WO2022079927A1 - Procédé de culture d'une cellule végétale - Google Patents

Procédé de culture d'une cellule végétale Download PDF

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WO2022079927A1
WO2022079927A1 PCT/JP2021/002140 JP2021002140W WO2022079927A1 WO 2022079927 A1 WO2022079927 A1 WO 2022079927A1 JP 2021002140 W JP2021002140 W JP 2021002140W WO 2022079927 A1 WO2022079927 A1 WO 2022079927A1
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plant
medium
cells
trehalose
culturing
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PCT/JP2021/002140
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Japanese (ja)
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浩史 塚本
千寿 柳原
祐二 石田
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株式会社カネカ
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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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • 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 for culturing plant cells and a method for regenerating plants.
  • Non-Patent Document 1 Culture medium Various improvements in culture medium have been tried in the transformation of maize with Agrobacterium. Selection of transformed cells in N6 basal medium (Non-Patent Document 1), addition of AgNO3 and carbenicillin to medium (Non-Patent Document 1, Non-Patent Document 2), addition of cysteine to coexisting medium (Non-Patent Document 3) As a result, the culture efficiency was improved. It has also been reported that the transformation efficiency in indicine is enhanced by modifying the composition of the co-culture medium and the gelling agent (Non-Patent Document 4).
  • Non-Patent Document 5 Pretreatment of plant tissue pieces Hiei et al. (2006) (Non-Patent Document 5) shows that the transformation efficiency is increased by heat-treating or centrifuging immature embryos of rice and maize before inoculation with Agrobacterium, and even varieties that could not be transformed so far. It was reported that a transformant was obtained. Further, Ishida and Hiei (WO2011 / 013764) (Patent Document 1) found that transformed plants can be obtained with high efficiency by removing hypocotyls from immature wheat embryos co-cultured with Agrobacterium.
  • the culture method for transforming rice, corn and wheat with Agrobacterium has significantly improved efficiency and expanded the number of applicable varieties. Has been made.
  • Non-Patent Document 6 As other methods, in recent years, as a method for producing a transformant, there are a method using a fertilized egg (Non-Patent Document 6) and an implanter particle gun method (Non-Patent Document 7).
  • Trehalose Small animals such as Polypedilum vanderplanki and plants such as Selaginella can sleep and survive in extremely dry environments such as deserts because trehalose is accumulated in the living body at a high concentration. It has been known. In addition, it has been reported that trehalose functions even during dry dormancy and imparts various stress-tolerant functionality to animals and plants (Non-Patent Document 8).
  • sucrose and / or maltose are used as carbon sources as a conventional method.
  • trehalose is rarely used in plant tissue culture.
  • PLBs protocomb-like spheres
  • PLB formation was promoted at concentrations of 5-40 g / l of sucrose and 10-80 g / l of trehalose, with concentrations of 20 g / l each being the most effective. Rooting from shoots was observed only in the trehalose-added group, and trehalose has been reported to be effective for rooting from shoots (Non-Patent Document 10).
  • Non-Patent Document 11 trehalose is known to inhibit the metabolism and growth of plants (Non-Patent Document 12, Non-Patent Document 13).
  • Non-Patent Document 14 trehalose has been reported to interfere with the regulation of carbohydrate-related genes (Non-Patent Document 14, Non-Patent Document 15). As a result, trehalose was not used for culturing gramineous plants in place of sucrose and / or maltose.
  • An object of the present invention is to provide a method for culturing plant cells and a method for regenerating plants.
  • the present inventors cultivate immature wheat embryos in a medium containing trehalose instead of the carbon source (sucrose or maltose) used in the conventional method. As a result, it was found that the plant regeneration rate was remarkably improved as compared with the conventional medium, and the present invention was completed. By using this method, it has become possible to efficiently produce transformed crops and genome-edited crops.
  • the present invention includes, but is not limited to, the following aspects.
  • a method for culturing plant cells which comprises culturing cells of a gramineous plant using a culture medium containing trehalose.
  • Aspect 2 The method according to aspect 1, wherein the plant cells of the Gramineae family are cultured to obtain a plant cell group and / or a regenerated plant.
  • Aspect 3 The method according to aspect 1 or 2, wherein the gramineous plant is selected from the group consisting of rice, corn, wheat, barley, rye, and sorghum.
  • Aspect 4 The method according to any one of aspects 1-3, wherein the gramineous plant is wheat or corn.
  • the present invention relates to a plant culturing method.
  • the method of the present invention comprises culturing grass cells using a culture medium containing trehalose.
  • Plant The "plant” in the above method is a gramineous plant.
  • the gramineous plant any plant belonging to the gramineous family (Poaceae) can be used.
  • the gramineous plant is selected from the group consisting of rice, corn, wheat, barley, rye, and sorghum, without limitation.
  • the term “one aspect” is intended to be non-limiting.
  • the above culture method can be used particularly for plants or varieties that are considered to be "difficult to culture”. Is.
  • Difficult-to-culture means that it is difficult to culture, specifically, for example, it is difficult to culture cells or tissues isolated from a plant, formation of callus by treatment such as dedifferentiation, or a plant from callus. It means that it is difficult to redifferentiate into.
  • the gramineous plant preferably belongs to the genus Wheat (Triticum) or the genus Corn (Zea).
  • “wheat” and “corn” mean plants of the genus Wheat and the genus Corn, respectively, unless otherwise specified.
  • T.I. aegilopodes As the genus Wheat, for example, as a one-grain wheat, T.I. aegilopodes, T.I. thaudar and T.I. Using monococcum (1 grain wheat) as a 2-grain wheat, T.I. dicoccoides, T.I. dicoccum (2 grains of wheat, emmer wheat), T.I. pyromidale, T.I. Orientale (Khorasan wheat), T.I. durum (durum wheat, macaroni wheat), T.I. turgidium (rivet wheat), T.I. Polishum (Polish wheat), and T.I. Persicum (Persian wheat), as well as three-grain wheat, T.I.
  • Zea mays As the genus Corn, five species are known: Zea mays, Zea diploperennis, Zea luxurians, Zea nicaraguensis and Zea perennis. Many cultivars have been developed for Zea mays.
  • the seeds and varieties of corn used in the present invention are not particularly limited, but are preferably varieties belonging to Zea mays.
  • Plant materials include, but are not limited to, immature embryos, ripe seeds, fertilized eggs, callus, protoplasts, or cell groups derived from them. Immature embryos or ripe seeds are preferred.
  • the term "immature embryo” refers to an embryo of an immature seed that is in the process of ripening after pollination.
  • the stage (ripening stage) of the immature embryos used in the method of the present invention is not particularly limited and may be collected at any time after pollination, but those 7 to 21 days after pollination are used. preferable.
  • "Ripe seeds” are those that have completed the ripening process after pollination and are fully ripe as seeds.
  • “Fertilized egg” means a cell in which a sperm cell and an egg cell are fused.
  • a "plant fertilized egg” is a fertilized egg cell isolated from a tissue containing a plant embryo sac, that is, a fertilized egg that has been fertilized and fertilized at the stage of a plant and isolated from the plant. May be good.
  • an egg cell and a sperm cell may be isolated from a plant before pollination and fertilization, and then fused to produce and obtain a fertilized egg cell.
  • a fertilized egg may be prepared and obtained by electrical fusion. Further, it may be a fertilized egg obtained by fusing egg cells and sperm cells of different kinds of plants.
  • a "protoplast” is a cell from which a cell wall has been removed from a plant cell.
  • the method for culturing a plant includes culturing cells of a gramineous plant using a culture medium containing trehalose. Limited, in one embodiment, the culture of plant cells is carried out in the step of regenerating the plant from the immature embryo.
  • the culture medium for plant cells may be one that is usually used for culturing plant cells, and is not particularly limited.
  • Non-limiting examples thereof include WLS medium, WLS-RES medium, MS medium, LS medium, N6 medium, B5 medium, R2 medium, and medium based on CC medium. These media may be concentrated from the basic concentration or diluted. The amount is preferably 1/10 to 2 times, more preferably 1/5 to 1 times.
  • the basic medium is a WLS medium or a WLS-RES medium. These media have the following composition.
  • WLS medium 1 x LS major inorganic salt, 100 ⁇ M FeEDTA, 1 x LS minor inorganic salt, 1 x MS vitamin, 0.5 mg / L 2,4-D, 2.2 mg / L picrolam, 4% maltose monohydrate , 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl 2, 0.195% MES, 57 ⁇ M ascorbic acid, 5 ⁇ M AgNO 3 , 5 g / L agarose, pH 5.8.
  • WLS-RES medium medium in which 2.2 mg / l picrolam of WLS medium is replaced with 2.5 mg / l dicamba
  • 1 ⁇ LS major inorganic salt 100 ⁇ M FeEDTA, 1 ⁇ LS minor inorganic salt, 1 ⁇ MS vitamin, 0 .5 mg / L 2,4-D, 2.5 mg / L medium, 4% maltose monohydrate, 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl 2, 0.195% MES, 57 ⁇ M ascorbic acid, 5 ⁇ M AgNO 3 , 5 g / L agarose, pH 5.8.
  • Trehalose is a type of disaccharide in which glucose is 1,1-glycosidic bonded. Trehalose is added to the appropriately prepared culture medium.
  • the concentration of trehalose added to the culture medium is not particularly limited. In one embodiment, the concentration of trehalose in the culture medium is 20 mM or more, 50 mM or more, 75 mM or more, 100 mM or more, 125 mM or more, 150 mM or more, 180 mM or more, 200 mM or more, 220 mM or more, 240 mM or more, 250 mM or more.
  • the trehalose concentration in the culture medium is 1000 mM or less, 800 mM or less, 600 mM or less, 500 mM or less, 450 mM or less, 400 mM or less, 350 mM or less, 300 mM or less, 280 mM or less.
  • the concentration of trehalose in the culture medium is 20 mM or more and 1000 mM or less, 50 mM or more and 800 mM or less, 100 mM or more and 600 mM or less, and 150 mM or more and 400 mM or less.
  • the culture medium containing trehalose does not contain a carbon source (for example, sucrose or maltose) used in a conventional method, or may have a reduced content, instead of containing trehalose.
  • the culture medium contains no carbon source other than trehalose.
  • the carbon source means sucrose, maltose, glucose and the like.
  • the carbon source means maltose.
  • long-term cultures on carbon sources containing no other than trehalose are excluded.
  • Non-limitingly long-term culture means, for example, 6 days or more, 7 days or more, 8 days or more, 10 days or more, 17 days or more.
  • the concentration of trehalose can be grasped as the content per unit volume of the medium when the trehalose is not always uniformly contained in the whole medium, for example, when the medium is a solid.
  • the time when trehalose is added to the medium is not particularly limited.
  • the addition time may be at or after the start of culturing.
  • the culture medium containing trehalose is used at any time of the step of culturing the plant cells until the time when the plant cells have the ability to regenerate and differentiate.
  • the culture medium containing trehalose is used for a short period of time after the immature embryos are prepared, for example, within 16 days, within 8 days, within 5 days, within 3 days, or within 2 days.
  • the culture medium containing trehalose is used for the period from the preparation of immature embryos to the introduction of the substance into the plant.
  • trehalose may be added to the culture medium at one time or in divided doses (for example, several times).
  • a culture involving substance introduction with a particle gun an immature embryo and a cell group derived from the immature embryo are placed and cultured on a culture medium containing trehalose from 1 day or 2 days before the particle gun treatment. Is good.
  • a culture involving introduction of a substance with agrobacterium when a cell group derived from an immature embryo is used as a material, the day before the agrobacterium inoculation treatment, one day before, or two days after the inoculation. It is better to place them on a culture medium containing trehalose and cultivate them. It is good to place it on the bed and inoculate it.
  • the medium may include, but is not limited to, a plant growth regulator.
  • Plant growth regulators are auxins. Specifically, for example, as auxins, indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), picrolam, daikamba, naphthalene acetic acid (NAA), naphthoxyacetic acid, phenylacetic acid, for example. , 2,4,5-Trichlorophenoxyacetic acid or other auxins can be added.
  • the auxins are plant hormones selected from the group consisting of 2,4-D, IAA, NAA.
  • other plant growth regulators such as kinetin and cytokinins such as 4PU can be added. These plant growth regulators may be used alone or in admixture of a plurality at different concentrations.
  • feeder cells may be added to the culture medium.
  • the type of feeder cell is not particularly limited.
  • liquid cultured cells of rice, liquid cultured cells of wheat, and the like can be used. It is possible to use known feeder cells and plant explants. Examples thereof include rice suspension cell culture (Oc, RIKEN BioResource Research Center) and wheat suspension culture cells (HT3-1). Examples of the plant explants include ovaries.
  • the timing of adding the feeder cells is not limited, and may be before, at the start, or after the start of culturing the plant cells.
  • the culture temperature can be appropriately selected, preferably 18 ° C.-35 ° C., more preferably 20-30 ° C., and most preferably 23-28 ° C. Further, the culture in this step is preferably performed in a dark place or a dim place, but the culture is not limited to this.
  • the above-mentioned culturing method includes culturing plant cells of the Gramineae family to obtain a plant cell group and / or a regenerated plant.
  • the cell group may be an embryoid body, a spherical embryo, a callus, or the like.
  • the process of inducing division of a fertilized egg and causing cell proliferation to form an embryoid body, a spherical embryo, a callus, or the like is not particularly limited because the optimum conditions differ depending on the plant.
  • the present invention is also used in a culture medium containing trehalose for culturing grass cells, use of trehalose in a culture medium for grass cells, and a culture medium for grass cells. Also includes trehalose.
  • the above-mentioned culturing method improves the culturing efficiency of plant cells as compared with the case of culturing in a medium containing no trehalose.
  • the culture efficiency of plant cells is not limited, and means, for example, the callus formation rate in culture.
  • the culture efficiency (for example, callus formation rate) is improved to, for example, 1.1 times or more, 2 times or more, 3 times or more, 5 times or more, 10 times or more, and 30 times or more.
  • "Improved culture efficiency" includes the case where callus formation occurs in Gramineae varieties in which callus was not formed when cultured in a medium containing no trehalose.
  • the present invention relates to a method for regenerating plants.
  • the regeneration method of the present invention is a method for regenerating a plant from cells of a gramineous plant. (1) The cells of Gramineae plants are cultured using a culture medium containing trehalose, and then (2) Culturing in redifferentiation medium, Including that.
  • the redifferentiation step that is, the culture step in the redifferentiation medium is not particularly limited.
  • the cultured cell group is transferred to an arbitrary medium, for example, a redifferentiation medium consisting of the LSF medium described in the examples of the present specification (for example, LSFCuP5 (—C) medium), and cultured.
  • the redifferentiation step may be performed by irradiating with light.
  • the redifferentiation medium may be, for example, LSF medium, MS medium, B5 medium, N6 medium, and may be a liquid medium containing no solid medium using a solidifying material such as agarose, agar, gellan gum, and gellite.
  • a plant growth regulator containing auxin and cytokinin may be added to the redifferentiation medium.
  • the redifferentiation medium comprises auxins.
  • auxins The meanings of terms such as “auxin” and “cytokinin” are as described in "1. Plant cell culture method”.
  • Gramineae plants regenerated plants
  • the regenerated plants include plants regenerated by culturing immature embryos and redifferentiation, cells, tissues, etc. obtained from the plants, and "T0 generation” which is the redifferentiation generation obtained by the above-mentioned plant regeneration method.
  • T1 generation progeny plants
  • progeny plants such as "T1 generation” derived from the self-fertilized seeds of T0 generation plants, hybrid plants crossed with them as one parent, and progeny plants.
  • the method for culturing plant cells and the method for regenerating plants of the present invention it has become possible to stably and efficiently obtain regenerated plants from the cell group obtained by culturing cells of grasses.
  • the regenerated plant into which the substance is introduced can be stably and efficiently obtained.
  • the transformant is introduced into the immature embryo.
  • the plant cells of the above-mentioned culture method and plant regeneration method are plant cells into which a substance has been introduced.
  • the plant cells of the culture method and the plant regeneration method of the present invention include a step of introducing a substance selected from the group consisting of nucleic acid, protein and nucleic acid protein complex into cells of grasses.
  • the substance to be introduced is selected from the group consisting of nucleic acid, protein and nucleic acid-protein complex without limitation.
  • the nucleic acid is not particularly limited, and may be DNA, RNA, a combination of both, or a mixture. It is preferably a circular DNA such as a vector, a linear DNA, a circular RNA or a linear RNA. Any length can be used depending on the transformation method used.
  • Protein is a general term for molecules in which various amino acids are linked in a fixed order by amide bonds (also referred to as "peptide bonds").
  • the protein contains a nuclease such as Cas9 nuclease for genome editing, a modifying enzyme, an antibody and the like.
  • a nucleic acid-protein complex is a complex formed by a nucleic acid and a protein.
  • deoxyribonuclear protein complex of DNA and VirD2 protein, etc.
  • ribonuclear protein complex of guide RNA and Cas9 protein, etc.
  • the like are included.
  • the nucleic acid may be two or more types of DNA or RNA, or a combination of DNA and RNA. Different types of substances such as nucleic acids and proteins may be introduced.
  • 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 a particle gun method, a polyethylene glycol method (PEG method), an electroporation method, a microinjection method, a whisker method, or a biological method such as an Agrobacterium method (agrobacterium method).
  • the method of indirect introduction of DNA can be preferably used.
  • the substance is introduced by the particle gun method or the Agrobacterium method.
  • Patent Document 2 International Publication WO2017 / 171092
  • Patent Document 3 International Publication WO2018 / 143480
  • transformation or genome editing may be performed by introducing a substance.
  • Genome editing is a technique for modifying a target gene as desired by using a site-specific nuclease.
  • site-specific nucleases include ZFN (Zinc finger nuclease), TALEN, and CRISPR / Cas9 (Crisper Cas9), which were developed and discovered after 2005.
  • Tissue culture of gramineous plants, for example, wheat and production of regenerated bodies are necessary techniques in the development of wheat varieties.
  • immature embryos or cultured cells that have been gene-introduced by the Agrobacterium method or the particle gun method are tissue-cultured, and regenerated bodies are produced from callus.
  • the method of acquisition is the standard method.
  • immature embryos or cultured cells into which ribonucleoprotein (RNP) has been introduced may be cultured to obtain a regenerated body.
  • RNP ribonucleoprotein
  • the above-mentioned culturing method improves the efficiency of introducing a substance as compared with the case of culturing in a medium containing no trehalose.
  • the introduction efficiency of a substance means, for example, a callus formation rate, a transformation rate (transformation frequency), a genome editing rate (genome editing frequency), and the like in culture without limitation.
  • high efficiency of substance introduction means that the target substance is introduced into plant cells with high efficiency, callus is induced with high efficiency from immature embryos, etc., and it is high from transformed callus. It is a concept that includes the fact that redifferentiation occurs with efficiency.
  • improved of introduction efficiency of a substance means improvement of introduction efficiency of a target substance into plant cells, improvement of callus induction rate from immature embryos, etc., and improvement of callus induction rate from transformed callus. It is a concept that includes improving the efficiency of redifferentiation.
  • the introduction efficiency (for example, transformation efficiency) of a substance is, for example, 1.3 times or more, 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 10 times or more, 25 times. It will be improved to the above. "Improvement of substance introduction efficiency" includes cases where transformation is not possible when cultured in a medium containing no trehalose, or these can be achieved in grass varieties that cannot be genome-edited.
  • Example 1 Callus induction from immature wheat embryos (1) Preparation and culture of immature embryos Part of the methods of WO2011 / 013764 (Patent Document 1) and Ishida (2015) (Non-Patent Document 16) are modified to produce wheat. Immature embryos were prepared. Nine varieties of Fielder, Claire, Mace, JTX001, Cadenza, Paragon, JTX002, Cronox and Jugger were tested.
  • Immature wheat seeds about 14 days after flowering were demineralized, surface sterilized in 70% ethanol for 45 seconds, and then surface sterilized with 1% sodium hypochlorite containing 1 drop of Tween 20 for 10 minutes.
  • the treated immature seeds were washed 4 times with sterile water, immature embryos were collected under a stereomicroscope and recovered in WLS-liq medium.
  • the immature embryos were washed once with WLS-liq medium and then centrifuged at 5,000 ⁇ g at 4 ° C. for 10 minutes.
  • each medium is as follows. WLS-liq medium: 0.1 ⁇ LS major inorganic salt, 10 ⁇ M FeEDTA, 0.1 ⁇ LS minor inorganic salt, 0.1 ⁇ MS vitamin, 1% glucose, 0.05% MES, pH 5.8
  • WLS90-RES medium 1 x LS major inorganic salt, 100 ⁇ M FeEDTA, 1 x LS minor inorganic salt, 1 x MS vitamin, 0.5 mg / L 2,4-D, 2.5 mg / L daikamba, 9% maltose water.
  • Japanese product (250 mM) 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl 2, 0.195% MES, 57 ⁇ M ascorbic acid, 5 ⁇ M 5 g / L agarose, pH 5.8.
  • WLS0 / 90-RES medium A medium in which maltose monohydrate of WLS90-RES medium is changed to trehalose dihydrate (250 mM) having a concentration of 9.46%.
  • SpCas9 (Cas9 derived from Streptococcus pyogenes) gene is shown in SEQ ID NO: 2.
  • SEQ ID NO: 2 is Svitashev et al. It is quoted from the report (Non-Patent Document 17) of (2015).
  • SpCas9 is inserted between the corn ubiquitin promoter with the first intron of corn ubiquitin and the Nos and 35S terminators, and in addition, the SV40-derived nuclear localization signal (9 amino acids) and carboxyl terminus (C) on the amino-terminal (N-terminal) side.
  • a plasmid inserted into the pUC19 vector was constructed so that the (terminal) side contained a sequence encoding a virD2-derived nuclear localization signal (18 amino acids).
  • the base sequence of the sgRNA transcription unit is shown in SEQ ID NO: 3.
  • a plasmid was constructed by inserting the sgRNA gene sequence containing the promoter and terminator of wheat U6 polymerase III into the pUC18 vector.
  • the target sequence of the wheat LOX2 gene was used as the sgRNA gene sequence (Non-Patent Document 18).
  • the prepared gold particle suspension (40 mg / ml) was vortexed for 5 minutes until it became uniform.
  • the plasmid DNA was coated on gold particles and adhered to microcarriers.
  • water was added to make a total of 40 ⁇ l, and then TransIT-. 2020 (registered trademark) (0.8 ⁇ l) was added. Then, after vortexing continuously for 2 to 3 minutes, the tube was allowed to stand still for 1 minute.
  • a gene was used with a particle gun (BioLISTIC PDS1000 / He, Bio-Rad) according to the Biolistic PDS-1000 / He Particure Delivery System instruction manual (BIO-RAD). Introduced. 5 ⁇ l of gold particle suspension was used per shot. The target distance was 5 cm from the stopping plate, and the shooting pressure was 650 psi. After the particle gun treatment, immature embryos on the plate medium were cultured at 28 ° C. for 1 day (dark place) and then at 25 ° C.
  • the table below summarizes the number of days, medium, and experimental content (process) of each process from immature embryo preparation to leaf sampling.
  • Composition of WLS60-P5 1 ⁇ LS major inorganic salt, 100 ⁇ M FeEDTA, 1 ⁇ LS minor inorganic salt, 1 ⁇ MS vitamin, 0.5 mg / L 2,4-D, 2.2 mg / L picrolam, 6% maltose 1 Hydrate, 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl 2 , 0.195% MES, 57 ⁇ M ascorbic acid, 5 ⁇ M AgNO 3 , 5 mg / L phosphinotricin, 5 g / L agarose, pH 5 8.8.
  • Composition of WLS-P10 1 ⁇ LS major inorganic salt, 100 ⁇ M FeEDTA, 1 ⁇ LS minor inorganic salt, 1 ⁇ MS vitamin, 0.5 mg / L 2,4-D, 2.2 mg / L picrolam, 4% maltose 1 Hydrate, 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl 2, 0.195% MES, 57 ⁇ M ascorbic acid, 5 ⁇ M AgNO 3 , 10 mg / L phosphinotricin, 5 g / L agarose, pH 5 8.8.
  • the callus formation rate was determined by (number of callus generated / number of immature embryos under test) ⁇ 100 (%) in step 6 of Table 1.
  • callus was replanted in a medium containing phosphinotricin for selection by the selection marker bar gene and cultured (number of premature embryos tested in which regenerated individuals were generated /). It was determined by the number of immature embryos to be tested) ⁇ 100 (%). The results are shown in Table 2.
  • Example 2 Effect of maltose / trehalose ratio on callus induction from wheat immature embryos (1) Preparation and culture of immature embryos Immature embryos of wheat (variety: Fielder) were prepared in the same manner as in Example 1. As the initial culture medium, WLS90-RES medium, or various trehalose-modified WLS media (WLS72 / 18-RES medium, WLS60 / 30-RES medium, WLS30 /) in which all or part of the maltose of WLS-RES medium is replaced with trehalose. 60-RES medium, WLS18 / 72-RES medium and WLS0 / 90-RES medium) were used to culture immature embryos as shown in Table 4.
  • WLS90-RES medium or various trehalose-modified WLS media (WLS72 / 18-RES medium, WLS60 / 30-RES medium, WLS30 /) in which all or part of the maltose of WLS-RES medium is replaced with trehalose. 60-RES medium
  • WLS72 / 18-RES medium Change maltose monohydrate in WLS90-RES medium to 7.2% maltose monohydrate (200 mM) and 1.89% trehalose dihydrate (50 mM).
  • Medium (maltose: trehalose 8: 2).
  • the table below summarizes the number of days, medium, and experimental content (steps) of each step of preparing immature embryos, inducing callus, and counting callus.
  • Example 2 Effect of maltose / trehalose ratio on callus formation rate and plant regeneration rate
  • the callus formation rate was examined for immature embryos that had been DNA-introduced by a particle gun.
  • the callus formation rate was determined by (number of callus generated / number of immature embryos under test) ⁇ 100 (%) in step 6 of Table 4.
  • Example 3 Effect of trehalose on callus induction and seedling formation from immature wheat embryos (1) Preparation and culture of immature embryos In the same manner as in Example 1, immature embryos of wheat (variety: Fielder) were prepared. As the initial culture medium, WLS40-RES medium, WLS90-RES medium, or WLS0 / 40-RES medium and WLS0 / 90-RES medium as trehalose-modified WLS medium were used for culturing.
  • WLS40-RES medium 1 x LS major inorganic salt, 100 ⁇ M FeEDTA, 1 x LS minor inorganic salt, 1 x MS vitamin, 0.5 mg / L 2,4-D, 2.5 mg / L Daikamba, 4% maltose water Japanese product (111 mM), 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl2, 0.195% MES, 57 ⁇ M ascorbic acid, 5 ⁇ M 5 g / L agarose, pH 5.8.
  • WLS90-RES medium 1 x LS major inorganic salt, 100 ⁇ M FeEDTA, 1 x LS minor inorganic salt, 1 x MS vitamin, 0.5 mg / L 2,4-D, 2.5 mg / L Daikamba, 9% maltose water Japanese product (250 mM), 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl2, 0.195% MES, 57 ⁇ M ascorbic acid, 5 ⁇ M 5 g / L agarose, pH 5.8.
  • WLS0 / 40-RES medium A medium in which maltose monohydrate of WLS40-RES medium is changed to trehalose dihydrate (111 mM) having a concentration of 4.2%.
  • WLS0 / 90-RES medium A medium in which maltose monohydrate of WLS90-RES medium is changed to trehalose dihydrate (250 mM) having a concentration of 9.46%.
  • the table below summarizes the number of days, medium, and experimental content (steps) of each step of preparing immature embryos, inducing callus, forming regenerated bodies, and counting.
  • the callus formation rate was determined by (number of callus generated / number of immature embryos under test) ⁇ 100 (%) in step 4 of Table 6.
  • the seedling rate was determined by counting the regenerated seedlings in step 6 of Table 6 (number of premature embryos tested / number of immature embryos tested) ⁇ 100 (%).
  • Table 7 shows the effect of the trehalose-added medium on the callus formation rate
  • Table 8 shows the effect of the trehalose-added medium on the seedling rate.
  • Example 4 Callus induction from immature maize embryos
  • the immature embryos of maize inbread B104 were aseptically collected, and Ishida et al. (2007) Agrobacterium tumefaciens was inoculated by the method of (Non-Patent Document 21).
  • heat treatment at 46 ° C. and centrifugation at 20,000 ⁇ g were performed (Non-Patent Document 4).
  • the T-DNA region of pLC41GWH of WO2014 / 157541 was regulated by the corn ubiquitin promoter and intron, and the GUS gene (Pubi-Iubi-Icat GUS-) mediated by the catalase intron of Hima. Tonos), and LBA4404 (pLC41 GUS-bar) having a binary vector modified to T-DNA having a Bar gene (P35S-bar-T35S) controlled by the cauliflower mosaic virus 35S promoter, and WO2014 / 157541 (Patent Document 4). ) LBA4404 (pLC41 GUS-Bar :: pVGW9) having a booster vector was used.
  • the GUS gene (P35S-Icat GUS-Tnos) regulated by the cauliflower mosaic virus 35S promoter in the T-DNA region and mediated by the catalase intron of Hima
  • the Bar gene (P35S-bar) regulated by the cauliflower mosaic virus 35S promoter.
  • LBA4404 (pSB131) (Non-Patent Document 22) having a super binary vector having -Tnos) was also subjected to the test.
  • the immature embryos after inoculation were placed on the LS-AS medium and cultured at 25 ° C. in the dark for 7 days.
  • 2% sucrose in the LS-AS medium was reduced to 1.5%
  • 1% glucose was reduced to 0.75%
  • 1.08% trehalose dihydrate was added.
  • the present invention it has become possible to facilitate the cultivation of gramineous plants, which are conventionally said to be difficult to culture, and to efficiently obtain plant regenerated cells from cultured cells.
  • a substance into a plant cell at any stage of the steps of the method for culturing the plant cell and the method for regenerating the plant cell, it is possible to stably and efficiently obtain the regenerated plant into which the substance has been introduced.
  • this method it becomes possible to efficiently produce transformed crops of grasses and genome-edited crops, which have been difficult in the past.
  • SEQ ID NO: 1 is the base sequence of the bar gene.
  • SEQ ID NO: 2 is the base sequence of the SpCas9 gene.
  • SEQ ID NO: 3 is the base sequence of the sequence of the sgRNA expression unit.

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Abstract

La présente invention concerne un procédé de culture d'une cellule végétale et un procédé de reproduction d'un corps végétal.　Le procédé de culture d'une cellule végétale selon la présente invention comprend la culture d'une cellule d'une plante appartenant à la famille des Gramineae à l'aide d'un milieu de culture contenant du tréhalose. Le procédé de reproduction d'un corps végétal selon la présente invention est un procédé de reproduction d'un corps végétal à partir d'une cellule d'une plante appartenant à la famille Gramineae, et comprend (1) la culture d'une cellule d'une plante appartenant à la famille Gramineae en utilisant un milieu de culture contenant du tréhalose et (2) la culture du produit résultant dans un milieu de culture de redifférenciation.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007306917A (ja) * 2006-04-20 2007-11-29 National Agriculture & Food Research Organization イネの病原菌に対する抵抗性を高める方法及び病原菌耐性イネ形質転換体
WO2011013764A1 (fr) * 2009-07-29 2011-02-03 日本たばこ産業株式会社 Procédé de transfert de gène dans un plant triticum à l'aide d'une bactérie agrobacterium et procédé de production d'un plant transgénique d'un plant triticum

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007306917A (ja) * 2006-04-20 2007-11-29 National Agriculture & Food Research Organization イネの病原菌に対する抵抗性を高める方法及び病原菌耐性イネ形質転換体
WO2011013764A1 (fr) * 2009-07-29 2011-02-03 日本たばこ産業株式会社 Procédé de transfert de gène dans un plant triticum à l'aide d'une bactérie agrobacterium et procédé de production d'un plant transgénique d'un plant triticum

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HIEI, YUKOH ET AL.: "Development of Agrobacterium-mediated transformation method for monocotyledonous plants", BREEDING RESEARCH, vol. 2, no. 4, 2000, pages 205 - 213 *
ISHIDA Y, ET AL.: "IMPROVED PROTOCOL FOR TRANSFORMTION OF MAIZE (ZEA MAYS L.) MEDIATED BY AGROBACTERIUM TUMEFACIENS", PLANT BIOTECHNOLOGY, JAPANESE SOCIETY FOR PLANT CELL AND MOLECULAR BIOLOGY, TOKYO,, JP, vol. 20, no. 01, 1 March 2003 (2003-03-01), JP , pages 57 - 66, XP009070864, ISSN: 1342-4580 *
PUOLIMATKA MATTI, PAUK JANOS: "Effect of Induction Duration and Medium Composition on Plant Regeneration in Wheat (Triticum aestivum L.) Anther Culture", JOURNAL OF PLANT PHYSIOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 156, no. 2, 1 February 2000 (2000-02-01), AMSTERDAM, NL, pages 197 - 203, XP055932943, ISSN: 0176-1617, DOI: 10.1016/S0176-1617(00)80306-5 *
REDHA AMINA, SULEMAN PATRICE: "Assessment of Polyamines and Trehalose in Wheat Microspores Culture for Embryogenesis and Green Regenerated Plants", AMERICAN JOURNAL OF PLANT SCIENCES, SCIENTIFIC RESEARCH PUBLISHING, INC., US, vol. 04, no. 11, 1 November 2013 (2013-11-01), US , pages 2218 - 2226, XP055932945, ISSN: 2158-2742, DOI: 10.4236/ajps.2013.411275 *

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