amount of protoplasts.
  • Non-Patent Documents 13 and 14 On the other hand, unlike leaves, cultured cells, callus, etc., fertilized eggs take time to produce and isolate, and a large amount of protoplast cannot be obtained. In addition, egg cells and sperm cells immediately before fertilization do not start differentiation unless they are subjected to fusion treatment such as electrical treatment or addition of calcium solution. Further, it is considered that the cells of the fertilized egg obtained by such an in vitro fertilization system may be damaged by an artificial fertilization operation such as the above-mentioned fusion treatment. Furthermore, regarding fertilized egg cells, a method for removing the cell wall from the fertilized egg while maintaining the cell activity capable of continuing cell division and growing into a plant after removing the cell wall was unknown. Under these circumstances, it is speculated that egg cells, sperm cells, and fertilized egg cells are not suitable as targets for gene transfer, and it has been reported that gene transfer was performed by a method such as the PEG method, leading to cell division. I wasn't.
  • Patent Document 1 WO2017. / 171092
  • Patent Document 2 WO2018 / 143480
  • the fertilized egg may adhere to laboratory equipment such as slide glasses, cover glasses, and patterned needles during isolation / culture and substance introduction.
  • fertilized eggs may aggregate with each other, and in such a case, the fertilized eggs obtained with great care may not be collected, and it may be difficult to use them for subsequent culture.
  • the problem has been raised that the culture is not started due to the scratches made at the time of recovery, the culture efficiency is significantly lowered, and the efficiency of gene transfer is lowered.
  • the PEG method when the purpose is to introduce a gene or the like into a plant cell, the PEG method has a major feature that a substance can be introduced into a large number of cells at the same time.
  • the advantages of the PEG method are lost because a large number of cells cannot be handled at one time and only a small amount of cells can be handled.
  • the phenomenon of adhesion and aggregation observed in fertilized egg cells was also observed in plant culture using protoplasts, which are isolated plant cells, and in the case of substance introduction, as in the case of fertilized egg cells.
  • An object of the present invention is to provide a method for suppressing aggregation of isolated plant cells and a method for introducing a substance into a plant.
  • the present inventors suppress the aggregation of plant cells by contacting the isolated plant cells with a composition containing at least one gelling agent. We found that we could do it, and came up with the present invention.
  • the present invention includes, but is not limited to, the following aspects.
  • a method for suppressing the aggregation of isolated plant cells which comprises contacting the isolated plant cells with a composition containing at least one gelling agent.
  • Aspect 2 The method according to aspect 1, wherein a part of the step of isolating and culturing the plant cells comprises isolating and culturing the isolated plant cells using a composition containing at least one gelling agent. ..
  • Aspect 3 The supplementary method according to aspect 1 or 2, comprising contacting the isolated plant cell with a composition containing at least one gelling agent prior to the step of culturing the plant cell.
  • Aspect 4 The method according to any one of aspects 1-3, wherein the isolated plant cell is a fertilized egg cell or an egg cell.
  • the isolated plant cells have the following steps: (1-i) A fertilized egg cell is isolated from a tissue containing a fertilized egg cell of a plant, and then the fertilized egg cell is treated with an enzyme solution containing a plant tissue degrading enzyme. (1-ii) A tissue containing fertilized egg cells of a plant is treated with an enzyme solution containing a plant tissue degrading enzyme, and then the enzyme-treated fertilized egg cells are isolated.
  • the tissue containing the fertilized egg cells of the plant is treated with an enzyme solution containing a plant tissue degrading enzyme under low titer conditions, and at the same time, the enzyme-treated fertilized egg cells are isolated.
  • An egg cell and a sperm cell are isolated from a plant body and fused to produce a fertilized egg, and then the fertilized egg cell is treated with an enzyme solution containing a plant tissue degrading enzyme, or
  • Tissues containing plant egg cells are treated with an enzyme solution containing a plant tissue degrading enzyme, then the enzyme-treated egg cells are isolated and further fused with the isolated sperm cells.
  • Aspect 8 The method according to any one of aspects 1-7, comprising adding plant cells to a composition containing at least one gelling agent, and then transferring to a culture medium for culturing.
  • the method according to any one of aspects 1-8 comprising introducing into a plant cell a substance selected from the group consisting of nucleic acids, proteins and peptides.
  • Aspect 10 Aspect 1-9, comprising contacting the plant cells with a composition comprising at least one gelling agent prior to the introduction of the substance, at the same time as the introduction of the substance and / or before culturing after the introduction of the substance.
  • a method of introducing substances into plants I) Isolate plant cells; (Ii) Introduce a substance selected from the group consisting of nucleic acids, proteins and peptides into plant cells; (Iii) Culturing plant cells into which supplies have been introduced, The method comprising contacting an isolated plant cell with a composition comprising at least one gelling agent in any step, or between steps.
  • Aspect 14 Includes contacting plant cells with a composition comprising at least one gelling agent prior to introduction of the substance, at the same time as the introduction of the substance and / or before culturing after introduction of the substance.
  • Aspect 15 (Iii') Aspect 13 or 14, wherein the cells into which the substance has been introduced are called or embryoid-like by culturing plant cells, and the callus-like or embryoid-like tissue is redifferentiated in a redifferentiation medium. The method described.
  • Aspect 16 The method according to any one of aspects 13-15, wherein the substance introduction is performed using a PEG method or an electroporation method.
  • the plant cells when handling isolated plant cells such as fertilized eggs and protoplasts, the plant cells are contacted with a composition containing a gelling agent such as agarose in order to avoid aggregation of the plant cells.
  • a gelling agent such as agarose
  • the culture efficiency can be improved.
  • the efficiency of introducing the substance can be improved.
  • the present invention relates to a method of suppressing aggregation of isolated plant cells.
  • the method of the present invention comprises contacting an isolated plant cell with a composition comprising at least one gelling agent.
  • Plants are not particularly limited. It may be either a dicotyledonous plant or a monocotyledonous plant, preferably a monocotyledonous plant, more preferably corn, wheat, barley, rice, sorghum, limewood, etc., and most preferably corn, wheat, rice.
  • the method of suppressing the aggregation of isolated plant cells can be used particularly for plants or varieties that are considered to be "difficult to culture".
  • "Difficult-to-culture” means that it is difficult to culture, specifically, for example, it is difficult to culture cells isolated from a plant, formation of callus by treatment such as dedifferentiation, or re-culture from callus to a plant. It means that it is difficult to differentiate.
  • Difficult to culture plants include, for example, soybeans, common beans, capsicum and the like.
  • “Difficult-to-cultivate varieties” means varieties that are more difficult to cultivate than general research varieties of the same species (such as A188 for corn), for example, corn elite varieties derived from corn B73 and B73. , Elite varieties of wheat (for example, AC Barrie, TAM, etc.), varieties other than GoldenPromise and Igri of corn, 296B, C401, SA281, P88812, Pioneer 8505, Tx430 of sorghum, and the like.
  • the plant is selected from the group consisting of corn, wheat, barley, rice, sorghum and rye.
  • an "isolated plant cell” is not a cell in which almost all cells other than plasmodesma, such as plant cells existing in a plant tissue, are surrounded by a cell wall, but one cell or a very small number. Is a cell that exists in a state of being separated and isolated from other cells. For example, it includes cells in which some or all of the cells are not surrounded by a cell wall (plant cells in which some or all are protoplastized) isolated by enzyme treatment of plant tissue or the like. Alternatively, the isolated plant germ cells also include gametes for sexual reproduction, including egg cells, sperm cells.
  • the term “egg cell” means a female gamete formed by meiosis of embryonic sac mother cells in a female sac.
  • the method for isolating the egg cell is not limited, but for example, the ovary can be cut in a solution having an appropriate osmotic pressure, and the egg cell emerged from the cut surface can be isolated under a microscope using a glass capillary.
  • the term “sperm cell” means a male gamete formed by meiosis of pollen mother cells in anthers of a stamen.
  • the term "fertilized egg cell” means a cell in which a sperm cell and an egg cell are fused, but is not limited to the term, and includes an egg cell that starts development without fusion without undergoing meiosis.
  • the isolated plant germ cell is an isolated fertilized egg cell or egg cell.
  • a fertilized egg cell formed by fertilization, in which cell wall formation has not started, or cell wall formation has started but has not yet been completed, and the fertilized egg cell or cell wall formation is incomplete. Contains fertilized egg cells that have been completed.
  • the cell wall formation of plant germ cells can be confirmed by known methods such as cellulose staining with calcoflor and aniline blue staining.
  • Calcoflor is a non-specific fluorescent dye that binds to cellulose and chitin contained in the cell walls of plant cells, fungi and the like.
  • the excitation is 300 to 440 nm (maximum 355 nm), and the maximum fluorescence of cellulose in 0.1 M phosphate buffer pH 7.0 is 433 nm.
  • the fluorescence brightness can be measured using a confocal laser scanning microscope, and the integrated brightness can be obtained by performing image analysis of the fluorescence intensity.
  • the "cell wall formation rate” is, for example, a ratio of brightness when compared with the brightness of a cell in which the cell wall formation of a plant cell is completely completed and the entire cell is covered with the cell wall. Can be expressed. “Isolated” means that the cell wall formation rate is preferably 80% or less, more preferably 70% or less, still more preferably 65% or less, still more preferably 63% or less, and particularly preferably. It means that it is 60% or less, particularly more preferably 50% or less, and most preferably 30% or less.
  • a fertilized egg cell in one aspect of the method of suppressing the aggregation of isolated plant cells, can be used as a plant germ cell.
  • the method for obtaining fertilized egg cells is not particularly limited.
  • a fertilized egg cell may be produced in a plant by a natural fertilization method, and the created fertilized egg cell may be obtained from the plant.
  • the method for obtaining fertilized egg cells using the natural fertilization method is, for example, a method in which the stigma is exposed, pollen is attached and pollinated, and then the fertilized egg cells are isolated from the tissue containing the embryo sac.
  • the ovary immediately after fertilization is removed from the pollinated plant, the ovary is cut in a solution of appropriate osmotic pressure, and the fertilized egg cell that emerges from the cut surface is removed. It can be isolated under a microscope using a glass capillary or the like.
  • tissues such as the pearl core can be dissected, excised, and isolated under a microscope using, for example, a glass needle or the like.
  • the natural fertilization method may be an artificial mating in which pollen is artificially attached to the stigma, or a natural mating.
  • the egg cell of the plant and the sperm cell may be fused in vitro to produce a fertilized egg cell. That is, an egg cell and a sperm cell may be first isolated from a plant body, and a fertilized egg cell may be produced in vitro by a known method such as an electrofusion method (also referred to as gamete fusion). In a non-limiting method for suppressing the aggregation of isolated plant cells, it is preferable to produce fertilized egg cells in vitro.
  • the electrical fusion method is a method in which two or more types of cells are fused in vitro by electrical stimulation.
  • the ovary before pollination was cut in a solution of appropriate osmotic pressure, and the egg cells emerged from the cut surface were isolated under a microscope using a glass capillary or the like, and into a solution of appropriate osmotic pressure.
  • Cell fusion can be triggered by submerging pollination and applying a pulse to the sperm cells released from the pollination under a microscope using a glass capillary or the like.
  • the DC voltage is not limited, and the lower limit is preferably 10 kV or more, and the upper limit is 17 kV or less. Is preferable.
  • the upper limit and the lower limit can be appropriately selected by those skilled in the art.
  • the distance between the electrodes is not limited, and the lower limit is preferably 1.5 times or more the sum of the diameters of the egg cells and sperm cells to be fused, and the upper limit is preferably 6 times or less.
  • a method of measuring the cell diameter there are a method of measuring the diameter using a microscopic eyepiece attached to a microscope, and a method of capturing an image taken by the microscope into a computer and measuring it with image analysis software.
  • the lower limit is preferably 80 ⁇ m or more
  • the upper limit is preferably 240 ⁇ m or less.
  • the upper limit and the lower limit of the distance between the electrodes can be appropriately selected by those skilled in the art.
  • the osmotic pressure of the solution used for electrically fusing sperm cells and egg cells to prepare one fused cell can be appropriately selected according to the type of plant used.
  • the lower limit 380mosmol / kg H 2 O or more rice more preferably, to 390mosmol / kg H 2 O or more, and is preferably not more than 470mosmol / kg H 2 O limit.
  • the lower limit is preferably 500 mosmol / kg H 2 O or more
  • the upper limit is preferably 700 mosmol / kg H 2 O or less.
  • the upper and lower limits of the osmotic pressure of the solution can be appropriately selected by those skilled in the art.
  • cell fusion methods such as calcium fusion method and PEG fusion method may be used for cell fusion of egg cells and sperm cells.
  • the "calcium fusion method” utilizes the property of the cell membrane that the fusion of the cell membrane is likely to occur depending on the calcium concentration.
  • the “PEG fusion method” utilizes the fact that cells are treated with polyethylene glycol (polyethylene glycol, PEG) to bind cell membranes, and when PEG is removed, cells are fused.
  • the isolated plant cells are subjected to the following steps: (1-i) A fertilized egg cell is isolated from a tissue containing a fertilized egg cell of a plant, and then the fertilized egg cell is treated with an enzyme solution containing a plant tissue degrading enzyme. (1-ii) A tissue containing fertilized egg cells of a plant is treated with an enzyme solution containing a plant tissue degrading enzyme, and then the enzyme-treated fertilized egg cells are isolated. (1-iii) The tissue containing the fertilized egg cells of the plant is treated with an enzyme solution containing a plant tissue degrading enzyme under low titer conditions, and at the same time, the enzyme-treated fertilized egg cells are isolated.
  • An egg cell and a sperm cell are isolated from a plant body and fused to produce a fertilized egg, and then the fertilized egg cell is treated with an enzyme solution containing a plant tissue degrading enzyme, or (1-v) Tissues containing plant egg cells are treated with an enzyme solution containing a plant tissue degrading enzyme, then the enzyme-treated egg cells are isolated and further fused with the isolated sperm cells. It may be obtained by any of the steps of.
  • a tissue containing an embryo sac (eg, an ovule) is cut in a solution of appropriate osmotic pressure, and (fertilized) egg cells emerged from the cut surface are isolated under a microscope using a glass capillary or the like. be able to.
  • the isolated (fertilized) egg cell is treated with an enzyme solution for a certain period of time to obtain an enzyme-treated fertilized egg.
  • the tissue containing the ovule such as the ovule
  • the tissue such as the ovule can be dissected under a microscope using, for example, a glass needle or the like, and mechanically removed and isolated. ..
  • an enzyme-treated (fertilized) egg can be obtained without subsequent enzyme treatment.
  • the enzyme treatment for obtaining a fertilized egg by fusing the isolated egg cell and the sperm cell may be performed at any stage before, at the same time, or after the fusion with the sperm cell.
  • the method of the present invention is characterized in that (fertilized) egg cells are treated with a low titer condition with an enzyme solution containing a plant tissue degrading enzyme from a tissue containing (fertilized) egg cells of a plant.
  • the enzyme treatment may be performed at any time before the (fertilized) egg cell is isolated from the tissue, at the same time as the isolation, or after the isolation, but preferably at the same time as the isolation or after the isolation. Is.
  • the cell wall of a plant has a basic skeleton made of cellulose embedded in a substrate (matrix, substrate gel) made of other polysaccharides and proteins.
  • the polysaccharides that make up the substrate are traditionally divided into pectin, which is extracted with hot water or an acidic buffer, and hemicellulose, which is an alkali-soluble component.
  • matrix polysaccharides matrix polysaccharides
  • plant tissue-degrading enzyme as used herein is a general term for enzymes that directly or indirectly act on and decompose pectin, cellulose, hemicellulose, other matrix polysaccharides, phospholipids, proteins, etc. around plant tissues and cells. is there.
  • plant tissue degrading enzyme for example, non-limitingly, protoplast preparation enzyme, phospholipase that decomposes cell membrane, tannase that is considered to be useful for tissue decomposition, rice and other components contained in the type II cell wall are decomposed. Ferrulic acid esterase, protease and the like are included. In particular, various protoplast preparation enzymes that are used to lyse the cell walls of plant cells to prepare protoplasts can be used.
  • pectinase for example, pectinase, cellulase, protease, hemicellulase (hemicellulase is a general term for enzymes that hydrolyze hemicellulose), glucuronidase, zymolidase, chitinase, glucanase, xylanase, galactanase, arabinase and ligninase. , Or a mixture thereof (a mixture of two or more of these enzyme groups) is included.
  • Pectinases include, for example, polygalacturonase (galacturonase), pectin lyase and pectin methyl esterase.
  • the isolated plant cell may be a fertilized egg cell that has not been treated with a plant tissue degrading enzyme as described above.
  • a plant tissue degrading enzyme for example, the isolated plant germ cell described in Patent Document 2 (WO2018 / 143480) can be used.
  • the isolated plant cell is a plant tissue-degrading enzyme that comprises a fertilized egg cell, a tissue containing a plant fertilized egg cell, or a tissue containing a plant egg cell, as described in Patent Document 1 (WO2017 / 171092). It may be treated under low titer conditions with an enzyme solution containing.
  • the isolated plant cell may be a protoplastized plant cell.
  • a "protoplast” is a cell from which a cell wall has been removed from a plant cell, which is generally spherical and weak, and has the property of being destroyed by a slight impact. Utilizing the properties of protoplasts, it is possible to introduce substances into cells and fuse them by treatment with PEG or electrical stimulation. Callus can be obtained by culturing and growing protoplasts. However, for example, when the purpose is to introduce a substance, problems such as protoplasts tending to aggregate with each other due to PEG treatment and adhesion to a test instrument may occur. In such a case, undesired aggregation can be suppressed by contacting the protoplast with a composition containing at least one gelling agent.
  • Gelling agent used in the method for suppressing the aggregation of plant cells may be a known gelling agent, and a gelling agent known in the art can be used.
  • Non-limiting examples of such gelling agents include, for example, agarose, agar and gellan gum, gellite, alginic acid, gelatin, fighter gel and the like.
  • the concentration of the gelling agent is appropriately selected depending on the type of the gelling agent.
  • the composition containing at least one gelling agent is selected to have a concentration that is liquid or semi-solid.
  • 0.05-2.0% is preferable, 0.1-1.0% is more preferable, 0.1-0.5% is further preferable, and 0.15-0.3% is preferable. Most preferred.
  • liquid state indicates that the composition containing the gelling agent (for example, a solution) is liquid, and indicates a state in which a fluid having no fixed shape is taken.
  • Gel-solid refers to a state that has both liquid and solid attributes, is defined as a semi-fluid that is closer to solid than liquid, and is characterized by being viscous and freely deformable, so-called gel-like. Show things.
  • a solid aspect such as agarose beads that can maintain a constant shape for a certain period of time is not included in the present invention.
  • composition Containing At least One Gelling Agent is not particularly limited as long as it is a composition containing a gelling agent.
  • the isolated plant cells are isolated using a composition containing at least one gelling agent.
  • Including culturing Non-limitingly comprising contacting the isolated plant cells with a composition comprising at least one gelling agent prior to the step of culturing the plant cells.
  • Plant cells may be added to a composition containing at least one gelling agent and then transferred to a culture medium for culturing.
  • the gelling agent contained in the composition may be one type or two or more types (for example, two types, three types, four types, or more).
  • the composition comprising at least one gelling agent is a solution that transfers isolated isolated plant cells prior to transplantation into a medium for culture.
  • solutions also referred to as MMG solutions
  • osmoregulators such as mannitol and sucrose
  • pH regulators such as MES and / or magnesium salts as main components.
  • a “medium” is a medium for culturing a plant cell, and contains a carbon source, a nitrogen source, a salt, etc. necessary for culturing the plant cell (limited). However, for example, it refers to M6 medium, B5 medium, N6 medium, ZMS medium, etc.).
  • the “medium” for culturing is distinguished from the above-mentioned solutions for treating isolated plant cells prior to the start of culturing, such as MMG solutions.
  • the plant tissues are enzymatically treated to form protoplasts, and then added to a solution containing mannitol or sucrose for isolation or washing with protoplasts.
  • the composition containing at least one gelling agent may be a solution containing the mannitol or sucrose.
  • the composition containing at least one gelling agent may contain a component that induces dedifferentiation (callus formation) or redifferentiation.
  • the plant cell when the plant cell is a fertilized egg cell, the fertilized egg enzymatically or mechanically isolated from the plant tissue or the fertilized egg obtained by electrically fusing the egg cell and the sperm cell is isolated or obtained.
  • a composition containing at least one gelling agent which is added to a solution containing mannitol or shoe cloth (such as, but not limited to, MMG solution) once for washing or incubation before transfer to the medium. May be the solution.
  • the composition containing at least one gelling agent is in liquid or semi-solid form.
  • the method of suppressing the aggregation of plant cells may include introducing a substance selected from the group consisting of nucleic acids, proteins and peptides into plant cells.
  • a substance is introduced into a plant cell using the PEG method
  • the composition containing at least one gelling agent is an isolated plant cell, a PEG solution containing the substance to be introduced, and / or the said. It may be a solution to be moved before the PEG solution.
  • a fertilized egg enzymatically or mechanically isolated from a plant tissue, or a fertilized egg obtained by electrically fusing an egg cell and a sperm cell may be subjected to either or both of the following (i) and (ii).
  • a gelling agent may be included.
  • both (i) and (ii) may contain a gelling agent.
  • (I) A solution containing mannitol or sucrose (such as, but not limited to, MMG solution) for once washing and incubation after isolation or acquisition of the fertilized egg and before transfer to the medium; / Or (Ii) A solution used for introducing a substance to be introduced and a substance by the PEG method containing PEG.
  • mannitol or sucrose such as, but not limited to, MMG solution
  • aggregation of isolated plant cells is suppressed by the method of the present invention.
  • “Aggregation of plant cells” means, for example, aggregation of plant cells, for example, aggregation of fertilized eggs or protoplasts, adhesion of plant cells to a test instrument (for example, cover glass), cell aggregation, adhesion, etc. It means that it includes close contact widely.
  • “Suppressing plant cell aggregation” means that the isolated plant cells are brought into contact with a composition containing at least one gelling agent, as compared to the case where the plant cells are not aggregated. Means that is suppressed. As a result, preferably, for example, the recovery rate of a single fertilized egg cell is improved, the culture efficiency of plant cells is improved, and the introduction efficiency of a substance when a substance is introduced is improved. ..
  • the method for suppressing aggregation of isolated plant cells may include a step of introducing a substance selected from the group consisting of nucleic acids, proteins and peptides into plant cells.
  • a substance introduced into a plant is a substance of a size and properties that can be supplied into a target cell. It may be naturally occurring or artificially manufactured. Examples include various biomolecules and compounds. Examples of biomolecules include nucleic acids, proteins, peptides, polysaccharides, lipids, organelles and the like. In one aspect, the substance is selected from the group consisting of nucleic acids, proteins and peptides. Nucleases such as Cas9 nuclease for genome editing and proteins such as modifying enzymes and antibodies can also be introduced. Two or more kinds of nucleic acids, proteins, peptides, polysaccharides, lipids and the like, and substances such as metal ions and compounds may be introduced in combination. For example, the nucleic acid may be two or more types of DNA or RNA, or a combination of DNA and RNA. Different species of substances such as nucleic acids and proteins may be introduced simultaneously or as a complex.
  • the method for introducing a substance into a plant is not particularly limited as long as it is a known method capable of introducing a desired substance into a plant, and can be appropriately selected depending on the type of plant.
  • a physicochemical method such as the PEG method, electroporation method, particle gun method, microinjection method, whisker method, or a biological method such as the Agrobacterium method (indirect introduction method of DNA).
  • a direct introduction method is preferable, and a PEG method or an electroporation method is more preferable.
  • the PEG method is more preferable.
  • the PEG method can be carried out with reference to a known method such as Non-Patent Document 13 (Yoo et al. (2007)).
  • the time when the plant cells are brought into contact with the composition containing at least one gelling agent is not particularly limited.
  • the method of suppressing the aggregation of isolated plant cells is to gel at least one type of plant cell prior to the introduction of the substance, at the same time as the introduction of the substance, and / or before culturing after the introduction of the substance. Includes contact with a composition containing an agent.
  • the present invention relates to a method of introducing a substance into a plant.
  • the method of the present invention Isolate plant cells; (Ii) Introduce a substance selected from the group consisting of nucleic acids, proteins and peptides into plant cells; (Iii) Culturing plant cells into which supplies have been introduced, Including the process.
  • the isolated plant cells are brought into contact with a composition containing at least one gelling agent in any of the steps (i)-(iii) above, or between steps. It is characterized by including.
  • plant cells The definitions of "plant cells”, “substances”, and “introduction of substances” are as described in the section "1. Method of suppressing aggregation of isolated plant cells”.
  • the time when the plant cells are brought into contact with the composition containing at least one gelling agent is not particularly limited.
  • the gelling agent may be contained in the solution used in any of the steps (i)-(iii) above.
  • a step of contacting the isolated plant cell with the composition containing at least one gelling agent may be included between the steps (i)-(iii) above.
  • the method of suppressing the aggregation of isolated plant cells is to gel at least one type of plant cell prior to the introduction of the substance, at the same time as the introduction of the substance, and / or before culturing after the introduction of the substance. Includes contact with a composition containing an agent.
  • the substance is introduced using the PEG method or the electroporation method.
  • the method of introducing a substance into a plant is to callus or embryoidize the cells into which the substance has been introduced by culturing (iii') plant cells, and then callus or embryoidize the tissue. It may include a step of redifferentiating with a redifferentiation medium.
  • the callus formation or embryoid body formation step and the redifferentiation step are not particularly limited, and a known method for regenerating a plant body from a plant cell can be used.
  • a known method for regenerating a plant body from a plant cell can be used.
  • the method described in Patent Document 2 (WO2018 / 143480) may be used.
  • the step of inducing division and proliferating the substance-introduced fertilized egg cell to form callus or embryo-like body is not particularly limited because the optimum conditions differ depending on the plant, but a nurse culture method in which Feeder cells are added is used. be able to.
  • the material introduced fertilized egg cell mannitol droplets (e.g., 450mosmol / kg H 2 O) were washed and put into the process before performing the sterilization, as well as transferring material introduced fertilized egg cell in the liquid medium, standing After that, the culture is shaken, and the culture in a liquid medium is included. It is preferable to add feeder cells to the liquid medium and perform co-culture (nurse culture method).
  • a liquid MS medium, B5 medium, N6 medium or the like to which auxin such as 2,4-dichlorophenoxyacetic acid or naphthalene acetic acid is added can be used.
  • spherical embryoid bodies having a diameter of about 50 to 200 ⁇ m are formed 4 to 14 days after the start of culturing the fertilized egg stick.
  • the redifferentiation step can also be carried out according to a known redifferentiation step.
  • the method described in Patent Document 2 (WO2018 / 143480) may be used.
  • the spherical embryoid body is transferred to the medium to which the feeder cells are not added, further cultured for about 10 to 14 days, and then placed in an arbitrary medium to which auxin is not added, for example, MS medium, and the plant is cultured.
  • the medium include MS medium, B5 medium, N6 medium, and solid medium using agarose, agar, gellan gum, gellite, and the like.
  • “Improved culture efficiency of plant cells” means that the isolated plant cells are brought into contact with a composition containing at least one gelling agent, as compared to the case where the plant cells are not contacted. Means that is cultivated more efficiently. For example, culturing fertilized eggs increases the proportion of fertilized eggs that normally initiate division (division initiation rate), increases the rate of formation of globular or callus-like embryos, and increases the probability of survival in subsequent culturing steps. It can be confirmed by events such as. For example, the split initiation rate is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30 as compared with the case where the composition containing at least one gelling agent is not added. It rises by more than%.
  • “Improved substance introduction efficiency” means that the isolated plant cells are brought into contact with the composition containing at least one gelling agent, as compared to the case where the substance is not brought into contact with the plant. It means that the probability of being introduced will increase.
  • the substance introduction efficiency is 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25, as compared with the case where the composition containing at least one gelling agent is not added. % Or more, 30% or more, 35% or more, increase.
  • Substance-introduced plants The present invention also includes substance-introduced plants obtained by methods of introducing substances into plants. Prior to the present invention, it was difficult or impossible to obtain a substance-introduced plant, especially for plants and varieties that are considered to be “difficult to culture”. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to efficiently obtain a substance-introduced plant for such plants and varieties by a simple method.
  • Example 1 Isolation of fertilized maize egg cells
  • fertilized maize egg cells were isolated.
  • the ears of corn grown in a greenhouse at the appropriate mating time were collected, the foreskin of the ears was removed, the ovary was exposed, and the length of silk (corn whiskers, pistils) was trimmed to about 12 cm. Then, the ears were divided into two in the vertical direction, and the tip of the silk was pollinated with pollen collected from the ears of corn. The mating time was around 9 am.
  • the ears after mating were placed on a sugar-free MS agar medium and placed in an environment of about 25 ° C.
  • the ears were moved from 5 ° C to room temperature. From ovules ears were excised and nucellus sections containing the embryo sac, was placed in a 10% mannitol solution 1mL in 3.5cm plastic petri dish (650mosmol / kg H 2 O) . 0.5 mL of the enzyme mixture was placed in a 3.5 cm plastic petri dish to prepare a 1.5 mL enzyme solution, which was left at room temperature for 5 to 45 minutes.
  • the enzyme solution is a 3-fold dilution of a 650 mosmol / kg mannitol aqueous solution containing 1% cellulase (manufactured by Worthington), 0.3% macerozyme (manufactured by Yakult Honsha), and 0.05% pectriase (manufactured by Shengshin Pharmaceutical Co., Ltd.). I used the one that I did.
  • the fertilized egg was isolated using two glass needles. Fertilized egg cells were isolated by fixing the bead core section with one glass needle to immobilize it and scraping the tissue in the area where the fertilized egg cell is presumed to be present with the other glass needle. As for the estimation of the region, when fertilization was performed, the one invaded by the pollen tube among the two existing helper cells was denatured and turned dark brown, so that was used as a marker. The isolated fertilized egg cells were transferred to droplets on a cover glass or a glass-bottomed charley after washing the micropipette with mannitol solution.
  • the droplets on the cover glass were created by the following method.
  • Droplets on the glass bottom charley were created by the following method.
  • Example 2 Nucleic acid introduction into fertilized egg
  • nucleic acid was introduced into the fertilized egg obtained in Example 1.
  • the fertilized egg was brought into contact with the composition containing the gelling agent.
  • Example 1 the fertilized egg cells isolated in Example 1 were transferred to droplets (about 2 ⁇ L) of MMG solution (15 mM MgCl 2 , 4 mM MES (pH 5.7), 650 mosmol / kg H 2 O mannitol), and then transferred.
  • Nucleotide sequence to be introduced into MMG, 35S promoter :: signal sequence :: GFP :: endoplasmic reticulum residual signal (HDEL) :: transferred to a droplet to which a plasmid containing a nosterminator (Non-Patent Document 11) was added.
  • MMG solution 15 mM MgCl 2 , 4 mM MES (pH 5.7), 650 mosmol / kg H 2 O mannitol
  • liquid droplet and the PEG solution containing fertilized egg cells (12.5 mL mannitol solution (650mosmol / kg H 2 O) , so as to 25mg with distilled water added 1M calcium chloride PEG4000,2.5mL of 7.5g Droplets (about 2 ⁇ L) of (adjusted) were mixed and stirred 30-50 times with a glass capillary.
  • Example 3 Culturing of fertilized egg cells subjected to nucleic acid introduction treatment
  • the fertilized egg cells subjected to nucleic acid introduction treatment in Example 2 were cultured in this example.
  • the fertilized egg cells subjected to the nucleic acid introduction treatment were transferred to 2 ⁇ l of the prepared medium for fertilized cells and statically cultured in a dark place.
  • the medium for fertilized cells is ZMS medium (Kranz, 1993).
  • MS medium and of changes 165mg / L NH 4 NO3, as organic, 1.0 mg / L nicotinic acid, 10.0 mg / L thiamine ⁇ H 2 O, 1mg / L pyridoxine ⁇ HCl, 750mg / L glutamine, 150 mg / L Proline, 100 mg / L asparagine, and 100 mg / L myo-inositol were added.
  • the 2mg / L 2,4-D was added as a plant hormone, was further adjusted adding osmotic glucose to 600mosmol / kg H 2 O.
  • the pH was 5.7.
  • the prepared medium for fertilized cells was placed in a Millicell CM insert (manufactured by Millipore) having a diameter of 12 mm, and placed in a 3.5 cm plastic petri dish containing 2 mL of the medium. Further, 40 to 60 ⁇ L of a floating rice cell culture (Line Oct, manufactured by RIKEN BioResource Center) was added to the charley as a feeder cell.
  • the isolated embryo cells were put into fresh 9% mannitol droplets (600mosmol / kg H 2 O) , then on a membrane in the CM insert containing the fertilized cell medium Moved to.
  • the fertilized egg cells were allowed to stand in a dark place at 26 ° C. for 1 day, and then shake culture was started according to WO2018 / 143480.
  • the number of fertilized eggs that normally started to divide as a result of shaking culture is shown below.
  • Example 4 Isolation of fertilized egg cells of wheat
  • fertilized egg cells of wheat were isolated.
  • Non-Patent Document 15 From wheat (variety Fielder) grown in a greenhouse, Kumlehn et al. Fertilized egg cells were isolated according to (1997), Plant Journal, 12 (6): 1473-1479 (Non-Patent Document 15). Males were removed 1 to 3 days before flowering, and 4 to 6 days later, the stigmas of the removed florets were artificially crossed by contacting anthers of the same variety. Three to four hours after mating, the ears were sterilized by immersing them in 1-2% sodium hypochlorite for 6 minutes and then washing them three times with sterile distilled water.
  • the florets were aseptically disassembled and the ovary was collected on a 35 mm diameter plastic petri dish containing 4 mL of 0.55 M mannitol solution. While submerging the ovary, a scalpel was used at the bottom of the plastic petri dish to cut the bottom of the ovary.
  • the ovule section was removed from the ovule, the ovule section was fixed on one of the two glass needles, and the fertilized egg cell was removed by gently pressing the tissue in the area where the fertilized egg cell was presumed to be present on the other side. When fertilization was performed, the region was estimated because the pollen tube invaded the two existing helper cells denatured and turned dark brown, which was used as a marker.
  • Example 5 Washing and culturing of fertilized wheat egg cells
  • the fertilized wheat egg cells isolated in Example 4 were washed and cultured in this example.
  • the fertilized egg was brought into contact with the composition containing the gelling agent.
  • 0.55 M mannitol solution control group
  • 0.15% agarose Type
  • 200 ⁇ L of 0.55 M mannitol solution test group
  • IX Agarose A2576 manufactured by Sigma Co., Ltd.
  • the fertilized wheat cells isolated in Example 4 were sequentially transferred to the cover glass using a glass capillary and allowed to stand (washing step).
  • Non-Patent Document 16 a 12 mm diameter Millicell-CM insert (manufactured by Millipore) was placed in a 35 mm diameter plastic petri dish, and the 2,4-D concentration was changed to 0.02 mg / L inside the Millicell-CM insert. 0.2 mL of the modified N6Z liquid medium (Non-Patent Document 16) was added, and 2 mL of the above-mentioned modified N6Z medium was added to the outside.
  • the collected fertilized egg cells were transferred to the inside of the Millicell-CM insert.
  • the number of fertilized egg cells that could be cultured was counted by collecting them in a glass capillary as a single fertilized egg cell without adhesion between multiple fertilized egg cells. The results are shown in Table 2.

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Abstract

La présente invention concerne un procédé pour supprimer l'agrégation de cellules végétales isolées, et un procédé pour introduire une substance dans une plante. Le procédé selon la présente invention comprend la mise en contact de cellules végétales isolées avec une composition contenant au moins un type d'agent gélifiant.
PCT/JP2020/014111 2019-03-29 2020-03-27 Procédé de suppression de l'agrégation de cellules végétales isolées WO2020203806A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07502880A (ja) * 1990-12-28 1995-03-30 デカルブ　ジェネティックス　コーポレイション 電気穿孔によるトウモロコシ細胞の安定した形質転換方法
JP2005328780A (ja) * 2004-05-21 2005-12-02 Institute Of Physical & Chemical Research 形質転換単子葉植物の選抜方法
WO2017171092A1 (fr) * 2016-03-31 2017-10-05 日本たばこ産業株式会社 Procédé d'induction de substance dans une plante
WO2018143480A1 (fr) * 2017-01-31 2018-08-09 日本たばこ産業株式会社 Procédé d'introduction dune substance dans une plante

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07502880A (ja) * 1990-12-28 1995-03-30 デカルブ　ジェネティックス　コーポレイション 電気穿孔によるトウモロコシ細胞の安定した形質転換方法
JP2005328780A (ja) * 2004-05-21 2005-12-02 Institute Of Physical & Chemical Research 形質転換単子葉植物の選抜方法
WO2017171092A1 (fr) * 2016-03-31 2017-10-05 日本たばこ産業株式会社 Procédé d'induction de substance dans une plante
WO2018143480A1 (fr) * 2017-01-31 2018-08-09 日本たばこ産業株式会社 Procédé d'introduction dune substance dans une plante

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