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WO2025129109A1 - Compositions et procédés pour réduire l'édulcoration induite par le froid dans la pomme de terre - Google Patents

Compositions et procédés pour réduire l'édulcoration induite par le froid dans la pomme de terre Download PDF

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WO2025129109A1
WO2025129109A1 PCT/US2024/060189 US2024060189W WO2025129109A1 WO 2025129109 A1 WO2025129109 A1 WO 2025129109A1 US 2024060189 W US2024060189 W US 2024060189W WO 2025129109 A1 WO2025129109 A1 WO 2025129109A1
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potato plant
potato
tuber
mutation
vinvin2en
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Jiming Jiang
Xiaobiao ZHU
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Michigan State University MSU
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Michigan State University MSU
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    • 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
    • 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
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1225—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold or salt resistance
    • 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
    • A01H5/04—Stems
    • 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
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • A01H6/827—Solanum tuberosum [potato]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • Reducing sugars are the primary determinant for the acrylamide content in fried potato products (Amrein et al., 2003; Becalski et al., 2004; Zhu et al., 2016).
  • developing methods to minimize reducing sugars in cold-stored tubers has been an important research focus to reduce acrylamide in fried potato products.
  • Described herein are potato (Solanum tuberosum) plants, potato plant cells, potato tubers or a portion of tubers, and potato plant seeds comprising at least one mutation in a 200-bp transcription enhancer, VInvIn2En, in the nucleic acid sequence of SEQ ID NO: 1 that modulates expression of a potato vacuolar invertase gene (VInv). Also provided are potato plants comprising the aforementioned potato plant cells. Cold storage temperatures of potatoes induce high transcription levels of the VInv gene, which causes the accumulation of reducing sugars and consequent problematic cold-induced sweetening of the potatoes.
  • the mutations in one or more DNA motifs of VInvIn2En abolished the function of VInvIn2En as a transcriptional enhancer, thereby reducing VInv transcription during cold storage temperature.
  • biological samples comprising a nucleic acid containing at least one mutation in the VInvIn2En enhancer of SEQ ID NO: 1 or an allelic variant thereof.
  • Polynucleotides comprising the at least one mutation in the VInvIn2En enhancer of SEQ ID NO: 1 are provided. In some embodiments, the aforementioned polynucleotides are isolated.
  • Guide RNA molecules comprising a spacer RNA molecule which target the VInvIn2En enhancer of SEQ ID NO: 1 or an allelic variant thereof are provided.
  • Guide RNA molecules comprising a spacer RNA encoded by SEQ ID NO: 37-46 are also provided.
  • Gene editing systems comprising a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to the VInvIn2En enhancer of SEQ ID NO: 1 or allelic variant thereof are provided.
  • Expression cassettes comprising a polynucleotide encoding CRISPR-Cas effector protein comprising a cleavage domain and a guide RNA molecule of the disclosure are also provided.
  • MSU TEC2024-0055 PCT // MVS: P15076WO01
  • Methods of cultivating such potato plant seeds, potato tubers or a portion of tubers, and potato plants are also described herein that include, for example, harvesting the potato plants, potato tubers, and potato seeds.
  • Methods for determining whether a potato plant cell, potato tuber or a portion of tuber, or potato plant comprises at least one mutation in the VInvIn2En enhancer of SEQ ID NO: 1 or an allelic variant thereof are provided.
  • Figs.1A-1B Discovery of a cold-responsive intronic enhancer in VInv gene.
  • Fig.1A is a map of DNaseI hypersensitive sites (DHSs) associated with VInv gene developed from tuber tissue of DM1-3 potato. Two DHSs (bars 1 and 2), one at the 5′ of the gene and one in the second intron, were detected.
  • Fig. 1B shows images of Katahdin tetraploid potato tubers expressing a GUS reporter gene in the second intron of VInv gene. Constructs using a mini35S and a full-length 35S promoters were used as negative and positive controls.
  • FIGs.2A-2F Identification of transcriptional enhancers in intron 2 of VInv gene.
  • Fig. 2A shows a diagram illustrating the sizes and positions of 10 sub-fragments derived from intron 2 of the VInv gene. The 1327-bp intron was divided into ten fragments (#1 to #10) using five breaks (b1 to b5).
  • Fig.2B is a diagram illustrating the sizes and positions of the 13 fragments derived from the DNA fragment #11.
  • FIG. 2C shows images of A. thaliana plants expressing a GUS reporter gene in the intron 2 in A. thaliana. Constructs using a mini35S and a full-length 35S promoters were used as negative and positive controls.
  • Fig. 2D shows images of GUS expression patterns in representative A. thaliana transgenic seedlings derived from each of the ten constructs consisting of a fragment ligated with the mini35S promoter and the GUS reporter gene.
  • Fig.2E shows images of GUS staining of 20 A. thaliana transgenic seedlings derived from constructs #11, #17, #19, and #21, respectively.
  • Fig.2F shows images of GUS reporter gene assays of the 200 bp VInvIn2En enhancer in “Katahdin” potato tubers. Tubers from three independent transgenic lines showed minimal GUS signals under 22°C but strong signals from tubers after 4 weeks of cold storage under 4°C. All numbers above bars/lines in Figs. 2A and 2B indicate base pairs. The scale bar represents 2 mm in Figs. 2C and 2D and 1 cm in Figs. 2E and 2F. Fragment numbers with arrows in Figs.
  • FIGS.3A-3B Distribution and function of DNA motifs in intron 2 and the VInvIn2En enhancer.
  • Fig. 3A provides maps showing the distribution of DNA motifs related to transcription factors (TFs) involved in response to cold stress. Each vertical bar represents a potential TF-binding site. Solid bars indicate that the binding sites of a relevant TF are enriched or exclusively located within the 200-bp enhancer.
  • Fig. 3B shows transgenic assays of VInvIn2En with mutated DNA motifs related to five different TFs. Nucleotides with arrows indicate the replaced sequence(s) in each construct.
  • TCP 1 CCAAT Binding Factor/ Nuclear factor Y
  • GATA 2 GATA 2 .
  • the TCP family of transcription factors is named after the first 4 characterized members, namely TEOSINTE BRANCHED1 (TB1) from maize (Zea mays), CYCLOIDEA (CYC) from snapdragon (Antirrhinum majus), as well as PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR1 (PCF1) and PCF2 from rice (Oryza sativa).
  • Figs.4A-4G Functional validation of the VInvIn2En enhancer using genome editing.
  • Fig.4A is a diagram illustrating the positions of all sgRNAs within and outside of intron 2 of VInv gene.
  • Fig.4B is an image of gel electrophoresis of PCR products amplified from the three homozygous CRISPR/Cas9 deletion lines (2-2-8, 13-1-3, and 13-2-1) developed from DMF5-73-1 (WT).
  • Fig.4C is a graph showing qRT-PCR-based transcription analysis of the VInv gene in cold-stored potato tissues from the three homozygous deletion lines (2-2-8, 13-1-3, and 13-2-1).
  • Fig. 4D shows images of chipping of tubers from deletion line 13-1-3 and from the wild type DMF5-73-1. Note: (1) the dark color toward one end of each chip is caused by the “jelly end” problem (two examples are indicated by arrows) associated with both 13-1-3 and WT.
  • FIG.4E is an image of gel electrophoresis of PCR products amplified from the genomic DNA of 3 T0 CRISPR/Cas9 lines (KV78, KV87, and KV108) developed from tetraploid potato cultivar Katahdin. Arrows indicate fragments resulted from deletions within VInvIn2En.
  • Fig. 4F shows sequencing of PCR products amplified from cDNAs of the 3 CRISPR/Cas9 lines.
  • Fig. 4G is a graph showing qRT-PCR-based analysis of VInv expression relative to the Actin97 gene of the three CRISPR/Cas lines. Expression was analyzed using tubers after 2 wks of storage at 22 °C and 4 °C, respectively. The y axis represents the relative expression level normalized by setting VInv expression in 22 °C-stored tubers of the WT ‘Katahdin’ to 1. Data are presented as mean ⁇ SD from 3 biological replicates and were tested by using PROC GLM ANOVA.
  • Fig.5 is a schematic illustration of the composition of introns and exons of VInv genes from different plant species.
  • Figs.6A-6B are maps showing genotyping of homozygous CRISPR/Cas deletion lines developed from DMF5-73-1.
  • the 200-bp VInvIn2En enhancer is shown with the positions of the four sgRNAs (R1, R2, R3, R4) within and outside of VInvIn2En.
  • the positions of the six sgRNAs (1a, 2a, 3a, 1b, 2b, 3b) within and outside of VInvIn2En are also shown.
  • Fig. 6A shows a 394-bp homozygous deletion spanning sgRNA 1a and 3b which was detected in lines 13-1-3 and 13-2-1.
  • Fig.6B shows a 369- bp homozygous deletion spanning sgRNA 3a and 2b and a 5-bp deletion within sgRNA 2a which were detected in line 2-2-8. All three homozygous lines were generated from DMF5- 73-1 potato background by selfing a T0 event transformed with the CRISPR/Cas binary vector. Genotyping was conducted by PCR using primers VInv-mut-F1/R1 and Sanger sequencing. [0016] Fig.
  • FIG. 7 is a nucleic acid sequence alignment of three haplotypes associated with the middle portion of intron 2 of VInv gene from tetraploid potato Katahdin.
  • the 200-bp VInvIn2En from RH potato is indicated by bold underlining.
  • the positions of the four sgRNAs (R1, R2, R3 and R4) and protospacer adjacent motif (PAM) sequences are marked below the sequence.
  • the PAM sequences are identical nucleotides conserved among all haplotypes. Arrows to particular nucleotides are associated single nucleotide polymorphisms (SNPs) between haplotype A and haplotype C. [0017] Fig.
  • FIG. 8 shows maps of nucleic acid deletions within haplotype A and haplotype B in VInvIn2En of Katahdin. Sequence variants were detected in three T0 CRISPR/Cas9 lines (KV78, KV87 and KV108). Dotted lines represent deletions in different sizes. Insertions of single nucleotide are pointed by arrowheads. Percentages in brackets indicate the percentage of sequences with deletion/mutation in total number of sequences, including the wild type of haplotype A VInvIn2En or haplotype B VInvIn2En. The actual fragment lengths for different type of deletions are listed on the right. All numbers above lines indicate base pairs. [0018] Fig.
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • biological sample refers to either intact or non-intact (e.g., milled potato seed or potato plant tissue, chopped potato plant tissue, lyophilized tissue) potato plant tissue. It may also be an extract comprising intact or non-intact seed or potato plant tissue.
  • the biological sample can comprise flour, meal, syrup, oil, starch, and cereals manufactured in whole or in part to contain potato plant by-products.
  • the biological sample is “non-regenerable” (i.e., incapable of being regenerated into a potato plant or potato plant part).
  • the terms “correspond,” “corresponding,” and the like, when used in the context of an nucleotide position, mutation, and/or substitution in any given polynucleotide (e.g., an allelic variant of SEQ ID NO: 1) with respect to the reference polynucleotide sequence (e.g., SEQ ID NO: 1) all refer to the position of the nucleotide in the given sequence that has identity to the nucleotide in the reference nucleotide sequence when the given polynucleotide is aligned to the reference polynucleotide sequence using a pairwise alignment algorithm (e.g., CLUSTAL O 1.2.4 with default parameters).
  • a pairwise alignment algorithm e.g., CLUSTAL O 1.2.4 with default parameters.
  • the phrase “endogenous gene” refers to the native form of a gene unit in its natural location in the genome of an organism.
  • the term “enhancer” refers to a cis-acting sequence that increases the transcription from a eukaryotic promoter and that can function at a variable distance from the promoter. MSU: TEC2024-0055 PCT // MVS: P15076WO01
  • expression refers to the production of a functional end- product (e.g., an mRNA, guide RNA, or a protein) in either precursor or mature form.
  • the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.
  • the term “isomorphic allele” refers to an allele of a gene having wild- type gene activity.
  • the term “introduced” means providing a nucleic acid (e.g., expression construct) or protein into a cell.
  • Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell and includes reference to the transient provision of a nucleic acid or protein to the cell.
  • the term “introduced” includes reference to stable or transient transformation methods.
  • “introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/expression construct) into a cell means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., nuclear chromosome, plasmid, plastid, chloroplast, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • a nucleic acid fragment e.g., a recombinant DNA construct/expression construct
  • a “non-natural” or “non-naturally occurring” mutation refers to a mutation in a gene which is generated via human intervention or descended from the mutation generated via human intervention.
  • human intervention which can be used to generate a non-naturally occurring mutation include mutagenesis (e.g., chemical mutagenesis, ionizing radiation mutagenesis), mutagenesis followed by DNA sequence-based screening and selection (TILLING), and targeted genetic modifications (e.g., CRISPR-based methods, TALEN-based methods, zinc finger-based methods).
  • mutagenesis e.g., chemical mutagenesis, ionizing radiation mutagenesis
  • TILLING DNA sequence-based screening and selection
  • targeted genetic modifications e.g., CRISPR-based methods, TALEN-based methods, zinc finger-based methods.
  • potato plant is used in its broadest sense.
  • Such structures include, but are not limited to, a seed, a tuber, a tiller, a sprig, a stolen, a plug, a rhizome, a shoot, a stem, a leaf, a flower petal, a fruit, a petiole, et cetera.
  • plant part include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); grain; stover; a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., pollen, embryos, pods; flowers, fruits, shoots, leaves, roots, stems, and explants).
  • a plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit.
  • a plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant.
  • Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, or stalks.
  • some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non-regenerable” plant cells.
  • promoter refers to a regulatory region comprising the transcriptional start site) and binding sites for core transcription factor complexes that induce RNA polymerase II-mediated (RNA PolII-mediated) transcription of RNA PolII promoters.
  • promoter refers to a regulatory region comprising the transcriptional start site) and binding sites for core transcription factor complexes that induce RNA polymerase II-mediated (RNA PolII-mediated) transcription of RNA PolII promoters.
  • variety refers a group of similar plants that by one or more structural features, genetic features, and/or performance can be distinguished from other varieties within the same species.
  • the term variety refers to the botanical taxonomic designation whereby variety is ranked below species or subspecies, as well as the legal definition whereby the term “variety” refers to a commercial plant that is protected under the terms outlined in the International Convention for the Protection of New Varieties of Plants.
  • isolated means a nucleic acid or polypeptide has been removed from its natural or native cell.
  • the nucleic acid or polypeptide can be physically isolated from the cell or the nucleic acid or polypeptide can be present or maintained in another cell where it is not naturally present or synthesized.
  • transgenic when used in reference to a plant or leaf or fruit, tuber, seed, or plant biomass, for example a "transgenic plant,” transgenic leaf,” “transgenic fruit,” “transgenic fruit,” “transgenic seed,” “transgenic biomass,” or a “transgenic host cell” refers to a plant or leaf or fruit or seed or tuber or biomass that contains at least one heterologous MSU: TEC2024-0055 PCT // MVS: P15076WO01 or foreign gene in one or more of its cells.
  • transgenic plant material refers broadly to a plant, a plant structure, a plant tissue, a plant seed, a plant tuber or portion of tuber, or a plant cell that contains at least one heterologous gene in one or more of its cells.
  • transgene refers to a foreign gene that is placed into an organism (e.g. a plant) or host cell by the process of transfection.
  • wild-type when made in reference to a plant refers to the plant type common throughout an outbred population that has not been genetically manipulated to contain an expression cassette, e.g., any of the expression cassettes described herein.
  • selective hybridize includes hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence (e.g., any of the SEQ ID NOS: 1-4 nucleic acids) to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences. Such selective hybridization substantially excludes non target nucleic acids.
  • Selectively hybridizing sequences typically have about at least 40% sequence identity, or at least 50% sequence identity, or at least 60% sequence identity, or at least 70% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, or 60-99% sequence identity, or 70-99% sequence identity, or 80-99% sequence identity, or 90-95% sequence identity, or 90-99% sequence identity, or 95-97% sequence identity, or 97-99% sequence identity, or 100% sequence identity (or complementarity) with each other.
  • a selectively hybridizing sequence has about at least about 80% sequence identity or complementarity with SEQ ID NOS: 1-4.
  • the nucleic acids of the invention include those with about 50 of the same nucleotides as SEQ ID NOS: 1-4 or about 100 of the same nucleotides, or about 150 of the same nucleotides, or about 200 of the same nucleotides, or the same nucleotides as SEQ ID NO: 1-4.
  • the identical nucleotides or amino acids can be distributed throughout the nucleic acid, and need not be contiguous.
  • a value of a variable that is necessarily an integer, e.g., the number of nucleotides or amino acids in a nucleic acid or protein is described as a range, e.g., 90- 99% sequence identity what is meant is that the value can be any integer between 90 and MSU: TEC2024-0055 PCT // MVS: P15076WO01 99 inclusive, i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, or any range between 90 and 99 inclusive, e.g., 91-99%, 91-98%, 92-99%, etc.
  • stringent conditions include conditions under which a probe will hybridize to its target sequence to a detectably greater degree than other sequences (e.g., at least 2-fold over background).
  • Stringent conditions are somewhat sequence-dependent and can vary in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified with up to 100% complementarity to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of sequence similarity are detected (heterologous probing).
  • the probe can be approximately 20-500 nucleotides in length but can vary greatly in length from about 18 nucleotides to equal to the entire length of the target sequence. In some embodiments, the probe is about 10-50 nucleotides in length, or about 18-25 nucleotides in length, or about 18-50 nucleotides in length, or about 18-100 nucleotides in length.
  • stringent conditions will be those where the salt concentration is less than about 1.5 M Na+ ion (or other salts), typically about 0.01 to 1.0 M Na+ ion concentration (or other salts), at pH 7.0 to 8.3 and the temperature is at least about 30 °C for shorter probes (e.g., 10 to 50 nucleotides) and at least about 60 °C for longer probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide or Denhardt’ s solution.
  • Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 °C, and a wash in 1 x SSC to 2 x SSC (where 20 x SSC is 3.0 M NaCl, 0.3 M trisodium citrate) at 50 to 55 °C
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1M NaCl, 1 % SDS at 37 °C, and a wash in 0.5 x SSC to 1 x SSC at 55 to 60 °C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C, and a wash in 0.1 x SSC at 60 to 65 °C. Specificity is typically a function of post-hybridization washes, where the factors controlling hybridization include the ionic strength and temperature of the final wash solution. Thus, high stringency conditions can include a wash that includes 0.1 x SSC at 60 to 65 °C.
  • MSU TEC2024-0055 PCT // MVS: P15076WO01 [0047]
  • the Tm can be approximated from the equation of Meinkoth and Wahl (Anal. Biochem.
  • Tm 81.5 °C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% formamide) - 500/L
  • M is the molarity of monovalent cations
  • % GC is the percentage of guanosine and cytosine nucleotides in the DNA
  • % formamide is the percentage of formamide in the hybridization solution
  • L is the length of the hybrid in base pairs.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. The Tm is reduced by about 1 °C for each 1 % of mismatching.
  • the Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired sequence identity. For example, if sequences with greater than or equal to 90% sequence identity are sought, the Tm can be decreased 10 °C.
  • stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH.
  • severely stringent conditions can include hybridization and/or a wash at 1, 2, 3 or 4° C lower than the thermal melting point (Tm).
  • Moderately stringent conditions can include hybridization and/or a wash at 6, 7, 8, 9 or 10 °C lower than the thermal melting point (Tm).
  • Low stringency conditions can include hybridization and/or a wash at 11, 12, 13, 14, 15 or 20 °C lower than the thermal melting point (Tm).
  • Tm thermal melting point
  • those of ordinary skill can identify and isolate nucleic acids with sequences related to any of SEQ ID NOS disclosed herein.
  • Those of skill in the art also understand how to vary the hybridization and/or wash solutions to isolate desirable nucleic acids. For example, if the desired degree of mismatching results in a Tm of less than 45 °C (aqueous solution) or 32 °C (formamide solution), it may be preferred to increase the SSC concentration so that a higher temperature can be used.
  • MSU TEC2024-0055 PCT // MVS: P15076WO01
  • high stringency is defined as hybridization in 4 x SSC, 5 x Denhardt’s (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65 °C, and a wash in 0.1 x SSC, 0.1% SDS at 65 °C.
  • the reference sequence can be a nucleic acid sequence (e.g., any of SEQ ID NOS: 1-4).
  • a reference sequence may be a subset or the entirety of a specified sequence.
  • a reference sequence may be a segment of a full-length cDNA or of a genomic DNA sequence, or the complete cDNA or complete genomic DNA sequence, or a domain of a polypeptide sequence.
  • comparison window refers to a contiguous and specified segment of a nucleic acid or an amino acid sequence, wherein the nucleic acid/amino acid sequence can be compared to a reference sequence and wherein the portion of the nucleic acid/amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window can vary for nucleic acid and polypeptide sequences. Generally, for nucleic acids, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or more nucleotides.
  • the comparison window is at least about 10 amino acids, and can optionally be 15, 20, 30, 40, 50, 100 or more amino acids.
  • a gap penalty is typically introduced and is subtracted from the number of matches.
  • the BLAST family of programs that can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences.
  • GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-53, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP makes a profit of gap creation penalty number of matches for each MSU: TEC2024-0055 PCT // MVS: P15076WO01 gap it inserts.
  • GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty.
  • Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively.
  • the gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more.
  • GAP presents one member of the family of best alignments. There may be many members of this family. GAP displays four figures of merit for alignments: Quality, Ratio, Identity and Similarity. The Quality is the metric maximized to align the sequences.
  • Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold.
  • the scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).
  • sequence identity/similarity values provided herein can refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et ah, (1997) Nucleic Acids Res.25:3389-402).
  • BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments.
  • the SEG Wang and Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Ci-ayerie and States, (1993) Comput. Chem. 17:191-201) low-complexity filters can be employed alone or in combination.
  • a targeting vector can be used to introduce a transcriptional element, promotor, deletion, or modification of the genomic RFS chromosomal sites.
  • a "targeting vector” is a vector generally has a 5' flanking region and a 3' flanking region homologous to segments of the gene of interest.
  • the 5' flanking region and the 3' flanking region can be homologous to regions within the gene, or such flanking regions can flank the coding MSU: TEC2024-0055 PCT // MVS: P15076WO01 region of gene to be deleted, mutated, or replaced with the unrelated DNA sequence.
  • the targeting vector does not comprise a selectable marker.
  • DNA comprising the targeting vector and the native gene of interest are contacted under conditions that favor homologous recombination (e.g., by transforming plant cell(s) with the targeting vector).
  • a typical targeting vector contains nucleic acid fragments of not less than about 0.1 kb nor more than about 10.0 kb from both the 5' and the 3' ends of the genomic locus which encodes the gene to be modified (e.g. the genomic VInvIn2En site(s)). These two fragments can be separated by an intervening fragment of nucleic acid that includes the modification to be introduced. When the resulting construct recombines homologously with the chromosome at this locus, it results in the introduction of the modification, e.g. an insertion, substitution, or a deletion of a portion of the genomic RFS site(s).
  • the present disclosure describes the modification of one or more nucleotides in a transcriptional enhancer of an endogenous potato VInv gene for providing potato plant cells, potato plant seeds, potato tubers or a portion of tubers, and potato plants that exhibit reduced cold-induced sweetening (CIS).
  • VInvIn2En represents the first potato enhancer that is functionally dependent on sequence integrity for binding of multiple transcription factors (TFs).
  • TFs transcription factors
  • VInv gene transcription in tubers is maintained at a minimal level under room temperature. VInv is dramatically upregulated during cold storage in CIS-susceptible potato cultivars (Zrenner et al.1996; Bagnaresi et al.2008; Bhaskar et al.2010), causing rapid accumulation of reducing sugars.
  • vacuolar invertases have been found to be specific to the cell wall, vacuole, or cytosol, respectively. Both cell wall and vacuolar invertases are also known to contribute to defense responses to abiotic and biotic stresses (Wan et al., 2018). Vacuolar invertases play essential roles in cell expansion and sugar accumulation, which are related to plant growth and development (Ruan et al., 2010; Wan et al., 2018). Therefore, silencing of the vacuolar invertase gene can cause major developmental defects in plants. For example, silencing of the vacuolar invertase gene in tomtao (Solanum lycopersicum) resulted in significantly smaller fruits (Klann et al., 1996).
  • VInv gene may not play a similar MSU: TEC2024-0055 PCT // MVS: P15076WO01 developmental role in potato as compared to other plant species.
  • VInv expresses in non-tuber tissues, the expression of VInv are not upregulated by cold stress in several non-tuber tissues, including petiole, stem, and root (unpublished data).
  • the GUS signals in the transgenic A are not upregulated by cold stress in several non-tuber tissues, including petiole, stem, and root (unpublished data).
  • VInvIn2 and VInvIn2En constructs were not enhanced by cold stress. These results suggest that the VInv gene has adapted for a distinct role in the tuber-bearing species in response to cold stress. A high level of VInv expression at cold temperatures will generate more sugars in tuber cells. This in turn would affect the osmatic pressure and increase the freezing tolerance of tuber cells that contain a high percentage of water.
  • TFs including CBF/NF-Y, TCP, and GATA, may play a role in VInvIn2En- mediated regulation of VInv under cold conditions, since mutations of the predicted binding sites of these TFs abolished the function of VInvIn2En as a transcriptional enhancer in A. thaliana (Fig.3B).
  • CBF/NF-Y, TCP, and GATA are large TF families in plants and include 41, 31, and 49 genes, respectively, in the potato genome (Wang et al. 2019; Li et al.2021; Yu et al.2022).
  • VInvIn2En The cold-induced transcription of VInv is not controlled by its promoter (Ou et al., 2013).
  • the VInv promoter is required to respond to sucrose/glucose, indole-3-acetic acid MSU: TEC2024-0055 PCT // MVS: P15076WO01 (IAA), and gibberellic acid (GA3), but not in response to cold temperatures (Ou et al., 2013).
  • the present disclosure describes the discovery of a 200-bp transcriptional enhancer, VInvIn2En, located in the second intron of VInv. This enhancer is responsible for the cold- induced expression of the VInv gene.
  • TFs transcription factors
  • VInvIn2En deletion lines of VInvIn2En were developed in both diploid and tetraploid potato lines using CRISPR/Cas9- mediated genome editing. VInv transcription was significantly reduced in the deletion lines during cold storage.
  • the VInvIn2En sequence was found to be highly conserved among distantly related plant species, revealing an evolutionary trajectory of the VInv gene in response to cold stress in the tuber-bearing Solanum species.
  • SEQ ID NO: 1 The endogenous 200bp wild-type nucleic acid sequence of VInvIn2En enhancer from potato (Solanum tuberosum) is shown below as SEQ ID NO: 1 and allelic variants thereof.
  • allelic variants of an endogenous potato VInvIn2En enhancer also include variants which comprise genomic DNA having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 1.
  • allelic variants of the endogenous potato VInvIn2En enhancer are isomorphic alleles of the endogenous potato VInvIn2En enhancer.
  • Potato plant cells, tubers or portion of tubers, plant parts, and plants comprising at least one mutation in the potato VInvIn2En enhancer of SEQ ID NO: 1 or an allelic variant thereof are provided.
  • the at least one mutation is a non-natural mutation.
  • the at least one mutation reduces expression of the endogenous VInv gene (e.g., by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, MSU: TEC2024-0055 PCT // MVS: P15076WO01 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,
  • the at least one mutation eliminates expression of the endogenous VInv gene.
  • mutations can include a deletion, an insertion, and/or a substitution of one or more nucleotides of the potato VInvIn2En enhancer.
  • the insertion, deletion, and/or substitution can be made anywhere in the potato VInvIn2En enhancer.
  • the at least one mutation comprises, consists essentially of, or consists of a deletion, insertion, and/or substitution of at least one nucleotide (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, or 200 nucleotides) of the potato VInvIn2En enhancer of SEQ ID NO: 1 or an allelic variant thereof.
  • At least one nucleotide e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, or 200 nucleotides
  • One or more mutation(s) in the VInvIn2En transcriptional enhancer can be used to reduce cold-induced expression of the VInv gene in potato.
  • the one or more mutations in VInvIn2En can comprise mutations in the CBF/NF-Y, TCP, and/or GATA DNA motifs.
  • the one or more mutation(s) can comprise one or more nucleotide mutation(s) within the GATA motif, which results in a complete loss of function of the VInvIn2En enhancer.
  • An example mutation in the GATA motif of VInvIn2En that resulted in loss of expression of the VInv gene is shown below in SEQ ID NO: 2 (mutated nucleic acid is shown in bold underline; See Fig.3B and Example 4 below).
  • the one or more mutation(s) can comprise one or more nucleotide mutation(s) within the TCP motif.
  • a Cas9/ CRISPR system can be used to create a modification in genomic VInvIn2En site(s).
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • Cas CRISPR-associated systems
  • RNA-programmable genome editing see e.g., Marraffini & Sontheimer. Nature Reviews Genetics 11: 181-190 (2010); Sorek et al. Nature Reviews Microbiology 20086: 181-6; Karginov and Hannon. Mol Cell 20101 :7-19; Hale et al. Mol Cell 2010:45:292-302; Jinek et al. Science 2012 337:815-820; Bikard and Marraffini Curr Opin Immunol 201224:15-20; Bikard et al.
  • a CRISPR guide RNA can be used that can target a Cas enzyme to the desired location in the genome, where it generates a double strand break. This technique is available in the art and described, e.g. at Mali et al. Science 2013 339:823-6; which is incorporated by reference herein in its entirety and kits for the design and use of CRISPR- mediated genome editing are commercially available, e.g. the PRECISION X CAS9 SMART NUCLEASETM System (Cat No.
  • a cDNA clone encoding the mutant VInvIn2En enhancer nucleic acid sequence is isolated from plant tissue, for example, a root, stem, leaf, seed, tuber, or flower tissue.
  • cDNA clones from selected species are made from isolated mRNA from selected plant tissues.
  • a nucleic acid encoding a mutant or modified VInvIn2En enhancer can be prepared by available methods or as described herein.
  • the nucleic acid encoding a mutant or modified VInvIn2En enhancer can be any nucleic acid with a coding region that hybridizes to a segment of a SEQ ID NOS: 1-4 nucleic acid.
  • the expression cassette can also optionally include 3' nontranslated plant regulatory DNA sequences that act as a signal to terminate transcription and allow for the polyadenylation of the resultant mRNA.
  • the 3' nontranslated regulatory DNA sequence preferably includes from about 300 to 1,000 nucleotide base pairs and contains plant transcriptional and translational termination sequences.
  • 3' elements that can be used include those derived from the nopaline synthase gene of Agrobacterium tumefaciens (Bevan et ah, Nucleic Acid Research.
  • the 3’ nontranslated regulatory sequences can be operably linked to the 3’ terminus of the RFS nucleic acids by standard methods.
  • Selectable and Screenable Marker Sequences To improve identification of transformants, a selectable or screenable marker gene can be employed with the VInvIn2R nucleic acids. "Marker genes” are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker.
  • Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can ‘select’ for by chemical means, e.g., by use of a selective agent (e.g., an herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify through observation or testing, i.e., by ‘screening’ (e.g., the R-locus trait).
  • a selective agent e.g., an herbicide, antibiotic, or the like
  • screening e.g., the R-locus trait
  • Such a secreted antigen marker can employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that imparts efficient expression and targeting across the plasma membrane and MSU: TEC2024-0055 PCT // MVS: P15076WO01 can produce protein that is bound in the cell wall and yet is accessible to antibodies.
  • a normally secreted wall protein modified to include a unique epitope would satisfy such requirements.
  • proteins suitable for modification in this manner include extensin or hydroxyproline rich glycoprotein (HPRG).
  • HPRG extensin or hydroxyproline rich glycoprotein
  • the maize HPRG (Stiefel et al., The Plant Cell. 2:785-793 (1990)) is well characterized in terms of molecular biology, expression, and protein structure and therefore can readily be employed.
  • acetolactate synthase gene which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals
  • ALS acetolactate synthase gene
  • European Patent Application 154,204 (1985) a mutant acetolactate synthase gene which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals
  • a methotrexate-resistant DHFR gene Thillet et al., J. Biol. Chem.263:12500-12508 (1988)
  • a dalapon dehalogenase gene that confers resistance to the herbicide dalapon
  • a mutated anthranilate synthase gene that confers resistance to 5 -methyl tryptophan.
  • PPT phosphinothricin acetyl transferase
  • genes from the maize R gene complex can be used as screenable markers.
  • the R gene complex in maize encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissue.
  • Maize strains can have one, or as many as four, R alleles that combine to regulate pigmentation in a developmental and tissue specific manner.
  • a gene from the R gene complex does not harm the transformed cells.
  • the R gene regulatory regions may be employed in chimeric constructs to provide mechanisms for controlling the expression of chimeric genes. More diversity of phenotypic expression is known at the R locus than at any other locus (Coe et al., in Corn and Corn Improvement, eds. Sprague, G.F. & Dudley, J.W. (Am. Soc. Agron., Madison, WI), pp. 81-258 (1988)). It is contemplated that regulatory regions obtained from regions 5' to the structural R gene can be useful in directing the expression of genes, e.g., insect resistance, drought resistance, herbicide tolerance or other protein coding regions.
  • any of the various R gene family members may be successfully employed (e.g., P, S, Fc, etc.).
  • Sn is a dominant member of the R gene complex and is functionally similar to the R and B loci in that Sn controls the tissue specific deposition of anthocyanin pigments in certain seedling and plant cells, therefore, its phenotype is similar to R.
  • a further screenable marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene.
  • An expression cassette of the invention can also further comprise plasmid DNA.
  • This binary Ti vector can be replicated in prokaryotic bacteria such as E. coli and Agrobacterium.
  • the Agrobacterium plasmid vectors can be used to transfer the expression cassette to dicot plant cells, and under certain conditions to monocot cells, such as rice cells.
  • the binary Ti vectors preferably include the nopaline T DNA right and left borders to provide for efficient plant cell transformation, a selectable marker gene, unique multiple cloning sites in the T border regions, the co/El replication of origin and a wide host range replicon.
  • the binary Ti vectors carrying an expression cassette of the invention can be used to transform both prokaryotic and eukaryotic cells but is preferably used to transform dicot plant cells.
  • Methods such as microprojectile bombardment or electroporation can be carried out with “naked” DNA where the expression cassette may be simply carried on any E. coli-derived plasmid cloning vector.
  • viral vectors it is desirable that the system retain replication functions, but lack functions for disease induction.
  • One method for dicot transformation involves infection of plant cells with Agrobacterium tumefaciens using the leaf-disk protocol (Horsch et ak, Science 227:1229- MSU: TEC2024-0055 PCT // MVS: P15076WO01 1231 (1985).
  • Monocots such as Zea mays can be transformed via microprojectile bombardment of embryogenic callus tissue or immature embryos, or by electroporation following partial enzymatic degradation of the cell wall with a pectinase-containing enzyme (U.S. Patent No. 5,384,253; and U.S. Patent No. 5,472,869).
  • embryogenic cell lines derived from immature Zea mays embryos can be transformed by accelerated particle treatment as described by Gordon-Kamm et al. (The Plant Cell.2:603- 618 (1990)) or U.S. Patent No.5,489,520; U.S. Patent No.5,538,877 and U.S. Patent No. 5,538,880, cited above.
  • Excised immature embryos can also be used as the target for transformation prior to tissue culture induction, selection and regeneration as described in U.S. application Serial No.08/112,245 and PCT publication WO 95/06128. Furthermore, methods for transformation of monocotyledonous plants utilizing Agrobacterium tumefaciens have been described by Hiei et al. (European Patent 0604662, 1994) and Saito et al. (European Patent 0672752, 1995). [00107] Methods such as microprojectile bombardment or electroporation are carried out with “naked” DNA where the expression cassette may be simply carried on any E. coli-derived plasmid cloning vector.
  • tissue source for transformation will depend on the nature of the host plant and the transformation protocol.
  • Useful tissue sources include callus, suspension culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like.
  • the tissue source is selected and transformed so that it retains the ability to regenerate whole, fertile plants following transformation, i.e., contains totipotent cells.
  • Type I or Type II embryonic maize callus and immature embryos are preferred Zea mays tissue sources. Similar tissues can be transformed for softwood or hardwood species.
  • Electroporation Where one wishes to introduce DNA by means of electroporation, it is contemplated that the method of Krzyzek et al. (U.S. Patent No. 5,384,253) may be advantageous. In this method, certain cell wall-degrading enzymes, such as pectin- degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells can be made more susceptible to transformation, by mechanical wounding.
  • friable tissues such as a suspension cell cultures, or embryogenic callus
  • the cell walls of the preselected cells or organs can be partially degraded by exposing them to pectin-degrading enzymes (pectinases or pectolyases) or mechanically wounding them in a controlled manner.
  • pectinases or pectolyases pectinases or pectolyases
  • Such cells would then be receptive to DNA uptake by electroporation, which may be carried out at this stage, and transformed cells then identified by a suitable selection or screening protocol dependent on the nature of the newly incorporated DNA.
  • Microprojectile Bombardment A further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment.
  • microparticles may be coated with DNA and delivered into cells by a propelling force.
  • Exemplary particles include those comprised of tungsten, gold, platinum, and the like.
  • DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment.
  • non-embryogenic BMS cells were bombarded with intact cells of the bacteria E. coli or Agrobacterium tumefaciens containing plasmids with either the b-glucoronidase or bar gene engineered for expression in maize.
  • Bacteria were inactivated by ethanol dehydration prior to bombardment. A low level of transient expression of the b-glucoronidase gene was observed 24-48 hours following DNA delivery. In addition, stable transformants containing the bar gene were recovered MSU: TEC2024-0055 PCT // MVS: P15076WO01 following bombardment with either E. coli or Agrobacterium tumefaciens cells. It is contemplated that particles may contain DNA rather than be coated with DNA. Hence it is proposed that particles may increase the level of DNA delivery but are not, in and of themselves, necessary to introduce DNA into plant cells.
  • the microprojectile bombardment is an effective means of reproducibly stably transforming monocots that avoids the need to prepare and isolate protoplasts (Christou et al., PNAS.84:3962-3966 (1987)), avoids the formation of partially degraded cells, and the susceptibility to Agrobacterium infection is not required.
  • An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with maize cells cultured in suspension (Gordon-Kamm et al., The Plant Cell. 2:603-618 (1990)).
  • the screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectile aggregate and may contribute to a higher frequency of transformation, by reducing damage inflicted on the recipient cells by an aggregated projectile.
  • cells in suspension are preferably concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium.
  • the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded.
  • the number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from about 1 to 10 and average about 1 to 3.
  • bombardment transformation one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment can influence transformation frequency. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the path and velocity of either the MSU: TEC2024-0055 PCT // MVS: P15076WO01 macro- or microprojectiles.
  • Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmid DNA.
  • TRFs trauma reduction factors
  • An exemplary embodiment of methods for identifying transformed cells involves exposing the bombarded cultures to a selective agent, such as a metabolic inhibitor, an antibiotic, herbicide or the like. Cells which have been transformed and have stably integrated a marker gene conferring resistance to the selective agent used, will grow and divide in culture. Sensitive cells will not be amenable to further culturing. [00120] To use the har-bialaphos or the EPSPS-glyphosate selective system, bombarded tissue is cultured for about 0-28 days on nonselective medium and subsequently transferred to medium containing from about 1-3 mg/1 bialaphos or about 1-3 mM glyphosate, as appropriate.
  • a selective agent such as a metabolic inhibitor, an antibiotic, herbicide or the like.
  • An example of a screenable marker trait is the red pigment produced under the control of the R-locus in maize. This pigment may be detected by culturing cells on a solid support containing nutrient media capable of supporting growth at this stage and selecting cells from colonies (visible aggregates of cells) that are pigmented. These cells may be cultured further, either in suspension or on solid media.
  • the R-locus is useful for selection of transformants from bombarded immature embryos. In a similar fashion, the introduction of the Cl and B genes will result in pigmented cells and/or tissues.
  • the enzyme luciferase is also useful as a screenable marker in the context of the present invention.
  • cells expressing luciferase emit light which can be detected on photographic or X-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. All of these assays are nondestructive and transformed cells may be cultured further following identification.
  • the photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells which are expressing luciferase and manipulate those in real time. It is further contemplated that combinations of screenable and selectable markers may be useful for identification of transformed cells.
  • a growth inhibiting compound such as bialaphos or glyphosate at concentrations below those providing 100% inhibition followed by screening of growing tissue for expression of a screenable marker gene such as luciferase would allow one to recover transformants from cell or tissue types that are not amenable to selection alone.
  • a growth inhibiting compound such as bialaphos or glyphosate at concentrations below those providing 100% inhibition followed by screening of growing tissue for expression of a screenable marker gene such as luciferase
  • embryogenic Type II callus of Zea mays L. can be selected with sub-lethal levels of bialaphos. Slowly growing tissue was subsequently screened for expression of the luciferase gene and transformants can be identified.
  • Regeneration and Seed Production Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, are cultured in media that supports regeneration of plants.
  • the transformed cells identified by selection or screening and cultured in an appropriate medium that supports regeneration, can then be allowed to mature into plants.
  • Developing plantlets are transferred to soilless plant growth mix, and hardened, e.g., in an environmentally controlled chamber at about 85% relative humidity, about 600 ppm CO2, and at about 25-250 microeinsteins/sec-m of light.
  • Plants can be matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue.
  • cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are petri dishes and Plant ConTM. Regenerating plants can be grown at about 19 °C to 28 °C.
  • Mature plants are then obtained from cell lines that are known to express the trait.
  • the regenerated plants are self-pollinated.
  • pollen obtained from the regenerated plants can be crossed to seed grown plants of agronomically important inbred lines.
  • pollen from plants of these inbred lines is used to pollinate regenerated plants.
  • the trait is genetically characterized by evaluating the segregation of the trait in first and later generation progeny. The heritability and expression in plants of traits selected in tissue culture are of interest if the traits are to be commercially useful.
  • Regenerated plants can be repeatedly crossed to inbred plants to introgress the VInvIn2R nucleic acids into the genome of the inbred plants. This process is referred to as backcross conversion.
  • backcross conversion When a sufficient number of crosses to the recurrent inbred parent have been completed to produce a product of the backcross conversion process that is MSU: TEC2024-0055 PCT // MVS: P15076WO01 substantially isogenic with the recurrent inbred parent except for the presence of the introduced VInvIn2R nucleic acids, the plant is self-pollinated at least once to produce a homozygous backcross converted inbred containing the RFS nucleic acids. Progeny of these plants are true breeding.
  • seed from transformed monocot plants regenerated from transformed tissue cultures is grown in the field and self-pollinated to generate true breeding plants.
  • Seed from the fertile transgenic plants can then be evaluated for the presence and/or expression of the VInvIn2R nucleic acid.
  • Transgenic plant and/or seed tissue can be analyzed for VInvIn2R transcriptional enhancement using standard methods such as SDS polyacrylamide gel electrophoresis, liquid chromatography (e.g., HPLC) or other means of detecting a product of RFS activity (e.g., increased glucan content and/or good growth).
  • the seed can be used to develop true breeding plants.
  • the true breeding plants are used to develop a line of plants with a reduced CIS while still maintaining other desirable functional agronomic traits. Adding the trait of reduced CIS and normal growth of the plant can be accomplished by back-crossing with this trait and with plants that do not exhibit this trait and studying the pattern of inheritance in segregating generations. Those plants expressing the target trait in a dominant fashion are preferably selected.
  • Back-crossing is carried out by crossing the original fertile transgenic plants with a plant from an inbred line exhibiting desirable functional agronomic characteristics while not necessarily expressing the trait of reduced CIS.
  • the resulting progeny are then crossed back to the parent that expresses the increased reduced CIS trait.
  • the progeny from this cross will also segregate so that some of the progeny carry the trait and some do not.
  • This back-crossing is repeated until an inbred line with the desirable functional agronomic traits, and with expression of the trait involving reduced CIS.
  • Such expression of reduced CIS can be expressed in a dominant fashion.
  • Such assays include, for example, molecular biological assays available to those of skill in the art, such as Southern and MSU: TEC2024-0055 PCT // MVS: P15076WO01 Northern blotting and PCR; biochemical assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf, seed or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
  • DNA analysis techniques may be conducted using DNA isolated from any part of a plant, RNA may only be expressed in particular cells or tissue types and so RNA for analysis can be obtained from those tissues.
  • PCR techniques may also be used for detection and quantification of RNA produced from introduced VInvIn2R nucleic acids. PCR also be used to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then this DNA can be amplified by use of conventional PCR techniques. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and also demonstrate the presence or absence of an RNA species.
  • Southern blotting and PCR may be used to detect the RFS nucleic acid in question, they do not provide information as to whether the preselected DNA segment is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced RFS nucleic acids or evaluating the phenotypic changes brought about by their expression.
  • Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange, liquid chromatography or gel exclusion chromatography.
  • the unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques.
  • MSU TEC2024-0055 PCT // MVS: P15076WO01
  • the expression of a gene product can also be determined by evaluating the phenotypic results of its expression.
  • These assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of the plant. Chemical composition may be altered by expression of preselected DNA segments encoding storage proteins which change amino acid composition and may be detected by amino acid analysis.
  • Example 1 Materials and Methods
  • Example 2 This example describes some of the materials and methods used in developing the technology.
  • Enhancer validation using transgenic assays in potato [00141] An intronic DHS within intron 2 of VInv gene was identified from the DHS data published previously (Zeng et al., 2019). The entire intron 2 from the VInv gene of RH potato was used for enhancer validation using a GUS reporter system (Zhu et al., 2015).
  • VIT-F6/R6 and VIT-F8/R8 primers VIT-F6/R6 and VIT-F8/R8 (Table 1), respectively, and were subsequently cloned into pKGWFS 7.0 vector containing a minimal 35S promoter (-50 to - 2 bp) (mini35S) and the GUS reporter (Zhu et al., 2015). Constructs were transferred into Agrobacterium tumefaciens strain GV3101 (pMP90), followed by transformation of potato variety Katahdin using methods described previously (Bhaskar et al., 2008; Bhaskar et al., 2010).
  • Tuber slices were placed in a plastic plate (70 x 15 mm) and soaked in GUS-staining solution (100 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.1% [v/v] Triton X-100, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and 0.05% [w/v] XGluc), with vacuum infiltration for 30 min and incubation in dark at 37°C overnight. Tuber slices were washed in 80% ethanol several times. Images of tuber slices were captured using an EPSON Perfection 4180 scanner. [00144] 2. Enhancer dissection using transgenic assays in A. thaliana. [00145] Seeds of A.
  • thaliana ecotype Col-0 were germinated in one-half-strength Murashige and Skoog (0.5 x MS) medium, and the seedlings were transplanted in potting soil and grown in greenhouses with 16/8 h light/dark cycles at 23°C and light intensity of 150 ⁇ mol m-2 s-1 until flowering.
  • the VInvIn2 and VInvIn2R constructs were initially used to transform A. thaliana ecotype Col-0 using the floral dip method (Clough and Bent, 1998).
  • Transgenic seedlings were screened on solid 0.5 x MS medium containing kanamycin (50 ⁇ g mL-1) and were grown in an illumination incubator with the same light-dark condition described above and were examined for GUS activity according to published potocols (Zhu et al., 2015).
  • To map the position of the enhancer within the intron 2 of VInv we divided the intron 2 into ten DNA fragments (#1 to #10) using five breaks (b1 to b5) for transgenic assays. The stem/petiole-specific enhancer (within DNA fragment #11) was further divided into 14 (#11 to #24) sub-fragments.
  • pHNCas9::VInvIn2En was introduced into A. tumefaciens GV3101 (pMP90) and was used to transform Katahdin according to published protocols (Bhaskar et al., 2008). Positive transformants were screened using PCR with primers Kan-F3/R3, Cas-F1/R1, and VInv- Edit-F/R (Table 1). Transgenic lines containing additional smaller bands (2% agarose gel) were further confirmed by Sanger sequencing. PCR products were purified by using QIAquick PCR Purification Kit (Qiagen) and were cloned into E. coli using pMD TM 19-T vector (TaKaRa).
  • MSU TEC2024-0055 PCT // MVS: P15076WO01 [00155]
  • Each of 10 seed tubers of RH potato was planted in potting soil under normal greenhouse conditions as described above. Standard cultivation and management practices were followed throughout the growing period. Tubers were harvested 120 days after seedling emergence when leaves senesced naturally. Tubers harvested from two pots were combined together as one biological replicate. Tubers of five biological replicates were stored in dark at RT (22°C, 50%-70% humidity) for 10 d and then divided into two groups. Each group was stored in dark at RT (22°C, 50%-70% humidity) and cold (4°C, 60%-70% humidity) for 0, 2, 4, 8, and 16 weeks, respectively.
  • T0 CRISPR/Cas deletion lines Three T0 CRISPR/Cas deletion lines (three plants for each line) developed from Katahdin were grown under normal greenhouse conditions as described above. Tubers harvested from the same line were combined together and stored under dark at room temperature (RT, 22°C) for 10 days, and then divided into two groups for RT (22°C, 50%- 70% humidity) and cold (4°C, 60%-70% humidity) treatments, and each group of tubers with three replicates were treated for 2 and 4 weeks, respectively. [00157] Tuber samples of 1.5-mm thick slices (1-3 slices for each tuber) prepared from apical to basal end of the tuber were taken for chipping analysis. The remaining tuber samples were frozen in liquid nitrogen and used for analysis of VInv expression.
  • RNAs were extracted from tuber tissues using Plant RNA Isolation Mini Kit (Agilent) following the manufacturer’s instructions and were reverse transcribed to cDNAs using Invitrogen SuperScriptTM III Reverse Transcriptase Kit (Invitrogen) with oligo(dT)20 primer.
  • VInv transcripts were quantified by quantitative real time-PCR (qRT-PCR) using the SYBR Advantage qPCR Premix (Clontech) with the specific primers for VInv and the reference gene Actin97 described previously (Zhu et al., 2014; Zhu et al., 2016).
  • qRT-PCR was performed on the CFX96 Touch TM Real-Time PCR Detection System (Bio-Rad) with a program of 30 s at 95°C, 40 cycles of 10 s at 95°C, 20 s at 60°C for VInv and Actin97, and 30 s at 72°C, followed by a plate read.
  • VInv gene thaliana, cucumber (C. sativus), and soybean (G. max), were selected for evolutionary analysis of the VInv gene.
  • Information on evolutionary timescale of life for all 31 species were collected from the TimeTree 5 database (http://www.timetree.org/) (Kumar et al., 2022) and visualized in MEGA X software (Kumar et al., 2018).
  • Protein sequences of VInv gene from the 31 different plant species were extracted and aligned to that from RH potato using the NCBI BLASTp program (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
  • Example 2 Discovery of a cold-responsive intronic enhancer within VInv gene [00165] Genomic regions containing active cis-regulatory elements (CREs), such as promoters and transcriptional enhancers, can be identified as DNase I hypersensitive sites (DHSs) (Zhang et al.2012; Jiang 2015; Zhao et al.2018).
  • DHSs DNase I hypersensitive sites
  • the intron was cloned into the pKGWFS 7.0 vector containing a minimal 35S promoter ( ⁇ 50 to ⁇ 2 bp) (m35S) and the ⁇ -glucuronidase (GUS) reporter gene (Zhu et al.2015).
  • m35S minimal 35S promoter
  • GUS ⁇ -glucuronidase reporter gene
  • Example 3 Dissection of intronic enhancers via reporter gene assays in A. thaliana [00168] Since the entire intron 2 from RH potato was used for GUS reporter assays, the precise size and position of the predicted enhancer within intron 2 could not be determined. We attempted to fine-map the enhancer using reporter gene assay in A. thaliana. We first examined the GUS signal profiles of transgenic A. thaliana plants using the VInvIn2 and VInvIn2R constructs.
  • VInvIn2En spans 678 to 877 bp in the intron and is located within the 475 bp DHS, which spans 597 to 1,071 bp in the intron (Fig.1A).
  • Example 4 Identification of DNA motifs related to VInvIn2En function [00173] We speculated that VInvIn2En contains DNA motifs bound by TFs involved in plant response to cold stress. We identified putative DNA motifs related to a total of 15 TFs in the intron 2 sequence of RH (Zhou et al.2020). These motifs were consistently detected by 2 independent programs using both CIS-BP (Weirauch et al.2014) and PlantPAN 3.0 (Chow et al. 2019).
  • TF motifs were enriched in the 200 bp VInvIn2En, including bHLH and CBF/NF-Y.
  • motifs related to TCP and GATA were found only in the 200 bp enhancer region (Fig.3A).
  • the target motif(s) were mutated by replacing 1 to 3 nucleotide(s) within the sequence (Fig. 3B, Table 3).
  • thaliana plants using VInvIn2En with a mutated B3 motif showed similar GUS signal patterns as those from wild-type (WT) VInvIn2En.
  • Reduced GUS signals were detected from transgenic plants using VInvIn2En with 2 mutated bHLH motifs.
  • a single-nucleotide mutation within the GATA motif resulted in a complete loss of function of the VInvIn2En enhancer (Fig.3B).
  • Table 3 Sequences and mutated nucleotides within the 200-bp VInvIn2En of SEQ ID NO: 1.
  • VInvIn2En (b ) MSU: TEC2024-0055 PCT // MVS: P15076WO01 [00176]
  • Example 5 Genome editing of VInvIn2En in diploid potato [00177]
  • the in vivo function of a predicted enhancer can be validated by mutation or deletion using genome editing (Meng et al.2021; Zhao et al.2022; Fang et al.2023).
  • This clone was self-pollinated for 5 generations from a self-compatible diploid hybrid DM1-3 ⁇ M6 (Endelman and Jansky 2016).
  • DMF5-73-1 is amenable to Agrobacterium- mediated transformation (Butler et al.2020).
  • Five sgRNAs flanking VInvIn2En (1a (SEQ ID NO:37), 2a (SEQ ID NO:38), 3a (SEQ ID NO:39), 1b (SEQ ID NO:42), and 2b (SEQ ID NO:41)) and a single sgRNA (3b (SEQ ID NO:40)) targeting VInvIn2En (Fig. 4A, Table 2) were designed and assembled into a single construct.
  • DMF5-73-1 is not susceptible to CIS and expresses a weak CIS genotype. Tubers harvested from the three deletion lines were stored at 12.8°C for 6 weeks followed by at 6.7°C for nine additional weeks, a storage procedure used to maximize the CIS phenotype. Tuber tissues were then sampled for RNA extraction and qRT-PCR analysis. We found that the expression of VInv gene was reduced by 54% for 2-2-8, 45% for 13-1-3, and 41% for 13-2-1, respectively, compared to the wild-type DMF5-73-1 (Fig.4C).
  • DMF5-73-1 is not an ideal line to accurately evaluate the impact of VInvIn2En on CIS since it is resistant to CIS and has poor tuber traits. In addition, DMF5-73-1 retains a significant level of heterozygosity. Hence, the homozygous deletion lines developed from this clone are phenotypically different from the parental DMF5-73-1 (Fig. 4D).
  • Fig. 4D The homozygous deletion lines developed from this clone are phenotypically different from the parental DMF5-73-1.
  • We first amplified and sequenced intron 2 of VInv from ‘Katahdin.’ We identified 3 haplotypes: A (2 copies), B, and C.
  • Potato chips processed from tubers stored under 22 °C showed a similar color from all 3 lines as well as WT ‘Katahdin’ (Fig.9). After the 4 wks of storage of the tubers under 4 °C, chips from KV78, KV87, and KV108 all showed a lighter color than those from ‘Katahdin’ (Fig.9).
  • thaliana, cucumber (Cucumis sativus), and soybean (Glycine max), were used as outgroups in evolutionary analysis.
  • the VINV protein of potato shared 92% to 99% sequence similarity with those from tomato and wild Solanum species.
  • the structure of the VInv genes is also highly conserved among different species (Fig.5).
  • the distinct small exon 2 (9 bp) was detected in all Solanaceous species, as well as in several distantly related plant species.
  • a large intron 2 was identified following the small exon 2 in all species (Fig.5), with sizes ranging from 780 bp to 2,997 bp.
  • VInvIn2En represents a conserved enhancer sequence in Solanum species.
  • MSU TEC2024-0055 PCT // MVS: P15076WO01 2.
  • a tissue culture of regenerable cells comprising the modified potato plant cell of embodiment 12.
  • a modified potato plant part containing a chromosome comprising the targeted modification(s) in the endogenous VInvIn2En enhancer nucleic acid sequence set forth in any one of embodiments 1 to 11.
  • a method for generating a potato tuber from a potato plant seed or potato plant, wherein the potato tuber exhibits reduced cold-induced sweetening comprising: introducing at least one mutation into a VInvIn2En enhancer nucleic acid sequence of a VInv gene in the genome of the potato plant seed or potato plant, wherein the VInvIn2En enhancer has a nucleic acid sequence of SEQ ID NO: 1 or an allelic MSU: TEC2024-0055 PCT // MVS: P15076WO01 variant thereof, and wherein the mutation reduces expression of the VInv gene a potato tuber derived from the potato plant seed or potato plant relative to a potato tuber derived from a potato plant seed or potato plant without the VInvIn2En enhancer mutation in conditions where the temperature is less than about 15°C; and cultivating the potato plant tuber, potato plant seed, or potato plant to produce a mature potato plant.
  • the at least one mutation in the VInvIn2En enhancer nucleic acid sequence of SEQ ID NO: 1 comprises one or more mutation(s) in a CBF/NF-Y, TCP, and/or GATA DNA binding motif.
  • the one or more mutation(s) in the GATA DNA motif comprises a mutation of nucleic acid at position 21 of SEQ ID NO: 1 from A to T. 21.
  • the one or more mutation(s) in the GATA DNA motif comprises the DNA molecule set forth in SEQ ID NO: 2 or at least 90% sequence identity thereto. 22.
  • the one or more mutation(s) in the TCP DNA motif comprises a mutation of one or more nucleic acids at position 128, 130, 165, 166, and/or 167 of SEQ ID NO:1.
  • the one or more mutation(s) in the TCP DNA motif comprises the DNA molecule set forth in SEQ ID NO: 3 or at least 90% sequence identity thereto.
  • the one or more mutation(s) in the CBF/NF- Y DNA motifs comprise a mutation of one or more nucleic acids at positions 62, 63, 84, 85, and/or 111 of SEQ ID NO:1. 25.
  • a method of identifying a biological sample comprising a DNA molecule containing the targeted modification(s) in the endogenous VInvIn2En enhancer nucleic acid sequence set forth in any one of embodiments 1 to 11, comprising the step of detecting the presence of the DNA molecule in the biological sample.
  • a method of reducing the expression of an endogenous VInv gene in a potato plant comprising introducing the targeted modification(s) in the endogenous VInvIn2En enhancer nucleic acid sequence set forth in any one of embodiments 1 to 11.
  • MSU TEC2024-0055 PCT // MVS: P15076WO01 35.
  • the at least one mutation is introduced by: (i) directing both: (a) a guide RNA (gRNA) molecule comprising a spacer RNA molecule which targets the VInvIn2En enhancer nucleic acid sequence set forth in SEQ ID NO: 1 or an allelic variant thereof, or a spacer RNA molecule comprising the RNA encoded by any of SEQ ID NO: 33-42; and (b) an RNA dependent endonuclease (RDE) which recognizes the gRNA molecule to the genome of a target potato plant cell; and (ii) isolating a potato plant cell or potato plant comprising the at least one mutation in the VInvIn2En enhancer nucleic acid sequence of SEQ ID NO: 1 or an allelic variant thereof.
  • gRNA guide RNA
  • RDE RNA dependent endonuclease
  • directing of the gRNA and the RDE to the genome of the target potato plant cell comprises introducing the gRNA, the RDE, a gRNA/RDE complex, a nucleic acid encoding the gRNA, and/or a nucleic acid encoding the RDE into the target potato plant cell.
  • a gene editing system comprising: a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to the VInvIn2En enhancer nucleic acid sequence set forth in SEQ ID NO: 1 or an allelic variant thereof.
  • References in the present disclosure are as follows: Amrein, T.M., Bachmann, S., Noti, A., Biedermann, M., Barbosa, M.F., Biedermann-Brem, S., Grob, K., Keiser, A., Realini, P., Escher, F., and Amado, R. (2003). Potential of acrylamide formation, sugars, and free asparagine in potatoes: A comparison of cultivars and farming systems. J Agr Food Chem 51, 5556-5560. Bagnaresi, P., Moschella, A., Beretta, O., Vitulli, F., Ranalli, P., and Perata, P.
  • MSU TEC2024-0055 PCT // MVS: P15076WO01
  • PlantPAN3.0 a new and updated resource for MSU: TEC2024-0055 PCT // MVS: P15076WO01 reconstructing transcriptional regulatory networks from ChIP-seq experiments in plants. Nucleic Acids Res 47, D1155-D1163.
  • Floral dip a simplified method for Agrobacterium- mediated transformation of Arabidopsis thaliana. Plant J.16, 735-743. Dahro, B., Wang, Y., Khan, M., Zhang, Y., Fang, T., Ming, R.H., Li, C.L., and Liu, J.H. (2022). Two AT-Hook proteins regulate A/NINV7 expression to modulate sucrose catabolism for cold tolerance in Poncirus trifoliata. New Phytol 235, 2331-2349. Dale, M.F.B., and Bradshaw, J.E. (2003). Progress in improving processing attributes in potato. Trends Plant Sci 8, 310-312.
  • MSU TEC2024-0055 PCT // MVS: P15076WO01 He, F., Li, H.G., Wang, J.J., Su, Y.Y., Wang, H.L., Feng, C.H., Yang, Y.L., Niu, M.X., Liu, C., Yin, W.L., and Xia, X.L. (2019).
  • PeSTZ1 a C2H2-type zinc finger transcription factor from Populus euphratica, enhances freezing tolerance through modulation of ROS scavenging by directly regulating PeAPX2. Plant Biotechnol J 17, 2169-2183.
  • GSDS 2.0 an upgraded gene feature visualization server. Bioinformatics 31, 1296-1297.
  • Rapid and user-friendly open-source CRISPR/Cas9 system for single- or multi-site editing of tomato genome Hortic Res-England 6, 7. Klann, E.M., Hall, B., and Bennett, A.B. (1996).
  • Antisense acid invertase (TIV1) gene alters soluble sugar composition and size in transgenic tomato fruit. Plant Physiol.112, 1321-1330. Kumar, S., Stecher, G., Li, M., Knyaz, C., and Tamura, K. (2016). MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35, 1547-1549. Kumar, S., Suleski, M., Craig, J.M., Kasprowicz, A.E., Sanderford, M., Li, M., Stecher, G., and Hedges, S.B. (2022). TimeTree 5: an expanded resource for species divergence times. Molecular Biology and Evolution 39, msac174.
  • CRISPR-P 2.0 an improved CRISPR-Cas9 tool for genome editing in plants.
  • StvacINV1 a cold-responsive member
  • StInvInh2 as an inhibitor of StvacINV1 regulates the cold-induced sweetening of potato tubers by specifically capping vacuolar invertase activity. Plant Biotechnol J 11, 640-647.
  • CRISPR-Cas9 can deliver potato cultivars with reduced browning and acrylamide. Plants 12, 379. Ma, X.L., Zhu, Q.L., Chen, Y.L., and Liu, Y.G. (2016). CRISPR/Cas9 platforms for genome editing in plants: developments and applications. Mol Plant 9, 961-974.
  • MSU TEC2024-0055 PCT // MVS: P15076WO01 Mckenzie, M.J., Chen, R.K.Y., Harris, J.C., Ashworth, M.J., and Brummell, D.A. (2013).
  • Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers.
  • Cold sweetening in diploid potato Mapping quantitative trait loci and candidate genes.
  • MSU TEC2024-0055 PCT // MVS: P15076WO01 Xie, X.B., Li, S., Zhang, R.F., Zhao, J., Chen, Y.C., Zhao, Q., Yao, Y.X., You, C.X., Zhang, X.S., and Hao, Y.J. (2012).
  • the bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples. Plant Cell Environ 35, 1884-1897. Ye, J., Shakya, R., Shrestha, P., and Rommens, C.M. (2010).
  • Tuber-specific silencing of the acid invertase gene substantially lowers the acrylamide forming potential of potato.
  • Antisense suppression of an acid invertase gene (MAI1) in muskmelon alters plant growth and fruit development. J. Exp. Bot.59, 2969-2977.
  • Soluble acid invertase determines the hexose-to-sucrose ratio in cold-stored potato tubers. Planta 198, 246-252.
  • All cited patents and patent publications referred to in this application are incorporated herein by reference in their entirety. All of the materials and methods disclosed and claimed herein can be made and used without undue experimentation as instructed by the above disclosure and illustrated by the examples. Although the materials and methods of this disclosure have been described in terms of embodiments and illustrative examples, it will be apparent to those of skill in the art that substitutions and variations can be applied to the materials and methods described herein without departing from the concept, spirit, and scope of the disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the disclosure as encompassed by the embodiments of the disclosures recited herein and the specification and appended claims.

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Abstract

La divulgation concerne de nouvelles cellules de plants de pomme de terre, ainsi que de nouveaux tubercules végétaux, plants et procédés utiles pour réduire l'édulcoration induite par le froid dans les pommes de terre. La divulgation concerne en outre l'utilisation des plants de pomme de terre et des graines dans la production de graines de pomme de terre et de tubercules de pomme de terre.
PCT/US2024/060189 2023-12-14 2024-12-13 Compositions et procédés pour réduire l'édulcoration induite par le froid dans la pomme de terre Pending WO2025129109A1 (fr)

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US20160194648A1 (en) * 2009-02-03 2016-07-07 Wisconsin Alumni Research Foundation Control of cold-induced sweetening and reduction of acrylamide levels in potato or sweet potato
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CN109337913B (zh) * 2018-10-31 2021-05-04 西南大学 马铃薯cisr1基因及其在抗低温糖化中的应用

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WO2018140899A1 (fr) * 2017-01-28 2018-08-02 Inari Agriculture, Inc. Nouvelles cellules végétales, plantes et semences

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