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WO2025206265A1 - Construction d'acide nucléique exprimant l'ipt - Google Patents

Construction d'acide nucléique exprimant l'ipt

Info

Publication number
WO2025206265A1
WO2025206265A1 PCT/JP2025/012676 JP2025012676W WO2025206265A1 WO 2025206265 A1 WO2025206265 A1 WO 2025206265A1 JP 2025012676 W JP2025012676 W JP 2025012676W WO 2025206265 A1 WO2025206265 A1 WO 2025206265A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
ipt
regenerated
genome editing
promoter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2025/012676
Other languages
English (en)
Japanese (ja)
Inventor
創 山田
祐二 石田
晴康 濱田
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kaneka Corp
Original Assignee
Kaneka Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kaneka Corp filed Critical Kaneka Corp
Publication of WO2025206265A1 publication Critical patent/WO2025206265A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)

Definitions

  • the present invention relates to a nucleic acid construct comprising a polynucleotide encoding isopentenyltransferase (IPT) and a promoter operably linked to the polynucleotide.
  • the present invention also relates to an agent for promoting the production of regenerated plants derived from plant tissue explants, comprising the nucleic acid construct; a method for promoting the production of regenerated plants derived from plant tissue explants; and a method for producing regenerated plants derived from plant tissue explants.
  • the present invention further relates to an agent for improving the efficiency of plant genome editing, comprising the nucleic acid construct; a method for improving genome editing efficiency in plants; and a method for producing genome-edited plants.
  • Non-Patent Document 2 describes co-introducing a target gene and an IPT gene into a plant and selecting genetically modified organisms using morphological abnormalities as an indicator. Furthermore, Patent Document 1 describes obtaining genome-edited individuals in which the foreign gene has not been integrated into the genome by removing individuals in which the foreign gene has been integrated into the genome using morphological abnormalities as an indicator when regenerating individuals derived from plant cells into which the IPT gene and a genome editing enzyme gene have been introduced.
  • the objective of the present invention is to provide a means for shortening the time required to regenerate a plant from a plant tissue fragment, or a means for improving the efficiency of genome editing in plants.
  • FIG. 1 Figures showing the T-DNA structures of the vectors prepared in Production Examples 1 to 5.
  • A Genome-editing vector for non-IPT expression.
  • B Genome-editing vector for PNative-IPT expression, genome-editing vector for PPcUbi-IPT expression, genome-editing vector for P35S-IPT expression, and genome-editing vector for P2x35S-IPT expression.
  • A Agrobacterium carrying a non-IPT expression genome editing vector or
  • a PPcUbi-IPT expression genome editing vector was inoculated into potato leaf pieces, and the leaf pieces were cultured on coculture medium PCM for 3 days, then on callus formation medium PCM for 3 days, and then on regeneration medium PSM.
  • the present invention relates to a nucleic acid construct comprising a polynucleotide encoding isopentenyltransferase (IPT) and a promoter operably linked to the polynucleotide (hereinafter referred to as the "IPT-expressing nucleic acid construct of the present invention").
  • the IPT-expressing nucleic acid construct of the present invention can be used to promote the generation of regenerated plants from plant tissue explants and/or improve the efficiency of plant genome editing.
  • plasmids include, but are not limited to, pUC series (pUC57, pUC18, pUC19, pUC9, etc.), pLC series (pLC41, etc.), pAL series (pAL51, pAL156, etc.), pBI series (pBI121, pBI101, pBI221, pBI2113, pBI101.2, etc.), pPZP series, pSMA series, and intermediate vector series (pLGV23Neo, pNCAT, etc.).
  • the IPT-expressing nucleic acid construct of the present invention comprises, as essential components, a polynucleotide encoding isopentenyltransferase (IPT) and a promoter operably linked to the polynucleotide.
  • IPT isopentenyltransferase
  • IPT isopentenyl transferase
  • IPT is an enzyme involved in the biosynthesis of cytokinin.
  • Isopentenyl transferase may be derived from plants or bacteria such as Agrobacterium.
  • Agrobacterium refers to bacteria of the genus Agrobacterium.
  • the isopentenyl transferase may be, for example, an isopentenyl transferase derived from Agrobacterium tumefaciens.
  • the isopentenyl transferase may be, for example, a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • the isopentenyl transferase may also be a protein having isopentenyl transferase activity and consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been added, deleted, and/or substituted in the amino acid sequence shown in SEQ ID NO: 1.
  • amino acid substitution preferably refers to substitutions within conservative amino acid groups that have similar properties, such as charge, side chain, polarity, and aromaticity, among the 20 types of amino acids that make up naturally occurring proteins. Examples include substitutions within uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because they are known to be less likely to cause changes in the properties of polypeptides.
  • amino acid sequence identity refers to a numerical value indicating the percentage of sites containing the same type of amino acid residue within the comparison range of two amino acid sequences. Even when the lengths of the two amino acid sequences are different, amino acid sequence identity can be calculated by aligning the sequences to maximize the degree of amino acid identity within the comparison range.
  • a representative algorithm for such analysis is BLAST, although this is not limited to it. BLAST is available in a variety of software and web services.
  • amino acid sequence identity can be easily calculated using the genetic information processing software GENETYX (https://www.genetyx.co.jp/) or the NCBI BLAST server (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
  • GENETYX https://www.genetyx.co.jp/
  • NCBI BLAST server https://blast.ncbi.nlm.nih.gov/Blast.cgi.
  • FASTA FASTA
  • the polynucleotide encoding isopentenyl transferase may be, for example, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2.
  • the polynucleotide encoding isopentenyl transferase may also be a polynucleotide consisting of a nucleotide sequence in which 1 to 10, 1 to 7, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 nucleotide has been added, deleted, and/or substituted in the nucleotide sequence shown in SEQ ID NO: 2, and which encodes a protein having isopentenyl transferase activity.
  • the polynucleotide encoding isopentenyl transferase may also be a polynucleotide consisting of a nucleotide sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 2, and which encodes a protein having isopentenyl transferase activity.
  • nucleotide sequence identity is a numerical value that indicates the percentage of sites with the same type of base within the comparison range of two nucleotide sequences, similar to the amino acid sequence identity described above. Even if the lengths of the two nucleotide sequences are different, nucleotide sequence identity can be calculated by aligning them so that the degree of base identity within the comparison range is highest. While not limited to the aforementioned BLAST and FASTA, analysis algorithms such as MUMmer can also be used to analyze nucleotide sequence identity.
  • polynucleotide refers not only to double-stranded nucleic acids, but also to single-stranded nucleic acids such as the positive strand (sense strand) or complementary strand (antisense strand) that constitute them, and unless otherwise specified, includes both DNA and RNA.
  • the promoter operably linked to the polynucleotide encoding isopentenyl transferase may be of plant or non-plant origin, as long as it is capable of inducing transcription in plant cells.
  • the promoter may be the native promoter of the isopentenyl transferase gene or a promoter of another gene.
  • the promoter may also be a naturally occurring promoter or a modified promoter.
  • the promoter may be, for example, a constitutive promoter.
  • constitutive promoter refers to a promoter that can induce transcription in the entire plant (e.g., all tissues or all cells of the plant) regardless of the developmental stage.
  • the promoter may also be, for example, a high-expression promoter.
  • a "high-expression promoter” refers to a promoter having activity greater than that of the promoter shown in SEQ ID NO: 16 (the native promoter of the Agrobacterium tumefaciens IPT gene), for example, a promoter having activity 1.5 times or more (e.g., 2 times or more, 4 times or more, 6 times or more, 8 times or more, 10 times or more, 20 times or more, 40 times or more, 60 times or more, 80 times or more, 100 times or more, 200 times or more, 400 times or more, 600 times or more, 800 times or more, or 1000 times or more) that of the promoter shown in SEQ ID NO: 16.
  • Promoter activity can be confirmed by constructing a vector in which various reporter genes, such as ⁇ -glucuronidase (GUS) or luciferase (LUC) genes, are linked downstream of the promoter, transforming a plant with the vector using the Agrobacterium method or the like, and then measuring the expression of the reporter gene.
  • GUS ⁇ -glucuronidase
  • LOC luciferase
  • promoters include the isopentenyl transferase (IPT) gene promoter (e.g., the Agrobacterium tumefaciens IPT gene promoter), ubiquitin promoters (e.g., the parsley ubiquitin promoter, the maize ubiquitin promoter), the cauliflower mosaic virus (CaMV) 35S promoter, modified CaMV 35S promoter promoters (e.g., the 2x35S promoter, the El2-35S omega promoter), actin promoters (e.g., the rice actin promoter), the nopaline synthase (NOS) gene promoter, the alcohol dehydrogenase (ADH) gene promoter, the tobacco-derived infection-specific protein (PR protein) promoter, the RuBisco promoter, and the U6 promoter (e.g., the Arabidopsis thaliana U6 promoter).
  • IPT isopentenyl transferase
  • ubiquitin promoters e.g., the
  • the promoter may be, for example, a polynucleotide consisting of the base sequence shown in any one of SEQ ID NOs: 16 to 19 and 44.
  • the promoter may also be a polynucleotide having promoter activity and consisting of a base sequence in which one to two or one base has been added, deleted, and/or substituted in the base sequence shown in any one of SEQ ID NOs: 16 to 19 and 44.
  • the promoter may also be a polynucleotide having promoter activity and consisting of a base sequence having 90% or more, 95% or more, or 100% sequence identity with the base sequence shown in any one of SEQ ID NOs: 16 to 19 and 44.
  • the IPT-expressing nucleic acid construct of the present invention may include an expression control region for isopentenyltransferase other than a promoter (e.g., an enhancer, an insulator, a terminator, a poly(A) addition signal, etc.).
  • a promoter e.g., an enhancer, an insulator, a terminator, a poly(A) addition signal, etc.
  • terminals include, but are not limited to, the Pea3A terminator, a heat shock protein (HSP) gene terminator, a nopaline synthase (NOS) gene terminator, an octopine synthase (OCS) gene terminator, and a CaMV 35S terminator.
  • HSP heat shock protein
  • NOS nopaline synthase
  • OCS octopine synthase
  • the IPT-expressing nucleic acid construct of the present invention may or may not contain a drug resistance gene.
  • drug resistance gene refers to a gene that confers resistance to a specific drug (a selective drug as described later in this specification) to a target plant.
  • drug resistance genes include, but are not limited to, the bar gene for phosphinothricin (PPT) (glufosinate), the hygromycin B resistance gene (hpt gene) for hygromycin B, the chloramphenicol resistance gene for chloramphenicol, the neomycin resistance gene for neomycin, and the kanamycin resistance gene for kanamycin.
  • the IPT-expressing nucleic acid construct of the present invention can be prepared by those skilled in the art using known techniques. For example, as shown in the Examples below, it can be prepared by cleaving the gene cassette with an appropriate restriction enzyme and ligating it to a restriction enzyme site or a multicloning site of an appropriate backbone vector.
  • the IPT-expressing nucleic acid construct of the present invention can also be prepared using techniques such as In-Fusion cloning, TA cloning, and double crossover recombination.
  • the base sequence of the gene encoded in the IPT-expressing nucleic acid construct of the present invention may be optimized to use codons appropriate for the plant cell into which it is introduced, in order to efficiently express its translation product in the cell.
  • the IPT expression nucleic acid construct of the present invention can promote the generation of regenerated plants from plant tissue explants and can also improve genome editing efficiency in plants.
  • the present invention provides an agent for promoting the production of regenerated plants derived from plant tissue fragments (hereinafter, sometimes referred to as the "agent for promoting the production of regenerated plants of the present invention"), which comprises the IPT-expressing nucleic acid construct of the present invention.
  • tissue explant refers to a section cut out from a part of a plant.
  • the plant tissue explant may be any tissue explant capable of regenerating a plant, and includes, for example, sections of leaves, stems, roots, or seeds.
  • the plant tissue explant may be, for example, a tissue explant containing a dividing cell or a cell capable of regeneration, such as, but not limited to, a tissue explant containing the hypocotyl, cotyledonary node, shoot apex, or terminal bud of a young plant.
  • Regenerated plants include plants from which adventitious buds, stems, and leaves have been regenerated or regenerated, plants from which roots have been regenerated or regenerated, and plants from which leaves, stems, and roots have been regenerated or regenerated.
  • regenerated plants whose generation is promoted by the present invention are preferably plants from which leaves and stems have been regenerated or regenerated, for example, plants from which morphologically normal leaves and stems have been regenerated or regenerated.
  • a polynucleotide encoding IPT may or may not be integrated into the genome of a regenerated plant.
  • the term "agent for promoting the production of regenerated plants” refers to an agent that, when introduced into a plant tissue fragment or cultured cells derived therefrom, promotes the production of regenerated plants derived from the plant tissue fragment.
  • Promotion of regenerated plant production can be, for example, promotion of the production of regenerated plants from a plant tissue fragment, or promotion of the production of regenerated plants from callus formed from a plant tissue fragment.
  • "promoting the production of regenerated plants” means increasing the amount of regenerated plants produced compared to the amount of regenerated plants produced when regenerated plants are produced from a plant tissue fragment without introducing a polynucleotide encoding isopentenyl transferase (IPT).
  • promotion of the production of regenerated plants can be, for example, promotion of the growth of regenerated plants. Therefore, the agent for promoting the production of regenerated plants of the present invention can be, for example, a growth promoter for regenerated plants.
  • the term "agent for promoting the growth of regenerated plants” refers to an agent that, when introduced into a plant tissue fragment or cultured cells derived therefrom, promotes the growth of regenerated plants derived from plant tissue fragments.
  • "promoting the growth of regenerated plants” includes promoting the growth rate of leaves and/or stems.
  • promoting the growth of a regenerated plant means increasing the growth rate of a regenerated plant compared to the growth rate of a plant regenerated from a plant tissue explant without introducing a polynucleotide encoding isopentenyl transferase (IPT).
  • IPT isopentenyl transferase
  • the IPT-expressing nucleic acid construct of the present invention has the effect of promoting the production of regenerated plants derived from plant tissue explants can be evaluated, for example, as described below.
  • the IPT-expressing nucleic acid construct of the present invention is introduced into plant tissue explants or cultured cells derived therefrom, and these are cultured for a predetermined period of time to regenerate plants.
  • the number of tissue explants that form shoots (number of shoot-forming tissue explants) is counted, and the ratio of the number of shoot-forming tissue explants to the total number of tissue explants is calculated.
  • the same procedure is performed using an identical nucleic acid construct except that it does not contain the IPT gene.
  • the ratio of the number of shoot-forming tissue explants to the total number of tissue explants when the IPT-expressing nucleic acid construct of the present invention is introduced is 1.5 times or more (e.g., 2 times or more, 3 times or more, 4 times or more, or 5 times or more) higher than the ratio of the number of shoot-forming tissue explants to the total number of tissue explants when a nucleic acid construct not containing the IPT gene is introduced, it can be determined that the IPT-expressing nucleic acid construct of the present invention has the effect of promoting the production of regenerated plants.
  • the IPT-expressing nucleic acid construct of the present invention can be introduced into plant tissue explants or cultured cells derived therefrom, which are then cultured to form callus, and the callus can then be cultured to regenerate a plant. Then, for example, the number of tissue explants that have grown to the point where the regenerated plant has formed morphologically normal leaves and stems (sometimes referred to as "stems and leaves" in this specification) after a specified period of time can be counted as the number of shoot-forming tissue explants, and the ratio of the number of shoot-forming tissue explants to the total number of tissue explants can be calculated.
  • the promoter for regenerated plant production of the present invention comprises the IPT-expressing nucleic acid construct of the present invention.
  • the IPT-expressing nucleic acid construct of the present invention is as described in "1. IPT-expressing nucleic acid construct.”
  • the regenerated plant production promoter of the present invention can be introduced into plant cells, for example, plant tissue explants (e.g., leaf, stem, root, or seed segments, tissue explants containing dividing cells or cells capable of regeneration, etc.), or cultured cells derived therefrom (e.g., callus or protoplasts).
  • plant tissue explants e.g., leaf, stem, root, or seed segments, tissue explants containing dividing cells or cells capable of regeneration, etc.
  • cultured cells derived therefrom e.g., callus or protoplasts.
  • callus can be formed from plant tissue explants into which the regenerated plant production promoter of the present invention has been introduced, and plants can be regenerated and grown from the callus, thereby promoting the generation of regenerated plants derived from plant tissue explants.
  • the regenerated plant production promoter of the present invention can be introduced into callus derived from plant tissue explants, and plants can be regenerated and grown from the callus, thereby promoting the generation of regenerated plants derived from plant tissue explants.
  • the regenerated plant production promoter of the present invention can be used in the regenerated plant production promoting method and regenerated plant production method of the present invention described below. Details of how to use the regenerated plant production promoting agent of the present invention are as described in "4. Regenerated plant production promoting method” and “5. Regenerated plant production method.”
  • the plant to which the agent for promoting the production of regenerated plants of the present invention is introduced may be either a herbaceous plant or a woody plant.
  • the plant may also be, for example, an agriculturally important plant, such as a crop plant such as a grain, vegetable, or fruit, or an ornamental plant.
  • the plant may be any of mosses, ferns, gymnosperms, or angiosperms, but is preferably an angiosperm, such as a monocotyledonous or dicotyledonous plant.
  • monocotyledonous plants include plants of the Poaceae family (e.g., rice, wheat, barley, corn, sugarcane, sorghum, sorghum, and turfgrass), plants of the Musaceae family (e.g., bananas), plants of the Amaryllidaceae family (e.g., leeks, onions, garlic, and chives), and plants of the Liliaceae family (e.g., lilies and tulips).
  • dicotyledonous plants examples include Brassicaceae plants (e.g., cabbage, radish, Chinese cabbage, rapeseed), Asteraceae plants (e.g., lettuce, burdock, chrysanthemum), Fabaceae plants (e.g., soybean, peanut, pea, kidney bean, lentil, chickpea, broad bean, licorice), Solanaceae plants (e.g., tomato, eggplant, potato, tobacco, bell pepper, chili pepper, petunia), and Rosaceae plants (e.g., strawberry, apple, pear, peach, loquat, almond, plum, rose, plum).
  • Brassicaceae plants e.g., cabbage, radish, Chinese cabbage, rapeseed
  • Asteraceae plants e.g., lettuce, burdock, chrysanthemum
  • Fabaceae plants e.g., soybean, peanut, pea, kidney bean, lentil, chickpea, broad bean, lic
  • Cucurbitaceae plants e.g., cucumber, gourd, pumpkin, melon, watermelon
  • Anacardiaceae plants e.g., mango, pistachio, cashew nut
  • Lauraceae plants e.g., avocado
  • Rutaceae plants e.g., mandarin orange, grapefruit, lemon, yuzu
  • Convolvulaceae plants e.g., sweet potato
  • Theaceae plants e.g., tea plant
  • Vitaceae plants e.g., grape).
  • the plant is preferably an angiosperm, more preferably a dicotyledon, even more preferably a solanaceae plant (e.g., tomato, eggplant, potato, tobacco, bell pepper, chili pepper, or petunia) or a legume (e.g., soybean, peanut, pea, kidney bean, lentil, chickpea, broad bean, or licorice), and most preferably potato (Solanum tuberosum) and soybean (Glycine max).
  • solanaceae plant e.g., tomato, eggplant, potato, tobacco, bell pepper, chili pepper, or petunia
  • a legume e.g., soybean, peanut, pea, kidney bean, lentil, chickpea, broad bean, or licorice
  • potato Solanum tuberosum
  • soybean Glycine max
  • Potato varieties include, but are not limited to, varieties such as “Sassy,” “Danshakuimo,” “Kitaakari,” “May Queen,” “Haruka,” “Cynthia,” and “Northern Ruby.”
  • Soybean varieties include, but are not limited to, varieties such as “Maple Arrow,” “Fukuyutaka,” “Enrei,” “Tachinagaha,” and “Yukihomare.”
  • the promoter for regenerated plant production of the present invention can promote the generation of regenerated plants from plant tissue fragments, thereby shortening the time required to regenerate plants from plant tissue fragments.
  • the promoter for regenerated plant production of the present invention can promote the generation of regenerated plants from plant tissue fragments, thereby shortening the time required to regenerate plants from plant tissue fragments.
  • it takes 2 to 3 months to form callus from plant tissue fragments, regenerate a plant from the callus, and grow the plant until leaves unfold.
  • the promoter for regenerated plant production of the present invention it takes only 1 to 2 months to form callus from plant tissue fragments, regenerate a plant from the callus, and grow the plant until leaves unfold.
  • composition for Promoting the Production of Regenerated Plants 3-1 provides a composition for promoting the production of regenerated plants derived from plant tissue fragments (hereinafter, sometimes referred to as the "composition for promoting the production of regenerated plants of the present invention"), which comprises the IPT-expressing nucleic acid construct of the present invention.
  • the regenerated plants whose production is promoted by the present invention may preferably be plants whose leaves and stems have been regenerated or redifferentiated.
  • composition for promoting the production of regenerated plants of the present invention contains the IPT-expressing nucleic acid construct of the present invention as an essential active ingredient.
  • the composition for promoting the production of regenerated plants of the present invention may further contain a polynucleotide encoding a genome editing system as an optional component. Each component is described below.
  • the IPT-expressing nucleic acid construct used in the composition for promoting the production of regenerated plants of the present invention may be a promoter for promoting the production of regenerated plants of the present invention.
  • the IPT-expressing nucleic acid construct or the promoter for promoting the production of regenerated plants of the present invention is as described in "1. IPT-Expressing Nucleic Acid Construct" and "2. Promoters for Promoting the Production of Regenerated Plants.”
  • the composition for promoting the production of regenerated plants of the present invention is preferably introduced into plant cells directly or via bacteria such as Agrobacterium so that the IPT-expressing nucleic acid construct of the present invention is in an amount effective for plant cells.
  • the term "genome editing system” is not particularly limited as long as it is an artificial restriction enzyme system that can cause modifications at specific sites in genomic DNA.
  • artificial restriction enzyme systems include the CRISPR/Cas system, the TALEN system, the ZFN system, and the PPR system.
  • the CRISPR/Cas system uses the Cas protein, a nuclease (RGN; RNA-guided nuclease), and guide RNA (gRNA, single-guide RNA (SgRNA)).
  • RGN RNA-guided nuclease
  • gRNA single-guide RNA
  • the guide RNA binds to the target site, and the Cas protein, guided to the binding site, can cleave the DNA (e.g., CRISPR-Cas9 (U.S. Patent No. 8,697,359, WO 2013/176772), CRISPR-Cpf1 (Zetsche B. et al., Cell, 163(3):759-71, (2015)).
  • the ZFN system uses artificial nucleases (ZFNs) that contain a nucleic acid cleavage domain conjugated to a DNA-binding domain containing a zinc finger array (e.g., U.S. Patents Nos. 6,265,196, 8,524,500, and 7,888,121; European Patent No. 1,720,995).
  • ZFNs artificial nucleases
  • a DNA-binding domain containing a zinc finger array e.g., U.S. Patents Nos. 6,265,196, 8,524,500, and 7,888,121; European Patent No. 1,720,995
  • the DNA-binding domain that binds to the target site can be designed according to known schemes.
  • PPR pentatricopeptiderepeat fused with a nuclease domain
  • PPR pentatricopeptiderepeat
  • the CRISPR/Cas system is preferred because it uses nucleic acids (guide RNAs) to recognize target sites, allowing for more flexible selection of target sites and simplifying its preparation.
  • the length of the guide RNA used in the CRISPR/Cas system is at least 15, 16, 17, 18, 19, or 20 bases.
  • the preferred upper limit of the base length is 30 or less, more preferably 25 or less, even more preferably 22 or less, and most preferably 20 or less.
  • the CRISPR/Cas system may use two or more guide RNAs (e.g., 2, 3, 4, or 5).
  • the Cas protein may contain one or more nuclear localization signals (NLS).
  • the Cas protein is preferably a type II CRISPR enzyme, such as the Cas9 protein.
  • the Cas9 protein may be Streptococcus pneumoniae, Streptococcus pyogenes Cas9, Streptococcus thermophilus Cas9, or a mutant thereof.
  • the Cas9 protein may be, for example, a protein consisting of the amino acid sequence shown in SEQ ID NO: 3.
  • the Cas9 protein may also be a protein consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been added, deleted, and/or substituted in the amino acid sequence shown in SEQ ID NO: 3, and which binds to DNA in a guide RNA-dependent manner.
  • the Cas9 protein may also be a protein consisting of an amino acid sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 3, and which binds to DNA in a guide RNA-dependent manner.
  • genes that can be targeted by genome editing there are no particular restrictions on the genes that can be targeted by genome editing, as long as the gene is one for which disruption, insertion, or replacement is desired.
  • the polynucleotide encoding the genome editing system may be contained in the IPT-expressing nucleic acid construct of the present invention.
  • the composition for promoting the production of regenerated plants of the present invention may contain the IPT-expressing nucleic acid construct of the present invention, which includes a polynucleotide encoding the genome editing system (hereinafter, sometimes referred to as the "IPT and genome editing system-expressing nucleic acid construct").
  • the polynucleotide encoding the genome editing system may be contained in a nucleic acid construct different from the IPT-expressing nucleic acid construct of the present invention.
  • the composition for promoting the production of regenerated plants of the present invention may contain, in addition to the IPT-expressing nucleic acid construct of the present invention, a nucleic acid construct containing a polynucleotide encoding the genome editing system (hereinafter sometimes referred to as a "genome editing system-expressing nucleic acid construct").
  • the IPT and genome editing system expression nucleic acid construct, and the genome editing system expression nucleic acid construct may further include expression control regions (promoter, enhancer, insulator, terminator, poly A addition signal, etc.) for the nuclease and/or guide RNA that constitute the genome editing system.
  • expression control regions promoter, enhancer, insulator, terminator, poly A addition signal, etc.
  • the promoters and terminators described in "1. IPT Expression Nucleic Acid Construct" can be used as the promoters and terminators for the nuclease and/or guide RNA.
  • composition for promoting the production of regenerated plants of the present invention may be in the form of a mixture in which two or more components (e.g., an IPT-expressing nucleic acid construct and a genome editing system-expressing nucleic acid construct) are present together, or in the form of a kit in which two or more components (e.g., an IPT-expressing nucleic acid construct and a genome editing system-expressing nucleic acid construct) are present separately.
  • two or more components e.g., an IPT-expressing nucleic acid construct and a genome editing system-expressing nucleic acid construct
  • composition for promoting the production of regenerated plants of the present invention may further contain a solvent such as water, a surfactant, a buffer, a pH adjuster, a solubilizing agent, a preservative, or the like.
  • a solvent such as water, a surfactant, a buffer, a pH adjuster, a solubilizing agent, a preservative, or the like.
  • composition for promoting the production of regenerated plants of the present invention can be introduced into plant cells, such as plant tissue explants (e.g., leaf, stem, root, or seed segments, tissue explants containing dividing cells or cells capable of regeneration, etc.), or cultured cells derived therefrom (e.g., callus or protoplasts).
  • plant tissue explants e.g., leaf, stem, root, or seed segments, tissue explants containing dividing cells or cells capable of regeneration, etc.
  • cultured cells derived therefrom e.g., callus or protoplasts
  • composition for promoting the production of regenerated plants of the present invention can be used in the method for promoting the production of regenerated plants and the method for producing regenerated plants of the present invention described below. Methods for using the composition for promoting the production of regenerated plants of the present invention are as described in "4. Method for promoting the production of regenerated plants” and “5. Method for producing regenerated plants.”
  • Method for promoting the production of regenerated plants of the present invention essentially comprises a construct introduction step and a regeneration step.
  • the plant cells may be plant tissue fragments (e.g., leaf, stem, root, or seed segments, tissue fragments containing dividing cells or cells capable of regeneration, etc.), or cultured cells derived therefrom (callus or protoplasts).
  • the plant cells may also be callus obtained in the callus formation step.
  • the regeneration process may involve culturing plant cells in a medium containing plant hormones, particularly auxin and/or cytokinin.
  • the medium used in the regeneration process may further contain gibberellin, a plant hormone that promotes elongation growth.
  • gibberellin a plant hormone that promotes elongation growth.
  • the type and concentration of plant hormones added to the medium can be adjusted appropriately depending on the type of plant and the type of tissue being cultured.
  • the medium used in the regeneration process may also further contain a selective agent.
  • the callus formation step is a step of culturing plant cells to form callus.
  • the plant cells may be plant tissue fragments (e.g., sections of leaves, stems, roots, or seeds, tissue fragments containing dividing cells or cells capable of regeneration, etc.), or cultured cells derived therefrom (e.g., protoplasts).
  • the plant cells may also be plant cells (e.g., plant tissue fragments or cultured cells derived therefrom) into which the composition for improving genome editing efficiency of the present invention has been introduced in the construct introduction step.
  • the callus formation process may involve culturing plant cells in a medium containing plant hormones, particularly auxins and cytokinins.
  • the amounts of auxin and cytokinin in the medium may be adjusted appropriately depending on the type of plant and the type of tissue to be cultured.
  • the auxin concentration may be 1 mg/L to 10 mg/L, 1 mg/L to 5 mg/L, or 1 mg/L to 3 mg/L
  • the cytokinin concentration may be 0.1 mg/L to 5 mg/L, 0.1 mg/L to 1 mg/L, or 0.3 mg/L to 0.7 mg/L.
  • the medium used in the callus formation step may also further contain a selective agent.
  • Culture conditions in the callus formation step may be static or shaking culture in the presence or absence of light, at 15 to 35°C, for example 25°C.
  • callus can be formed in either a medium containing or not containing plant hormones, but it is preferable to form callus in a medium containing plant hormones. By forming callus in a medium containing plant hormones, more callus can be obtained than when forming callus in a medium not containing plant hormones.
  • the regeneration step is a step of culturing the callus to induce regeneration and regenerate a plant body.
  • the callus may be the callus obtained in the callus formation step, or it may be callus into which the genome editing efficiency improving composition of the present invention has been introduced in the construct introduction step.
  • regeneration can be induced in either a medium containing or not containing plant hormones, but regeneration in a medium containing plant hormones is preferable.
  • regeneration in a medium containing plant hormones By regenerating in a medium containing plant hormones, more regenerated individuals can be obtained than when regenerating in a medium not containing plant hormones.
  • the shoot induction step is a step of culturing plant cells to induce shoot formation.
  • the plant cells may be plant tissue explants (e.g., leaf, stem, root, or seed segments, tissue explants containing dividing cells or cells capable of regeneration, etc.), or cultured cells derived therefrom (e.g., protoplasts).
  • the plant cells may be plant cells (e.g., plant tissue explants or cultured cells derived therefrom) into which the composition for improving genome editing efficiency of the present invention has been introduced in the construct introduction step.
  • the shoot induction step may involve culturing plant tissue explants in a medium containing a plant hormone, particularly cytokinin.
  • the amount of cytokinin in the medium may be adjusted appropriately depending on the type of plant and the type of tissue being cultured, but may be, for example, 0.1 mg/L to 10 mg/L, 0.5 mg/L to 5 mg/L, or 1 mg/L to 3 mg/L.
  • the medium used in the shoot induction step may further contain gibberellin, for example, in an amount of 0.1 mg/L to 0.9 mg/L, 0.1 mg/L to 0.7 mg/L, or 0.1 mg/L to 0.5 mg/L.
  • the medium used in the shoot induction step may also further contain a selective agent.
  • the culture in the shoot induction step can be carried out in the presence of light at 15 to 35°C, for example, 25°C.
  • the shoot elongation step is a step of elongating the shoot formed in the shoot induction step.
  • the shoot elongation step may include culturing the shoot in a medium containing a plant hormone, particularly cytokinin.
  • the amount of cytokinin in the medium may be appropriately adjusted depending on the type of plant and the type of tissue to be cultured, and may be, for example, 0.1 mg/L to 0.9 mg/L or 0.3 mg/L to 0.7 mg/L.
  • the medium used in the shoot elongation step may further contain gibberellin in an amount of, for example, 0.1 mg/L to 10 mg/L, 0.5 mg/L to 5 mg/L, or 1 mg/L to 3 mg/L.
  • the medium used in the shoot elongation step may also further contain a selective drug.
  • the culture in the shoot elongation step may be carried out in the presence of light at 15 to 35°C, for example, 25°C.
  • the present invention provides a method for producing a genome-edited plant (hereinafter, sometimes referred to as the "genome-edited plant production method of the present invention").
  • the genome-edited plant production method of the present invention includes a construct introduction step as an essential step.
  • the genome-edited plant production method of the present invention may include, as optional steps, a callus formation step and a regeneration step.
  • the genome-edited plant production method of the present invention may include, for example, (i) a construct introduction step, and (ii) a regeneration step consisting of a callus formation step and a regeneration step, in the order described.
  • the genome-edited plant production method of the present invention may also include, for example, a regeneration step consisting of (i) a callus formation step, (ii) a construct introduction step, and (iii) a regeneration step, in the order described.
  • the genome-edited plant production method of the present invention may also include, as optional steps, a shoot induction step and a shoot elongation step.
  • the genome-edited plant production method of the present invention may include, for example, (i) a construct introduction step, and (ii) a regeneration step consisting of a shoot induction step and a shoot elongation step, in the order described.
  • the genome-edited plant production method of the present invention may include a selection step as an optional step.
  • construct introduction process, regeneration process, callus formation process, redifferentiation process, shoot induction process, and shoot elongation process are as described for the method for improving genome editing efficiency of the present invention.
  • the “selection process” is a process in which genomic DNA is extracted from the regenerated plants obtained in the regeneration process, and genome-edited plants into which the desired modification has been introduced are selected.
  • “Desired modification” refers to the modification that the genome editing system introduced into the plant cells in the construct introduction process is intended to introduce into the genomic DNA. Whether the desired modification has been introduced into the genomic DNA can be evaluated by any known means. For example, using the extracted genomic DNA as a template, a nucleic acid amplification reaction is performed on the region of the genomic DNA containing the desired modification. The presence or absence of the desired modification can be determined by sequencing the base sequence of the obtained amplified product, or by treating the amplified product with a restriction enzyme, or by other methods.
  • regenerated plants with morphological abnormalities may be excluded from the targets for evaluation of the presence or absence of the desired modification.
  • Regenerated plants with morphological abnormalities are likely to have the IPT gene introduced into their genomic DNA.
  • ⁇ Production Example 1 Construction of IPT non-expressing genome editing vector> A pUC19 vector containing an expression cassette (PPcUbi-Cas9-Pea3A) linking the parsley ubiquitin promoter (PPcUbi) (SEQ ID NO: 17), the Streptococcus pyogenes-derived Cas9 gene (SEQ ID NO: 4), and the pea (Pisum sativum; P. sativum)-derived Pea3A terminator (SEQ ID NO: 20) was prepared by chemical synthesis at GenScrIPT.
  • PPcUbi-Cas9-Pea3A an expression cassette linking the parsley ubiquitin promoter (PPcUbi) (SEQ ID NO: 17), the Streptococcus pyogenes-derived Cas9 gene (SEQ ID NO: 4), and the pea (Pisum sativum; P. sativum)-derived Pea3A terminator (SEQ ID
  • This pUC19 vector and the pLC41 vector were treated with restriction enzymes BamHI-HF (New England Biolad), and then ligated using a Ligation Long kit (Takara). This resulted in a vector (pLC41-PPcUbi-Cas9 vector) in which PPcUbi-Cas9-Pea3A was inserted on the T-DNA between the left border (LB) and right border (RB) of the pLC41 vector.
  • This pZD-dxCas9 plasmid was digested with restriction enzymes PmeI and SbfI-HF (New England Biolad) to excise the gRNA expression cassette (SEQ ID NO: 6).
  • This gRNA expression cassette was ligated to the pLC41-PPcUbi-Cas9 vector, which had been opened by restriction enzyme digestion with PmeI and SbfI-HF, using a Ligation Long kit (Takara) to obtain the pLC41-gRNA-PPcUbi-Cas9 vector (hereinafter sometimes referred to as the "IPT non-expressing genome editing vector").
  • the structure of the T-DNA (SEQ ID NO: 21) of this vector is shown in Figure 1A.
  • the PPcUbi, Cas9, and Pea3A sequences were designed with reference to the pZH-FFCas9 vector described in Mikami et al., 2015, Plant Mol Biol, 88(6):561-572.
  • PNative-IPT expression genome editing vector Agrobacterium tumefaciens (Agrobacterium tumefaciens) derived IPT gene native promoter (PNative) (SEQ ID NO: 16), Agrobacterium tumefaciens derived IPT gene (SEQ ID NO: 2), Pea3A terminator (SEQ ID NO: 20) was linked to the expression cassette (PNative-IPT-Pea3A) pUC19 containing was prepared by chemical synthesis by GenScrIPT Co.
  • PNative is derived from the promoter sequence upstream of the IPT gene sequence (NCBI accession numbers: X14410 X17432) present in the DNA of the Agrobacterium tumefaciens A281 strain (patent number 3905607), which is registered with the DDBJ.
  • the sequence of PNative-IPT-Pea3A is a 1,408 bp sequence amplified by PCR using the forward primer: CCCGTTACAAGTATTGCACGTTTTGT (SEQ ID NO: 28) and the reverse primer: CTAATACATTCCGAACGGAT (SEQ ID NO: 29), with the Pea3A sequence linked downstream of the PNative-IPT sequence.
  • PPcUbi-IPT expression genome editing vector Construction of PPcUbi-IPT expression genome editing vector> PPcUbi (SEQ ID NO: 17), IPT gene (SEQ ID NO: 2), Pea3A (SEQ ID NO: 20) were amplified by PCR and introduced into the pLC41-gRNA-PPcUbi-Cas9 vector in the form of PPcUbi-IPT-Pea3A, which was treated with the restriction enzyme SpeI-HF using NEBuilder (New England Biolad).
  • pLC41-gRNA-PPcUbi-Cas9-PPcUbi-IPT vector hereinafter sometimes referred to as "PPcUbi-IPT expression genome editing vector").
  • the structure of the T-DNA (SEQ ID NO: 23) of this vector is shown in Figure 1B.
  • P35S-IPT expression genome editing vector The cauliflower mosaic virus-derived 35S promoter (P35S) (SEQ ID NO: 18), IPT gene (SEQ ID NO: 2), and Pea3A (SEQ ID NO: 20) were amplified by PCR and introduced into the pLC41-gRNA-PPcUbi-Cas9 vector in the form of P35S-IPT-Pea3A, which had been treated with the restriction enzyme SpeI-HF using NEBuilder, to obtain the pLC41-gRNA-PPcUbi-Cas9-P35S-IPT vector (hereinafter sometimes referred to as the "P35S-IPT expression genome editing vector").
  • the structure of the T-DNA (SEQ ID NO: 24) of this vector is shown in Figure 1B.
  • P35S was amplified (845 bp) using the forward primer: GCCTCGAGTCTAGAGATTAGCCT (SEQ ID NO: 32) and the reverse primer: TAGAGTCCCCCCGTGTTCTCT (SEQ ID NO: 33).
  • P2x35S-IPT expression genome editing vector Construction of P2x35S-IPT expression genome editing vector>
  • the 2x35S promoter (P2x35S) (SEQ ID NO: 19), a modified promoter with enhanced expression of P35S, the IPT gene (SEQ ID NO: 2), and Pea3A (SEQ ID NO: 20) were amplified by PCR and introduced into the pLC41-gRNA-PPcUbi-Cas9 vector in the form of P2x35S-IPT-Pea3A, which was treated with the restriction enzyme SpeI-HF using NEBuilder, to obtain the pLC41-gRNA-PPcUbi-Cas9-P2x35S-IPT vector (hereinafter sometimes referred to as the "P2x35S-IPT expression genome editing vector").
  • the structure of the T-DNA (SEQ ID NO: 25) of this vector is shown in Figure 1B.
  • P2x35S was amplified (759 bp) using pZD-dxCas9 (Kusano et al., 2018, Scientific Reports, 8, 13753) as a template with the forward primer: TTGCCAACATGGTGGAGCAC (SEQ ID NO: 34) and the reverse primer: ACTAGTCCGGCCTCTCCAAA (SEQ ID NO: 35).
  • Example 1 Effect of IPT on promoting the production of regenerated plants (1) To verify the effect of IPT in tissue culture, the following experiment was carried out.
  • Potatoes were transformed using Agrobacterium carrying the non-IPT expression genome-editing vector prepared in Production Example 1 or the PPcUbi-IPT expression genome-editing vector prepared in Production Example 3.
  • Potato transformation was carried out according to the Agrobacterium method described by Craze et al., Current Protocols in Plant Biology, 2018, Volume 3, Issue 1, pp. 33-41, with some modifications.
  • leaf pieces (area 0.3-0.5 cm 2 ) were aseptically excised from leaves of cultured potato (Solanum tuberosum) seedlings cultured from shoot tips in a culture room (25°C, 16 hours light/8 hours dark).
  • the Agrobacterium tumefaciens EHA105 strain into which the non-IPT-expressing genome-editing vector prepared in Production Example 1 or the PPcUbi-IPT-expressing genome-editing vector prepared in Production Example 3 had been introduced by electroporation, was spread on YP medium (Yeast Extract 5 g/L, Bacto Peptone 10 g/L, NaCl 5 g/L) containing 50 mg/L kanamycin and cultured at 28°C for 1 day. After that, the strain was transferred to liquid medium PCM (Craze et al., Current Protocols in Plant Biology, 2018, Volume 3, Issue 1, pp.
  • YP medium Yeast Extract 5 g/L, Bacto Peptone 10 g/L, NaCl 5 g/L
  • the PCM medium contains 2,4-dichlorophenoxyacetic acid (2,4-D) (synthetic auxin) 2 mg/L, zeatin (cytokinin) 0.5 mg/L, MS medium containing vitamins (Duchefa Biochemie) 4.4 g/L, MES 500 mg/L, sucrose 20 g/L, and agar 5 g/L, and is adjusted to pH 5.7 with NaOH.
  • 2,4-D 2,4-dichlorophenoxyacetic acid
  • zeatin cytokinin
  • MES 500 mg/L sucrose 20 g/L
  • agar 5 g/L agar 5 g/L
  • the leaf pieces were immersed in the Agrobacterium suspension and left to stand for 10 minutes. The leaf pieces were then transferred onto filter paper to remove excess suspension, and the leaf pieces were then placed with their surface facing upwards on co-cultivation medium PCM (Craze et al., Current Protocols in Plant Biology, 2018, Volume 3, Issue 1, pp. 33-41) containing 140 ⁇ M acetosyringone, and cultured at 25°C under illumination for 3 days.
  • PCM co-cultivation medium
  • PSM medium contains 0.5 mg/L zeatin (cytokinin), 2 mg/L gibberellic acid, 4.4 g/L MS medium containing vitamins (Duchefa Biochemie), 500 mg/L MES, 20 g/L sucrose, and 5 g/L agar, and is adjusted to pH 5.7 with NaOH.
  • the cultures were observed under a microscope and the number of leaf segments that were confirmed to have redifferentiated into plants (number of redifferentiated leaf segments) was counted, and the ratio of the number of redifferentiated leaf segments to the number of inoculated leaf segments was calculated as an index of regeneration efficiency.
  • the number of leaf segments that had grown until the redifferentiated individuals formed morphologically normal leaves (excluding multiple shoots) was counted as the number of shoot-forming leaf segments, and the ratio of the number of shoot-forming leaf segments to the number of inoculated leaf segments was calculated.
  • the ratio of the number of shoot-forming leaf segments to the number of inoculated leaf segments can be used as an index of the rate at which redifferentiated individuals are generated.
  • Figure 2 shows an example of cultured tissue four weeks after Agrobacterium inoculation.
  • Table 1 also shows the number of regenerated leaf segments, the number of leaf segments that formed shoots, and their ratio to the number of inoculated leaf segments.
  • the ratio of the number of regenerated leaf disks to the number of inoculated leaf disks was approximately 8% higher than in the test area where the non-IPT expression genome-editing vector was introduced. This indicates that regeneration is induced by expressing IPT.
  • the ratio of the number of leaf discs that formed shoots to the number of inoculated leaf discs was approximately eight times higher than in the test area where the non-IPT expression genome-editing vector was introduced. This indicates that expressing IPT can promote the generation of regenerated individuals (regenerated plants).
  • a vector containing T-DNA that includes a polynucleotide encoding a genome editing system is introduced into a plant, the following will occur: individuals in which the desired mutation has been introduced and the T-DNA has been integrated into the genome; individuals in which the desired mutation has been introduced and the T-DNA has not been integrated into the genome; individuals in which the desired mutation has not been introduced and the T-DNA has been integrated into the genome; and individuals in which the desired mutation has not been introduced and the T-DNA has not been integrated into the genome.
  • Plants with the IPT gene integrated into their genome typically exhibit morphological abnormalities such as multiple shoots. Therefore, in test plots where the PPcUbi-IPT expression genome editing vector (a vector containing a genome editing system and T-DNA containing a polynucleotide encoding IPT) was introduced, it is highly likely that individuals that formed normal leaves did not have T-DNA integrated into their genome. Therefore, it can be said that the above-mentioned effect of IPT in promoting the production of regenerated individuals also occurs in individuals that do not have the IPT gene integrated into their genome.
  • the PPcUbi-IPT expression genome editing vector a vector containing a genome editing system and T-DNA containing a polynucleotide encoding IPT
  • Example 2 Effect of IPT on promoting the production of regenerated plants (2) The following experiment was carried out to compare the promoting effect of IPT expression under different promoters on the production of regenerated plants.
  • Agrobacterium carrying the non-IPT expression genome editing vector, PNative-IPT expression genome editing vector, PPcUbi-IPT expression genome editing vector, P35S-IPT expression genome editing vector, or P2x35S-IPT expression genome editing vector prepared in Production Examples 1 to 5 was inoculated into potato leaf pieces, and the leaf pieces were cultured on coculture medium PCM for 3 days, then on callus formation medium PCM for 3 days, and then on regeneration medium PSM.
  • the number of leaf segments in which regeneration was confirmed was counted, and the ratio of the number of redifferentiated leaf segments to the number of inoculated leaf segments was calculated as an index of regeneration efficiency.
  • the number of leaf segments that had grown until the redifferentiated individuals had formed morphologically normal leaves (excluding multiple shoots) was counted as the number of shoot-forming leaf segments, and the ratio of the number of shoot-forming leaf segments to the number of inoculated leaf segments was calculated as an index of the growth rate of the redifferentiated individuals.
  • the ratio of the number of leaf segments that formed shoots to the number of inoculated leaf segments was 2.2%
  • the ratios of the number of leaf segments that formed shoots to the number of inoculated leaf segments were 46.2%, 57.7%, 70.8%, and 55.1%, respectively.
  • IPT can promote the generation of regenerated individuals (regenerated plants), and that the growth of regenerated individuals can be significantly promoted when IPT is expressed using high-expression promoters such as PPcUbi, P35S, and P2x35S.
  • Example 3 Effect of IPT on promoting the production of regenerated plants (3) An experiment similar to that of Example 2 was carried out, except that transformation was carried out using a potato variety, "Sassy,” different from that used in Example 2. The results are shown in Table 3.
  • the ratio of the number of regenerated leaf pieces to the number of inoculated leaf pieces was 32.0%
  • the ratio of the number of regenerated leaf pieces to the number of inoculated leaf pieces was 64.0%, 80.0%, 72.0%, and 76.0%, respectively. Therefore, the regeneration-inducing effect of the IPT gene was confirmed in the potato cultivar "Sassy.”
  • the ratio of the number of leaf segments that formed shoots to the number of inoculated leaf segments was 16.0%
  • the ratios of the number of leaf segments that formed shoots to the number of inoculated leaf segments were 48.0%, 56.0%, 48.0%, and 52.0%, respectively. Therefore, the effect of the IPT gene in promoting the production of regenerated plants was confirmed in the potato cultivar "Sassy.”
  • Example 4 Effect of IPT on promoting the production of regenerated plants (4) The following experiment was carried out to confirm whether the promotion of regenerated plant production by IPT observed in the potato tissue culture method in Examples 1 to 3 can also be observed in the soybean tissue culture method.
  • GUS-bar expression vector A vector (hereinafter referred to as "GUS-bar expression vector") having a GUS gene expression cassette and a bar gene expression cassette on the T-DNA of binary vector pLC41 (Genbank accession No. LC215698) was prepared.
  • the GUS gene expression cassette contained in the GUS-bar expression vector contains a cauliflower mosaic virus-derived 35S promoter (P35S) (SEQ ID NO: 38), a GUS gene mediated by a catalase intron from castor bean (Ricinus communis) (SEQ ID NO: 39) (Ohta et al., 1990, Plant Cell Physiol., 31(6); 805-813), and a Nos terminator (Tnos) (SEQ ID NO: 40).
  • the bar gene expression cassette contained in the GUS-bar expression vector contains a cauliflower mosaic virus-derived 35S promoter (P35S) (SEQ ID NO: 41), a bar gene (SEQ ID NO: 42), and a 35S terminator (T35S) (SEQ ID NO: 43).
  • the bar gene is a drug resistance gene that confers resistance to phosphinothricin to plants.
  • bar-IPT expression vector A vector (hereinafter referred to as "bar-IPT expression vector") having a bar gene expression cassette and an IPT gene expression cassette on the T-DNA of binary vector pLC41 (Genbank accession No. LC215698) was prepared.
  • the bar gene expression cassette contained in the bar-IPT expression vector is identical to the bar gene expression cassette contained in the GUS-bar expression vector.
  • the IPT gene expression cassette contained in the bar-IPT expression vector contains a maize ubiquitin promoter (PZmUbi) (SEQ ID NO: 44), an IPT gene (SEQ ID NO: 2), and a Nos terminator (Tnos) (SEQ ID NO: 45).
  • Agrobacterium tumefaciens EHA105 strains carrying the GUS-bar expression vector or bar-IPT expression vector were precultured on YP agar medium (Ishida et al., 2007, Nature Protocols, 2:1614-1621) for one day. The cultured Agrobacterium was then suspended in Agrobacterium suspension medium (Paz et al., 2006, Plant Cell Reports, 25(3):206-213) and used as the inoculum source.
  • the cotyledons were immersed in the inoculum and stored at room temperature for 15 minutes.
  • the Agrobacterium-inoculated cotyledons were then placed on a coculture medium (Paz et al., 2006, Plant Cell Reports, 25(3):206-213) (1/10 B5 salts, B5 vitamins, 7.5 ⁇ M 6-benzylaminopurine (BAP), 0.7 ⁇ M gibberellic acid ( GA ), 20 mM MES, 3% sucrose, 200 ⁇ M acetosyringone, 100-400 mg/L cysteine, 154 mg/L dithiothreitol, pH 5.4) and cultured at 25°C for 3 days under illumination.
  • the cotyledons were then placed on SI medium (shoot induction medium) (Paz et al., 2006, Plant Cell Reports, 25(3):206-213) (B5 salts, B5 vitamins, MSIII iron stock, 3% sucrose, 3 mM MES, 5.0 ⁇ M BAP, 50 mg/L timentin, 200 mg/L cefotaxime, 50 mg/L vancomycin, pH 5.7) so that the cotyledonary nodes were buried in the medium, and cultured at 25°C under illumination for 2 weeks.
  • the grown stems and leaves were removed, and the cotyledons were placed on SI medium containing 5 mg/L phosphinothricin and cultured at 25°C under illumination for 2 weeks.
  • Table 4 shows the number of cotyledonary nodes inoculated with Agrobacterium, the number of cotyledonary nodes that formed shoots after two weeks of culture on shoot elongation medium, and the total number of shoots formed.
  • Agrobacterium carrying either the non-IPT expression genome-editing vector prepared in Production Example 1 or the PPcUbi-IPT expression genome-editing vector prepared in Production Example 3 was inoculated onto potato leaf pieces, which were then cultured on coculture medium PCM for three days, then on callus formation medium PCM for three days, and then on regeneration medium PSM.
  • PRM rooting medium
  • DNA extraction was performed as follows: 300 ⁇ l of DNA extraction buffer (20 mM Tris-HCl (pH 8.0), 2.5 mM EDTA (pH 8.0), 25 mM Back, 0.05% SDS), 2-5 mm2 leaf segments of the plant to be analyzed, and two 5 mm stainless steel beads were added to each well of a 96-well plate, and the mixture was crushed at 1000 rpm for 1 minute and centrifuged at 3000 rpm for 10 minutes. 50 ⁇ l of the supernatant was then saved as the DNA extract.
  • DNA extraction buffer 20 mM Tris-HCl (pH 8.0), 2.5 mM EDTA (pH 8.0), 25 mM Back, 0.05% SDS
  • 2-5 mm2 leaf segments of the plant to be analyzed 2-5 mm2 leaf segments of the plant to be analyzed
  • two 5 mm stainless steel beads were added to each well of a 96-well plate, and the mixture was crushed at 1000 rpm for 1 minute and centrifuged at
  • CLEAVED AMPLIFIED POLYMORPHIC SEQUENCE (CAPS) analysis was used to evaluate whether mutations in the target genomic sequence due to genome editing had occurred in the regenerated individuals.
  • the target genomic sequence within the GBSS1 gene was amplified by PCR using primers GBSS1-Fw: CACTTTGTGTCAAGAAGCCAAAC (SEQ ID NO: 36) and GBSS1-Rv: TTTGACCTGCAGATAAAGTAGCG (SEQ ID NO: 37).
  • the PCR reaction mixture was prepared by adding sterile distilled water to a total volume of 10 ⁇ l, containing 0.8 ⁇ l of DNA extract, 2.5 pmol of each primer, 0.2 U of KOD FX NEO, 5 ⁇ l of 2x PCR Buffer for KOD FX Neo, and 2 ⁇ l of 2 mM dNTPs.
  • the PCR reaction was performed at 94°C for 2 minutes, followed by 35 cycles of 98°C for 15 seconds and 68°C for 1 minute.
  • the IPT gene was integrated into approximately 20% of the analyzed individuals.
  • the PPcUbi-IPT expression genome editing vector in the experimental group where the PPcUbi-IPT expression genome editing vector was introduced, there were 4 edited individuals in which the IPT gene was not integrated into their genome, accounting for 4.9% (4/102) of the analyzed individuals.
  • the PPcUbi-IPT expression genome editing vector in the experimental group where the PPcUbi-IPT expression genome editing vector was introduced, there were 11 edited individuals in which the IPT gene was not integrated into their genome, accounting for 4.0% (11/274) of the analyzed individuals. Therefore, improved genome editing efficiency was observed even in individuals in which IPT was not integrated into their genome.
  • Example 7 Genome editing efficiency improvement effect of IPT (2)> An experiment similar to that of Example 6 was carried out, except that a different potato variety, "Sassy,” was used. The results are shown in Table 8.

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Abstract

La présente invention concerne un moyen permettant de raccourcir le temps nécessaire à la régénération d'un corps végétal à partir d'un fragment de tissu végétal, et un moyen permettant d'améliorer l'efficacité de l'édition génomique pour une plante.　La présente invention concerne un agent favorisant la production d'un corps végétal régénéré issu d'un fragment de tissu végétal, l'agent favorisant comprenant une construction d'acide nucléique comprenant un polynucléotide codant pour l'isopentényltransférase (IPT) et un promoteur lié de manière fonctionnelle au polynucléotide. La présente invention concerne également un agent permettant d'améliorer l'efficacité de l'édition génomique chez les plantes, ledit agent comportant une construction d'acide nucléique comprenant un polynucléotide codant pour l'isopentényltransférase (IPT) et un promoteur lié de manière fonctionnelle au polynucléotide.
PCT/JP2025/012676 2024-03-29 2025-03-28 Construction d'acide nucléique exprimant l'ipt Pending WO2025206265A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019103034A1 (fr) * 2017-11-27 2019-05-31 国立研究開発法人理化学研究所 Procédé de production de plante à édition génique
WO2021022043A2 (fr) * 2019-07-30 2021-02-04 Pairwise Plants Services, Inc. Régulateurs morphogéniques et leurs procédés d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019103034A1 (fr) * 2017-11-27 2019-05-31 国立研究開発法人理化学研究所 Procédé de production de plante à édition génique
WO2021022043A2 (fr) * 2019-07-30 2021-02-04 Pairwise Plants Services, Inc. Régulateurs morphogéniques et leurs procédés d'utilisation

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