WO2016202927A1 - Plantes de tomate présentant une altération non transgénique dans le gène dhs - Google Patents

Plantes de tomate présentant une altération non transgénique dans le gène dhs Download PDF

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Publication number
WO2016202927A1
WO2016202927A1 PCT/EP2016/063904 EP2016063904W WO2016202927A1 WO 2016202927 A1 WO2016202927 A1 WO 2016202927A1 EP 2016063904 W EP2016063904 W EP 2016063904W WO 2016202927 A1 WO2016202927 A1 WO 2016202927A1
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Prior art keywords
tomato
plant
seq
acid sequence
mutation
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PCT/EP2016/063904
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English (en)
Inventor
Hendrik Willem Vriezen
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Nunhems B.V.
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Publication of WO2016202927A1 publication Critical patent/WO2016202927A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/08—Fruits
    • 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
    • A01H6/825—Solanum lycopersicum [tomato]
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8249—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
    • 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.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01046—Deoxyhypusine synthase (2.5.1.46)

Definitions

  • This invention relates to novel human-induced, nontransgenic mutations of the tomato deoxyhypusine synthase (DHS) gene and tomato plants having such non-transgenic mutations in at least one of their tomato deoxyhypusine synthase gene sequence.
  • This invention further relates to tomatoes that are firmer when ripe and soften more slowly post-harvest than wild type tomatoes as a result of human-induced, non-transgenic mutation in their tomato deoxyhypusine synthase gene.
  • This invention also relates to a method that utilizes non-transgenic means to create tomatoes having mutations in their tomato deoxyhypusine synthase gene.
  • This invention further concerns a method of creating non- transgenic tomato plants exhibiting an alteration in deoxyhypusine synthase activity.
  • this invention concerns novel nucleotide sequences and a novel protein sequence for deoxyhypusine synthases identified in tomato and the use of these sequences to modify softening in tomatoes.
  • DHS deoxyhypusine synthase
  • eIF-5A eukaryotic translation initiation factor 5 A
  • DHS is an enzyme that converts the inactive form of elF- 5 A to its active form, hypusine-modified eIF-5A.
  • eIF-5A This hypusine-modified eIF5A plays an important role in cell growth and differentiation. It has been suggested that eIF-5A, localized to the nuclear pore, directs the translocation of specific mRNAs from the nucleus to the cytoplasm thereby facilitating the translation of particular proteins. Studies in tomato (Wang et al., Journal of Biological Chemistry, 276(20): 17541- 17549, 2001) revealed that messenger RNAs for DH S and eIF-5A are increased in senescing flowers and fruit and in leaves from plants that have been environmentally stressed (e.g., osmotic and temperature stress).
  • WO2005048692 discloses tomato plants having an alteration in the deoxyhypusine synthase activity by a non-transgenic mutation in at least one deoxyhypusine synthase gene. The mutation however yielded a male sterile phenotype. Male sterile plants cannot be self-pollinated in order to create homozygous lines and need to be crossed to pollinator or restorer lines having wild type genes and thus render heterozygous lines.
  • the present invention relates to an isolated polynucleotide comprising a nucleic acid sequence comprising at least 70% sequence identity to SEQ I D NO:l and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3. It is understood that as a result of the mutation the nucleotides at position 429-431 do not encode a valine, i.e. the mutation is not a silent mutation.
  • the present invention further relates to an isolated polynucleotide comprising a nucleic acid sequence as defined by SEQ I D NO: 1.
  • the invention also relates to an isolated polynucleotide comprising a nucleic acid sequence comprising at least 70% sequence identity to SEQ ID NO: 1 and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3, wherein the mutation is a mutation at position 429 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3, preferably said mutation is a change of nucleotide 429 into adenine.
  • the present invention relates to an isolated polynucleotide comprising a nucleic acid sequence comprising at least 70% sequence identity to the nucleic acid sequence of the cDNA or mRNA encoding the S. lycopersicum deoxyhypusine synthase (DHS) protein as found in a plant deposited under number NCI M B 42270 and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3.
  • DHS S. lycopersicum deoxyhypusine synthase
  • the present invention relates to an isolated polynucleotide comprising the nucleic acid sequence of the cDNA or mRNA encoding the S. lycopersicum DHS protein as found in a plant deposited under number NCI MB 42270.
  • the present invention relates further to an isolated polypeptide comprising an amino acid sequence comprising at least 70%o sequence identity to SEQ ID NO: 2 and comprising a mutation at position 126 when compared to the wild type amino acid sequence defined by SEQ ID NO: 4, preferably said mutation is a change of an amino acid 126 into isoleucine.
  • the present invention relates also to an isolated polypeptide comprising an amino acid sequence as defined by SEQ ID NO: 2.
  • the present invention relates in addition, to an isolated polypeptide comprising an amino acid sequence comprising at least 70% sequence identity to the amino acid sequence of the S. lycopersicum DHS protein as found in a plant deposited under number NCI MB 42270 and comprising an isoleucine at position 126.
  • the present invention relates also to an isolated polypeptide comprising the amino acid sequence of the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCIMB 42270.
  • the present invention relates to an isolated polynucleotide comprising a genomic nucleic acid sequence comprising at least 70% sequence identity to SEQ ID NO: 5 and comprising a mutation at any of position 2882-2883 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 6.
  • the invention relates to an isolated polynucleotide comprising a genomic nucleic acid sequence encoding a polypeptide comprising an amino acid sequence comprising at least 70% (e.g. 75%, 80%, 85%, 90%, 95%, 98%, 99%) amino acid sequence identity to SEQ ID NO:2 and said polypeptide comprising a mutation at position 126 when compared to the wild type amino acid sequence defined by SEQ ID NO: 4, preferably said mutation is a change of an amino acid 126 into isoleucine.
  • a genomic nucleic acid sequence encoding a polypeptide comprising an amino acid sequence comprising at least 70% (e.g. 75%, 80%, 85%, 90%, 95%, 98%, 99%) amino acid sequence identity to SEQ ID NO:2 and said polypeptide comprising a mutation at position 126 when compared to the wild type amino acid sequence defined by SEQ ID NO: 4, preferably said mutation is a change of an amino acid 126 into isoleucine.
  • the present invention relates also to an isolated polynucleotide comprising a genomic nucleic acid sequence encoding a polypeptide comprising an amino acid sequence as defined by SEQ I D NO: 2.
  • the present invention relates to an isolated polynucleotide comprising a nucleic acid sequence as defined by SEQ ID NO: 5.
  • the present invention relates to an isolated polynucleotide comprising a nucleic acid sequence comprising at least 70% sequence identity to the nucleic acid sequence of the genomic polynucleotide encoding the S. lycopersicum DHS protein as found in a plant deposited under number NC 1 MB 42270 and comprising a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6, preferably said mutation is a change of nucleotide 2882 into adenine.
  • the present invention also relates to an isolated polynucleotide comprising a nucleic acid sequence of the genomic polynucleotide encoding the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NC!M B 42270.
  • the present invention relates to an isolated polynucleotide wherein the polynucleotide hybridizes with SEQ I D NO: l or SEQ I D NO: 5.
  • the polynucleotide hybridizing with SEQ I D NO:l or SEQ I D NO: 5 comprises nucleic acids that hybridize to a mutation at any of position 429- 430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 or hybridizes with SEQ I D NO: 5 comprising a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6.
  • the invention relates to an isolated polynucleotide which hybridizes with SEQ ID NO: I wherein the isolated polynucleotide hybridizes to a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3, and wherein nucleotides 429-43 1 do not encode for amino acid valine, i.e.
  • the polynucleotide comprises nucleotides corresponding to position 429-431 of S EQ I D NO: 3 and the polynucleotide sequence does not encode the wild type DH S sequence; or in another embodiment to an isolated polynucleotide w ich hybridizes with SEQ I D NO: 5 wherein the isolated polynucleotide hybridises to a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6 and wherein nucleotides 2882-2884 do not encode for amino acid valine and the polynucleotide comprises nucleotides corresponding to position 2882-2884 of SEQ I D NO:6; or in yet another embodiment the invention relates to a polynucleotide encoding for the polypeptide as defined by SEQ ID NO: 2.
  • the nucleotide sequence hybridizing to the wild type sequence is at least 300 nucleotides long, e.g. at least 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, or even at least 1610 nucleotides long.
  • the present invention relates further to a tomato plant or a tomato fruit comprising any of the polynucleotides of the invention and/or embodiments thereof.
  • the present invention relates to a tomato plant or a tomato fruit comprising any polypeptide of the invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate further to a tomato fruit having a shape selected from the group consisting of cherry, bell, blocky, currant, deep round oval or roma, flattened globe, grape, long blocky, long pointed, oxheart, pear, beefsteak, round, small pear, small pointed, stuffer, and plum.
  • the present invention and/or embodiments thereof relate further to a tomato plant producing a tomato fruit having a shape selected from the group consisting of cherry, bell, blocky, currant, deep round oval or roma, flattened globe, grape, long blocky, long pointed, oxheart, pear, beefsteak, round, small pear, small pointed, stuffer, and plum.
  • the present invention and/or embodiments thereof relate further to a tomato plant producing a tomato fruit having a having a skin colour selected from the group consisting of dark, bi-color. deep pink, golden, green, orange, pink, red, white, and yellow.
  • the present invention and/or embodiments thereof relate further to a tomato fruit having a having a skin colour selected from the group consisting of dark, bi-color, deep pink, golden, green, orange, pink, red, white, and yellow.
  • the present invention and/or embodiments thereof relate to a tomato plant that is detenriinate.
  • the present invention and/or embodiments thereof relate to a tomato plant that is indeterminate. [0031] In one embodiment, the present invention and/or embodiments thereof relate to a tomato plant that is heirloom, open-pollinated or hybrid. In another embodiment, the present invention and/or embodiments thereof relate to a hybrid tomato plant.
  • the present invention and/or embodiments thereof relate to a tomato plant, tomato fruit, cell of a tomato plant and/or part of a tomato plant, wherein the polynucleotide of the invention and/or embodiments thereof is present in at least one allele, preferably in two alleles.
  • the present invention and/or embodiments thereof relate to a tomato plant, tomato fruit, cell of a tomato plant and/or part of a tomato plant, wherein the polynucleotide of the invention and/or embodiments thereof is present in homozygous form.
  • the present invention and/or embodiments thereof relate to a tomato plant, tomato fruit, cell of a tomato plant and/or part of a tomato plant, wherein the polynucleotide of the invention and/or embodiments thereof is present in heterozygous form.
  • the present invention and/or embodiments thereof relate to a tomato fruit or a tomato plant producing a tomato fruit wherein the tomato fruit has slower softening and/or increased firmness and/or increased shelf life after harvest compared to a tomato fruit from a tomato plant comprising the wild type DHS polynucleotide as defined by SEQ I D NO 3 or 6, or the wild type DHS polypeptide as defined by SEQ ID NO: 4.
  • the present invention and/or embodiments thereof relate to tomato fruit or a tomato plant producing a tomato fruit wherein the tomato fruit has a firmness of at least 5.5 N/mm at 2 weeks after harvest, preferably at least 6 N/mm, more preferably at least 6.5 N/mm. It is understood that when reference is made to N/mm, this is according to the method as discussed in the examples.
  • the present invention and/or embodiments thereof relate to a tomato fruit or a tomato plant producing a tomato fruit wherein the tomato fruit has a firmness of at least 4.5 N/mm at 4 weeks after harvest, preferably at least 5 N/mm, more preferably at least 5.5 N/mm.
  • the present invention and/or embodiments thereof relate to tomato fruit or a tomato plant producing a tomato fruit wherein the tomato fruit has a firmness at 2-4 weeks after harvest that is at least 0.5 N/mm higher than a tomato fruit from a tomato plant comprising the wild type DHS polynucleotide as defined by SEQ I D NO 3 or 6, or the wild type DHS polypeptide as defined by SEQ ID NO: 4.
  • the present invention and/or embodiments thereof relate to a tomato plant according to the invention and/or any embodiment thereof comprising at least one desirable trait selected from the group consisting of total solids, pl l, Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin color, leaf length, internode length, fruit flesh color, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • the present invention and/or embodiments thereof relate to a tomato plant, tomato fruit, cell of a tomato plant and/or part of a tomato plant according to the invention and/or any embodiment thereof comprising a vector encoding for at least one desirable trait selected from the group consisting of total solids, pH, Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin color, leaf length, internode length, fruit flesh color, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • the present invention and/or embodiments thereof relate to pollen produced by a tomato plant according to the invention and/or any embodiment thereof.
  • the present invention and/or embodiments thereof relate to pollen produced by a tomato plant wherein the polynucleotide according to the invention and/or any embodiment thereof is present or wherein the polypeptide according to the invention and/or any embodiment thereof is present.
  • the present invention and/or embodiments thereof relate to seed produced by a tomato plant according to the invention and/or any embodiment thereof.
  • the present invention and/or embodiments thereof relate to seed produced by a tomato plant wherein the polynucleotide according to the invention and/or any embodiment thereof is present or wherein the polypeptide according to the invention and/or any embodiment thereof is present.
  • the present invention and/or embodiments thereof relate to seed from which a plant according to the invention and/or any embodiment thereof can be grown. In a preferred embodiment, the present invention and/or embodiments thereof relate to seed wherein the polynucleotide according invention and/or any embodiment thereof is present or wherein the polypeptide according to invention and/or any embodiment thereof is present. [0043] In another embodiment, the present invention and/or embodiments thereof relate to a tomato plant grown from seeds according to the present invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to food and/or a food product comprising the tomato fruit according to the present invention and/or embodiments thereof or parts thereof.
  • the present invention and/or embodiments thereof relate to food and/or a food product comprising the tomato fruit wherein the polynucleotide according to the present invention and/or embodiments thereof is present or wherein the polypeptide according to the present invention and/or embodiments thereof is present.
  • the present invention and/or embodiments thereof relate to a plant part of the tomato plant or tomato fruit according to the present invention and/or embodiments thereof, wherein the part is selected from the group consisting of a leaf, anther, pistil, stem, petiole, root, scion, rootstock, ovule, pollen, protoplast, tissue, seed, fruit, flower, cotyledon, hypocotyl, embryo and cell.
  • the present invention and/or embodiments thereof relate to a plant part of the tomato plant r tomato fruit according to the present invention and/or embodiments thereof, wherein the polynucleotide according to the present invention and/or embodiments thereof is present or wherein the polypeptide according to the present invention and/or embodiments thereof is present.
  • the present invention and/or embodiments thereof relate to a tomato plant comprising a scion according to the present invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to a tomato plant cell comprising a polynucleotide according to the present invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to a tomato plant cell comprising a polypeptide according to the present invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to a isolated tomato plant cell comprising a polynucleotide according to the present invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to an isolated tomato plant cell comprising a polypeptide according to the present invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to a tomato plant, tomato fruit, plant part and/or plant cell comprising a vector encoding for at least one desirable trait selected from the group consisting of total solids, pi I, Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin color, leaf length, internode length, fruit flesh color, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • the present invention and/or embodiments thereof relate to a vector comprising a polynucleotide according to the present invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to a vector comprising a polynucleotide comprising the nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of the cDNA or itiR A encoding the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NO MB 42270.
  • the present invention and/or embodiments thereof relate to a vector comprising a polynucleotide comprising the nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of the cDNA or mRNA encoding the S.
  • the present invention and/or embodiments thereof relate to a vector comprising a nucleotide sequence encoding a deoxyhypusine synthase protein according to the present invention and/or embodiments thereof.
  • the vector according present invention and/or embodiments thereof further comprises regulatory sequences operatively linked to the nucleic acid sequence such that the nucleic acid sequence is expressed in a plant cell into which it is transformed.
  • the present invention and/or embodiments thereof relate to a tomato plant, tomato fruit, plant cell or plant part according present invention and/or embodiments thereof comprising a vector according to the present invention and/or embodiments thereof.
  • the present invention further relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof, said method comprising the steps of
  • the present invention and/or embodiments thereof relate to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof wherein the introduction of a nucleic acid sequence according to the present invention and/or embodiments thereof into a plant cell is by crossing two tomato plants.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof wherein the introduction of a nucleic acid sequence according to the present invention and/or embodiments thereof into a plant cell is by crossing two tomato plants, wherein at least one tomato plant comprises a nucleic acid sequence according to the present invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato fruit, and/or a tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof wherein the introduction of a nucleic acid sequence according to the present invention and/or embodiments thereof into a plant cell is by introducing a vector according to the present invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato fruit, and/or a tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof wherein the introduction of a nucleic acid sequence according to the present invention and/or embodiments thereof into a plant cell is by introducing a mutation in a polynucleotide encoding a DH S protein in a tomato plant.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato fruit, and/or a tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof wherein the introduction of a nucleic acid sequence according to the present invention and/or embodiments thereof into a plant cell is by introducing a mutation in a polynucleotide encoding a DHS protein in a tomato plant by using a mutagen or radiation.
  • the present invention further relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit according to the invention and/or embodiments thereof comprising:
  • the present invention further relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit according to the invention and/or embodiments thereof comprising:
  • the present invention and/or embodiments relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato according to the invention and/or embodiments thereof wherein the second tomato plant is an inbred line.
  • the present invention and/or embodiments relate(s) to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit according to the invention and/or embodiments thereof wherein the first tomato plant comprises a vector encoding a desirable trait.
  • the present invention and/or embodiments relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit according to the invention and/or embodiments thereof comprising a further step of selecting tomato plants producing tomato fruit having an increased firmness, delayed softening and/or increased shelf life after harvest than a tomato fruit from a tomato plant comprising the wild type DHS polynucleotide as defined by SEQ ID NO 3 or 6, or the wild type DHS polypeptide as defined by SEQ I NO: 4.
  • the present invention and/or embodiments relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit according to the invention and/or embodiments thereof comprising a further step of selecting tomato plants producing tomato fruit having tomato fruit has a firmness of at least 5.5 N/mm at 2 weeks after harvest at full red stage, preferably at least 6 N/mm, more preferably at least 6.5 N/mm.
  • the present invention and/or embodiments relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit according to the invention and/or embodiments thereof comprising a further step of selecting tomato plants comprising a polynucleotide comprising a nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of the cDNA or mRNA polynucleotide of SEQ ID NO: 1 comprising a mutation at any of position 429- 430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e.
  • nucleotides at position 429-43 1 do not encode a valine; or a nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of the genomic polynucleotide of SEQ ID NO: 5 comprising a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine.
  • the present invention and/or embodiments thereof relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit according to the invention and/or embodiments thereof comprising a further step of selecting tomato plants comprising a polynucleotide hybridizing with SEQ ID NO:l comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e.
  • nucleotides at position 429-43 1 do not encode a valine; or a polynucleotide hybridizing with SEQ ID NO: 5 comprising a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ I NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine.
  • the polynucleotide hybridizing with SEQ I D NO:3 or SEQ I D NO: 6 is at least 300 nucleotides long. e.g. 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 or even at least 1600 nucleotides long.
  • the present invention and/or embodiments thereof relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit according to the invention and/or embodiments thereof comprising a further step of selecting tomato plants comprising a polynucleotide comprising the nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of the cDNA or mRNA encoding the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCI MB 42270 and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3; i.e.
  • the present invention and/or embodiments thereof relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato according to the invention and/or embodiments thereof comprising a further step of selecting tomato plants comprising a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 and comprising a mutation at position 126 when compared to the wild type amino acid sequence defined by SEQ ID NO: 4, preferably said mutation is a change of an amino acid 126 into isoleucine.
  • the present invention and/or embodiments thereof relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato according to the invention and/or embodiments thereof comprising a further step of selecting tomato plants comprising a polypeptide comprising the amino acid sequence having at least 70% sequence identity to the amino acid sequence of the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCI MB 42270 and said polypeptide comprising a mutation at position 126 when compared to the wild type amino acid sequence defined by SEQ I D NO: 4, preferably said mutation is a change of an amino acid 126 into isoleucine.
  • the present invention further relates to a method of producing a tomato plant, plant part and/or plant cell comprising a polynucleotide having a mutation in a nucleotide encoding for a deoxyhypusine synthase comprising the steps of:
  • the plant material in step (i) is a tomato seed.
  • the mutagen in step (ii) is EMS.
  • the present invention and/or embodiments thereof relates to a method of producing a tomato plant, tomato plant part and/or tomato plant cell comprising a polynucleotide having a mutation in a nucleotide encoding for a deoxyhypusine synthase wherein the mutation is a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e. nucleotides at position 429-431 do not encode a valine; or a mutation at any of position 2882- 2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine.
  • the present invention further relates to a method of producing a tomato plant comprising a mutation in the nucleic acid sequence encoding for deoxyhypusine synthase and further comprising a second trait, said method comprising the steps:
  • the present invention and/or embodiments thereof relates to a method of producing a tomato plant comprising a mutation in the nucleic acid sequence encoding for deoxyhypusine synthase and further comprising a second trait, wherein the trait is introduced into said tomato plant by a vector encoding for said trait.
  • the present invention and/or embodiments thereof relates to a method of producing a tomato plant comprising a mutation in the nucleic acid sequence encoding for deoxyhypusine synthase and further comprising a second trait, wherein the trait is introduced into said tomato plant by the steps of:
  • the present invention and/or embodiments thereof relates to a method of producing a tomato plant comprising a mutation in the nucleic acid sequence encoding for deoxyhypusine synthase and further comprising a second trait, wherein the trait is introduced into said tomato plant by further selecting a tomato plant having said mutation and having said second trait.
  • the present invention and/or embodiments thereof relates to a method of producing a tomato plant comprising a mutation in the nucleic acid sequence encoding for deoxyhypusine synthase and further comprising a second trait, wherein the trait is selected from the group consisting of total solids, pH. Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin color, leaf length, internode length, fruit flesh color, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof wherein the tomato fruit has a firmness of at least 5.5 N/mm at 2 weeks after harvest at full red stage, preferably at least 6 N/mm, more preferably at least 6.5 N/mm.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof wherein the tomato fruit has slower softening and/or increased firmness compared to a tomato fruit from a tomato plant comprising the wild type DHS polynucleotide as defined by SEQ ID NO 3 or 6. or the wild type DHS polypeptide as defined by SEQ I D NO: 4.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof wherein the tomato fruit has a firmness of at least 4.5 N/mm at 4 weeks after harvest, preferably at least 5 N/mm, more preferably at least 5.5 N/mm.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof wherein the tomato fruit has a firmness at 2-4 weeks after harvest that is at least 0.5 N/mm higher than a tomato fruit from a tomato plant comprising the wild type DHS polynucleotide as defined by SEQ ID NO 3 or 6, or the wild type DH S polypeptide as defined by SEQ ID NO: 4.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato plant according to the present invention and/or embodiments thereof wherein the tomato plant comprises at least one additional (second) desirable trait selected from the group consisting of total solids, pH, Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin color, leaf length, internode length, fruit flesh color, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • additional (second) desirable trait selected from the group consisting of total solids, pH, Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin color, leaf length, internode length, fruit flesh color, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato plant according to the present invention and/or embodiments thereof wherein the tomato plant comprises a vector encoding for at least one desirable trait selected from the group consisting of total solids, pi I. Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin color, leaf length, internode length, fruit flesh color, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof wherein the tomato fruit is having a shape selected from the group consisting of cherry, bell, blocky, currant, deep round oval or roma, flattened globe, grape, long blocky, long pointed, oxheart, pear, beefsteak, round, small pear, small pointed, stuffer, and plum, in one embodiment the plant of the invention produces tomato fruits for fresh market consumption (e.g. Fresh) .
  • the present invention and/or embodiments thereof relate to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof wherein the tomato fruit is having a skin colour selected from the group consisting of dark, bi-color, deep pink, golden, green, orange, pink, red, white, and yellow.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato plant according to the present invention and/or embodiments thereof wherein the tomato plant is determinate or indeterminate.
  • the present invention and/or embodiments thereof relate to a method of producing a tomato plant according to the present invention and/or embodiments thereof wherein the tomato plant is open-pollinated or hybrid. In a preferred embodiment, the present invention and/or embodiments thereof relate to a method of producing a tomato plant according to the present invention and/or embodiments thereof wherein the tomato plant is a hybrid tomato plant.
  • the present invention further relates to a method of screening for a tomato plant which produces tomato fruits that have an increased firmness and/or decreased softening and/or increases shelf life after harvest comprising:
  • the present invention and/or embodiments thereof relate to a method of screening for a tomato plant wherein the mutation is a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e. nucleotides at position 429-431 do not encode a valine; or a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine.
  • the present invention further relates to a method of screening tomato plants for the presence of a mutant DHS allele comprising the steps of
  • nucleotides at position 2882-2884 do not encode a valine; or having at least 70%o sequence identity to a nucleic acid sequence of the cDNA or niRNA encoding the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCIMB 42270 and having a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e. nucleotides at position 429-431 do not encode a valine.
  • the present invention and/or embodiments thereof relate to a method of screening tomato plants for the presence of a mutant DHS allele wherein said DNA from plant material from more than one tomato plant is pooled.
  • the present invention further relates to a method of screening tomato plants for the presence of a mutant DHS allele comprising the steps of
  • the present invention further relates to tomato plant, tomato seed, tomato fruit, tomato plant part, and/or tomato cell produced by any of the methods of aspect of the invention and/or embodiments thereof.
  • the present invention further relates to use of a tomato plant, tomato fruit, tomato plant part and/or tomato plan cell comprising a polynucleotide according to aspects of the invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to use of a tomato plant, tomato fruit, tomato plant part and/or tomato plan cell comprising a polynucleotide wherein the polynucleotide is having a sequence having at least 70%> sequence identity to a nucleic acid sequence as defined to SEQ I D NO 1 and having a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e.
  • nucleotides at position 429-43 1 do not encode a valine; or DNA having a sequence having at least 70% sequence identity to a nucleic acid sequence as defined to SEQ ID NO 5 and having a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine; or having at least 70% sequence identity to a nucleic acid sequence of the cDNA or mRNA encoding the S.
  • lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCI MB 42270 and having a mutation at any of position 2882- 2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine.
  • the present invention further relates to use of a tomato plant, tomato fruit, tomato plant part and/or tomato plan cell comprising a protein according to aspects of the invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to use of a tomato plant, tomato fruit, tomato plant part and/or tomato plan cell comprising a protein having a sequence having at least 70% sequence identity to the amino acid sequence as defined in SEQ ID NO: 2 and having a mutation at position 126 when compared to the wild type amino acid sequence as defined by SEQ ID NO: 4, or having at least 70% sequence identity to an amino acid sequence of the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCI MB 42270 and having a mutation at position 126 when compared to the wild type amino acid sequence as defined by SEQ I D NO: 4.
  • the present invention further relates to use of polynucleotide and/or polypetide according to any aspect of the invention and/or embodiments thereof for producing a tomato plant, tomato fruit, tomato plant cell or tomato plant pari.
  • the present invention and/or embodiments thereof relate to use of polynucleotide and/or polypetide according to any aspect of the invention and/or embodiments thereof for producing a tomato plant, tomato fruit, tomato plant cell or tomato plant part, wherein the tomato plant produces tomato fruit that has an increased firmness, and/or delayed softening, and/or increased shelflife when compared to a tomato fruit from a tomato plant comprising the wild type DI IS polynucleotide as defined by SEQ ID NO 3 or 6, or the wild type DHS polypeptide as defined by SEQ I D NO: 4.
  • the present invention and/or embodiments thereof relate to use of polynucleotide and/or polypeptide according to any aspect of the invention and/or embodiments thereof for producing a tomato plant, tomato fruit, tomato plant cell or tomato plant pan.
  • the use may comprise the following step: (i) Introducing into a plant cell a nucleic acid sequence according to aspects of the invention and/or embodiments thereof;
  • the present invention and/or embodiments thereof relate to use of polynucleotide and/or polypetide according to any aspect of the invention and/or embodiments thereof for producing a tomato plant, tomato fruit, tomato plant cell or tomato plant part wherein the introduction of the nucleic acid sequence is by introducing a vector comprising said nucleic acid sequence.
  • the present invention and/or embodiments thereof relate to use of polynucleotide and/or polypetide according to any aspect of the invention and/or embodiments thereof for producing a tomato plant, tomato fruit, tomato plant cell or tomato plant part wherein the introduction of the nucleic acid sequence is by introducing a mutation into the nucleic acid encoding S. lycopersicum deoxyhypusine synthase protein by a mutagen.
  • the present invention and/or embodiments thereof relate to use of polynucleotide and/or polypetide according to any aspect of the invention and/or embodiments thereof for producing a tomato plant, tomato fruit, tomato plant cell or tomato plant part wherein the introduction of the nucleic acid sequence is by crossing a tomato plant according to any aspect of the invention and/or embodiments thereof to a second tomato plant.
  • the present invention further relates to use of polynucleotide and/or polypetide according to the invention for screening for mutation according to any aspect of the present invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to use of polynucleotide and/or polypetide according to the invention for screening for mutation comprising the steps (i) provide plant material from a tomato plant;
  • nucleotides at position 2882-2884 do not encode a valine; or having at least 70%o sequence identity to a nucleic acid sequence of the cDNA or mRNA encoding the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCI MB 42270 and having a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e.
  • nucleotides at position 429-431 do not encode a valine; or detect the presence of a protein having a sequence hav ing at least 70% sequence identity to the amino acid sequence as defined in SEQ ID NO: 2 and having a mutation at position 126 when compared to the wild type amino acid sequence as defined by SEQ ID NO: 4, or having at least 70% sequence identity to an amino acid sequence of the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCIMB 42270 and having a mutation at position 126 when compared to the wild type amino acid sequence as defined by SEQ ID NO: 4.
  • the present invention and/or embodiments thereof relate to use of polynucleotide and/or polypetide according to the invention for screening for a mutation wherein said DNA or protein is from plant material from more than one tomato plant.
  • the present invention further relates to use of a polynucleotide for hybridising to a polynucleotide according to the invention and/or embodiments thereof for screening tomato plants and tomato plant material.
  • the present invention and/or embodiments thereof relate to use of polynucleotide for hybridising to a polynucleotide having a sequence having at least 70% sequence identity to a nucleic acid sequence as defined to SEQ I D NO 1 and having a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e.
  • nucleotides at position 429-431 do not encode a valine; or DNA having a sequence having at least 70% sequence identity to a nucleic acid sequence as defined to SEQ ID NO 5 and having a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine.
  • the present invention and/or embodiments thereof relate to use of polynucleotide for hybridizing to a polynucleotide having a sequence having at least 70% sequence identity to a nucleic acid sequence as defined to SEQ I D NO 1 and hybridizing to a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 or polynucleotide having a sequence having at least 70% sequence identity to a nucleic acid sequence as defined to SEQ I D NO 5 and hybridizing to a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6.
  • the present invention and/or embodiments thereof relate to the use of mutagen to producing a tomato plant, tomato seed, tomato plant part and/or tomato plant cell comprising a mutation in a nucleotide encoding for a S. lycopersicum deoxyhypusine synthase protein, comprising the steps of:
  • the present invention further relates to use of a tomato plant according to the present invention and/or embodiments thereof in a method to produce a transgenic tomato plant.
  • the present invention and/or embodiments thereof relate to use of a tomato plant according to the present invention and/or embodiments thereof in a method to produce a transgenic tomato plant producing a tomato fruit that has an increased firmness, and/or delayed softening, and/or increased shelflife when compared to a tomato fruit from a tomato plant comprising the wild type DHS polynucleotide as defined by SEQ ID NO 3 or 6, or the wild type DHS polypeptide as defined by SEQ I D NO: 4.
  • the present invention and/or embodiments thereof relate to use of a tomato plant according to the present invention and/or embodiments thereof in a method to produce a transgenic tomato plant comprising introducing nucleic acids encoding for a second trait into said tomato plant according to the present invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to use of a tomato plant according to the present invention and/or embodiments thereof in a method to produce a transgenic tomato plant comprising introducing nucleic acids encoding for a second trait into said tomato plant according to the present invention and/or embodiments thereof, wherein the introduction is by introduction of a vector encoding for a second trait.
  • the present invention and/or embodiments thereof relate to use of a tomato plant according to the present invention and/or embodiments thereof in a method to produce a transgenic tomato plant comprising introducing nucleic acids encoding for a second trait into said tomato plant according to the present invention and/or embodiments thereof, wherein the introduction is by crossing said tomato plant according to the present invention and/or embodiments thereof with a second tomato plant comprising said second trait.
  • the present invention further relates to tomato plant, tomato seed, tomato fruit, tomato plant part, and/or tomato cell produced by any of the use of the invention and/or embodiments thereof.
  • nucleic acid sequence or “nucleic acid molecule” or polynucleotide are used interchangeably and refer to a DNA or RNA molecule in single or double stranded form, particularly a DNA encoding a protein or protein fragment according to the invention.
  • isolated nucleic acid sequence refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome.
  • the term "gene” means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an in RNA or an RNAi molecule) in a cell, operably linked to suitable regulatory regions (e.g. a promoter).
  • a gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA ) and a 3' non-translated sequence comprising e.g. transcription termination sites.
  • a gene may be an endogenous gene (in the species of origin) or a chimeric gene (e.g. a transgene or cis-gene).
  • RNA which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide (or active peptide fragment) or which is active itself (e.g. in posttranscriptional gene silencing or RNAi).
  • the coding sequence may be in sense-orientation and encodes a desired, biologically active protein or peptide, or an active peptide fragment.
  • protein protein
  • amino acid sequence amino acid sequence
  • polypeptide polypeptide
  • a “fragment” or “portion” of DHS protein may thus still be referred to as a "protein”.
  • isolated protein is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
  • active protein or “functional protein” is a protein which has protein activity as measurable in vitro, e.g.
  • a wild type protein is a fully functional protein, as present in the wild type plant.
  • a “mutant protein” is herein a protein comprising one or more mutations in the nucleic acid sequence encoding the protein, whereby the mutation results in (the mutant nucleic acid molecule encoding) a protein having altered activity resulting in an increased firmness and/or slower softening of the tomato fruit, as e.g. measurable in vivo, e.g. by the phenotype conferred by the mutant allele.
  • “Functional derivatives” of the DHS protein as described herein are fragments, variants, analogues, or chemical derivatives of the DHS protein which retain at least a portion of the mutant DHS activity or immunological cross reactivity with an antibody specific for the mutant DHS.
  • a fragment of the mutant DHS protein refers to any subset of the molecule.
  • Variant peptides may be made by direct chemical synthesis, for example, using methods well known in the art.
  • An analogue of the mutant DHS refers to a non-natural protein substantially similar to either the entire protein or a fragment thereof.
  • Chemical derivatives of mutant DHS contain additional chemical moieties not normally a part of the peptide or peptide fragment. Modifications may be introduced into peptides or fragments thereof by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • a mutant DHS protein or peptide according to the invention may be produced by culturing a cell transformed with a nucleotide sequence of this invention allowing the cell to synthesize the protein and then isolating the protein, either as a free protein or as a fusion protein, depending on the cloning protocol used, from either the culture medium or from cell extracts.
  • the protein can be produced in a cell-free system. Ranu, et al., Meth. Enzymol, 60:459-484, (1979).
  • a "mutation" in a nucleic acid molecule is a change of one or more nucleotides compared to the wild type sequence, e.g. by replacement, deletion or insertion of one or more nucleotides.
  • a "point mutation” is the replacement of a single nucleotide, or the insertion or deletion of a single nucleotide.
  • a "silent mutation” is a mutation in a nucleic acid that does not change the amino acid sequence of the protein encoded by the nucleic acid.
  • a "mutation" in an amino acid molecule making up a protein is a change of one or more amino acids compared to the wild type sequence, e.g. by replacement, deletion or insertion of one or more amino acids.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the nucleic acid sequences being linked are typically contiguous.
  • Sequence identity and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned by for example the programs GAP or BE ST FIT or the Emboss program "Needle” (using default parameters, see below) share at least a certain minimal percentage of sequence identity (as defined further below). These programs use the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps.
  • the default scoring matrix used is DNAFULL and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915- 10919).
  • Sequence alignments and scores for percentage sequence identity may for example be determined using computer programs, such as EMBOSS
  • sequence similarity or identity may be determined by searching against databases such as FAST A, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity.
  • Two proteins or two protein domains, or two nucleic acid sequences have "substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more (as determined by Emboss "needle” using default parameters, i.e.
  • hybridisation is generally used to mean hybridisation of nucleic acids at appropriate conditions of stringency (stringent hybridisation conditions) as would be readily evident to those skilled in the art depending upon the nature of the probe sequence and target sequences.
  • Conditions of hybridisation and washing are well - known in the art, and the adjustment of conditions depending upon the desired stringency by varying incubation time, temperature and/or ionic strength of the solution are readily accomplished. See, for example, Sambrook. J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989.
  • the choice of conditions is dictated by the length of the sequences being hybridised, in particular, the length of the probe sequence, the relative G-C content of the nucleic acids and the amount of mismatches to be permitted.
  • Low stringency conditions are preferred when partial hybridisation between strands that have lesser degrees of complementarity is desired. When perfect or near perfect complementarity is desired, high stringency conditions are preferred.
  • the hybridisation solution contains 6X S.S.C., 0.01 M EDTA, I X Denhardt's solution and 0.5% SOS. hybridisation is carried out at about 68°C for about 3 to 4 hours for fragments of cloned ON A and for about 12 to about 16 hours for total eukaryotic ON A.
  • the temperature of hybridisation is reduced to about 42°C below the melting temperature (TM) of the duplex.
  • TM melting temperature
  • the TM is known to be a function of the G-C content and duplex length as well as the ionic strength of the solution.
  • hybridizes to a ON A or RNA molecule means that the molecule that hybridizes, e.g., oligonucleotide, polynucleotide, or any nucleotide sequence (in sense or antisense orientation) recognizes and hybridizes to a sequence in another nucleic acid molecule that is of approximately the same size and has enough sequence similarity thereto to effect hybridisation under appropriate conditions.
  • a 100 nucleotide long molecule from the 3' coding or non-coding region of tomato DHS will recognize and hybridize to an approximately 100 nucleotide portion of a nucleotide sequence within the 3' coding or non-coding region of the DHS gene or any other plant DHS gene so long as there is about 70% or more sequence similarity between the two sequences.
  • the size of the corresponding portion will allow for some mismatches in hybridisation such that the corresponding portion may be smaller or larger than the molecule which hybridizes to it, for example 20-30% larger or smaller, preferably no more than about 12- 15 % larger or smaller.
  • sequence comprising at least 70% sequence identity or "a sequence comprising at least 70% amino acid sequence identity” or "a sequence comprising at least 70% nucleotide sequence identity” means a sequence having at least 70%o e.g. at least 72%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even at least 99.1% e.g. at least 99.2%, 99.4%, 99.5%, or even 99.7%o or 99.8%) or 99.9%o sequence identity when compared with the reference sequence that is indicated. Sequence identity can be determined according the methods described herein.
  • allele(s) means any of one or more alternative forms of a gene at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus.
  • alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome.
  • loci plural locus on a chromosome.
  • One allele is present on each chromosome of the pair of homologous chromosomes.
  • a diploid plant species may comprise a large number of different alleles at a particular locus. These may be identical alleles of the gene (homozygous) or two different alleles (heterozygous).
  • locus means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found.
  • the DHS locus is thus the location in the genome where the DHS gene is found.
  • Wild type allele refers herein to a version of a gene encoding a fully functional protein (wild type protein).
  • a fully functional DHS protein is for example the wild type DHS cDNA (mRNA) sequence depicted in SEQ I D NO: 3, based on ( ion Bank Accession No. AF296077 or the wild type DHS genomic sequence depicted in SEQ I D NO: 6.
  • the protein sequence encoded by this wild type DHS mRNA is depicted in SEQ I NO: 4. It consists of 383 amino acids.
  • mutant allele refers herein to an allele comprising one or more mutations in the coding sequence (mRNA, cDNA or genomic sequence) compared to the wild type allele.
  • Such mutation(s) e.g. insertion, inversion, deletion and/or replacement of one or more nucleotide(s)
  • Such changes may lead to the protein having a different D conformation, being targeted to a different sub-cellular compartment, having a modified catalytic domain, having a modified binding activity to nucleic acids or proteins, etc.
  • Wild type plant and "wild type fruit” or “normal ripening” plants/ fruits” refers herein to a tomato plant or tomato fruit comprising two copies of a wild type (WT) DHS allele ( DHS DHS) en coding a fully functional DHS protein (e.g. i n contrast to "mutant plants", comprising a mutant DHS allele).
  • WT wild type
  • DHS DHS DHS allele
  • Such plants are for example suitable controls in phenotypic assays.
  • wild type and/or mutant plants are "cultivated tomato plants”.
  • the cuitivar Moneymaker is a wild type plant, as is cuitivar Ailsa Craig, cuitivar Tapa and many others.
  • “Azygous plant” and “azygous fruit” refers herein to a tomato plant having the same genetic background as the identified mutant plant however without the mutant DHS allele.
  • Other fully functional mutant DHS protein-encoding alleles may exist in other Solarium lycopersicum plants and may comprise substantial sequence identity with SEQ I D NO: 4 , i.e. having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% sequence identity with SEQ I D NO: 4.
  • Such fully functional mutant DHS proteins are herein referred to as "variants" of SEQ I D NO: 4.
  • nucleotide sequences encoding such fully functional mutant DHS proteins are referred to as variants of SEQ I D NO: 3 and SEQ I D NO: 6.
  • Mutant alleles or sequences code for a protein with a mutation as defined by SEQ I D NO: 2 and/or carry the mutation as defined by SEQ I D NO: 1 , and/or SEQ I D NO: 5.
  • Mutant alleles or sequences encode a DHS protein having an amino acid mutation at position 126 when referred to the wild type sequence [uniprot: Q9AXR0] or a DHS protein from a plant as deposited under NCI MB 42270 .
  • Mutant alleles or sequences encode a DHS protein having a isoleucine at position 126 when referred to the wild type sequence [uniprot: Q9AXR0]. Mutant alleles or sequences have a mutation at position 429 and/or 430 that change the amino acid in the encoded protein, when referred to the wild type cDNA sequence [NM_001247566]. In one embodiment, Mutant alleles or sequences have a mutation as present in the DHS gene of the plant deposited under NCI MB 42270.
  • nucleotide sequences referred to herein are cDNA, i.e. coding DNA sequences, encoding the proteins of SEQ I D NO: 2 and SEQ I D NO: 4.
  • cDNA is the coding region of the corresponding Solarium lycopersicum genomic DHS sequence, which, however, additionally contains introns and therefore the nucleotides have different numbering.
  • a tomato plant comprising an DHS sequence according to e.g.
  • any one of SEQ I D NO: 1 or SEQ I D NO: 3 it is, therefore, understood that the tomato plant comprising the genomic DHS sequence which comprises the coding DNA (cDNA), from which the mRN of SEQ I D NO: 1 or SEQ I D NO: 3 is transcribed (and which is in turn translated into protein).
  • the mRNA has the same nucleotide sequence as the cDNA, accept that Thymine (t) is Uracil (u) in the mRNA.
  • the plant comprises the genomic DHS sequence depicted in SEQ I D NO: 5 or a genomic DHS sequence substantially identical thereto (e.g.
  • mutant DHS alleles are exemplary of the increased firmness and/or slower softening and/or extended sheif-life conferring DHS mutations identified according to the present invention.
  • One exemplary mutant DHS allele conferring increased firmness and/or slower softening and/or extended shelf-life identified according to the present invention comprises a mutation.
  • the mutation is preferably resulting of the change of a valine (val or V) into a isoleucine (ile or I) at amino acid 126 when compared to the wild type protein (SEQ ID NO: 4).
  • the protein sequence of the DHS mutant is depicted in SEQ I D NO: 2.
  • the amino acid substitution is due to a guanine (G) to adenine (A) mutation at nucleotide 429 when compared to the wild type sequence of SEQ ID NO: 3.
  • the mutant cDNA is depicted in SEQ ID NO: 1.
  • the mutant genomic DNA is depicted in SEQ I D NO: 5. It is noted that due to the degeneracy of the nucleotides codons for amino acids, the nucleotide 430 and 431 may be mutated as well to give a isoleucine. Exemplary mutants are the change of 431T to 431C or 431 A.
  • the term "functional derivative" of a nucleic acid is used herein to mean a fragment, variant, homolog, or analogue of the gene or nucleotide sequence encoding DHS.
  • a functional derivative may retain at least a portion of the function of the DHS encoding DNA such as increased firmness, delayed softening and/or increased shelf life. Such function may include the ability to hybridize under low stringency conditions with native tomato DHS or substantially homologous DNA from another plant which encodes DHS or with an mRNA transcript thereof.
  • the functional derivative comprises the mutation of the invention.
  • a "fragment" of the gene or DNA sequence refers to any subset of the molecule, e.g., a shorter polynucleotide or oligonucleotide. In one aspect the fragment comprises the mutation as defined by the invention.
  • a “variant” refers to a molecule substantially similar to either the entire gene or a fragment thereof, such as a nucleotide substitution variant having one or more substituted nucleotides, but which maintains the ability to hybridize with the particular gene or to encode mRNA transcript which hybridizes with the native DNA.
  • the variant comprises the mutation as defined by the invention.
  • a “homologue” refers to a fragment or variant sequence from a different plant genus or species.
  • the homologue comprises the mutation as defined by the invention.
  • an "analogue” refers to a non-natural molecule substantially similar to or functioning in relation to either the entire molecule, a variant or a fragment thereof.
  • the analogue comprises the mutation as defined by the invention.
  • the term "plant” includes the whole plant or any parts or derivatives thereof, such as plant organs (e.g., harvested or non-harvested fruits, flowers, leaves, etc.), plant cells, plant protoplasts, plant cell or tissue cultures from which whole plants can be regenerated, regenerable or non- regenerabie plant cells, plant calli, plant cell clumps, and plant cells that are intact in plants, or parts of plants, such as embryos, pollen, ovules, ovaries, fruits (e.g., harvested tissues or organs, such as harvested tomatoes or parts thereof), flowers, leaves, seeds, tubers, clonally propagated plants, roots, stems, cotyledons, hypocotyls, root tips and the like.
  • plant organs e.g., harvested or non-harvested fruits, flowers, leaves, etc.
  • plant protoplasts e.g., plant protoplasts, plant cell or tissue cultures from which whole plants can be regenerated, regenerable or non- regenerabie plant cells, plant call
  • a “plant line” or “breeding line” refers to a plant and its progeny.
  • the term “inbred line” refers to a plant line which has been repeatedly selfed.
  • Plant variety is a group of plants within the same botanical taxon of the lowest grade known, which (irrespective of whether the conditions for the recognition of plant breeder's rights are fulfilled or not) can be defined on the basis of the expression of characteristics that result from a certain genotype or a combination of genotypes, can be distinguished from any other group of plants by the expression of at least one of those characteristics, and can be regarded as an entity, because it can be multiplied without any change.
  • plant variety cannot be used to denote a group of plants, even if they are of the same kind, if they are all characterized by the presence of 1 locus or gene (or a series of phenotypical characteristics due to this single locus or gene), but which can otherwise differ from one another enormously as regards the other loci or genes.
  • Fl, F2, etc. refers to the consecutive related generations following a cross between two parent plants or parent lines. The plants grown from the seeds produced by crossing two plants or lines is called the Fl generation. Selfing the Fl plants results in the F2 generation, etc.
  • Fl hybrid plant (or Fl seed) is the generation obtained from crossing two inbred parent lines.
  • An "Ml population” is a plurality of mutagenized seeds / plants of a certain plant line or cultivar.
  • M2, M3, M4, etc.” refers to the consecutive generations obtained following selfing of a first mutagenized seed / plant (Ml).
  • Tomato plants or “cultivated tomato plants” are plants of the Solanum lycopersicum, i.e.
  • Wild plants include for example ecotypes, PI (Plant Introduction) lines, landraces or wild accessions or wild relatives of a species.
  • heirloom varieties or cultivars i.e. open pollinated varieties or cultivars commonly grown during earlier periods in human history and often adapted to specific geographic regions, are in one aspect of the invention encompassed herein as cultivated tomato plants.
  • the tomato plant may, be any cultivated tomato, any commercial variety, any breeding line or other, it may be determinate or indeterminate, open pollinated or hybrid, producing fruit flesh of any color, fruits of any shape and size.
  • the tomato plant may be determinate or indeterminate, heirloom, or hybrid.
  • the tomato fruit may have different sizes and shapes, such as cherry, bell, blocky, currant, deep round oval or roma, flattened globe, grape, long blocky, long pointed, oxheart, pear, beefsteak, round, small pear, small pointed, stuffer, plum.
  • the skin color of the tomato may vary from deep red, purple, bright red, to yellow, and even pink. Recognized skin colors of tomatoes are for example, dark, bi-color, deep pink, golden, green, orange, pink, red, white, and yellow.
  • Table 2-9 give an exemplary overview of tomato varieties sorted by colours and table 10-19 give an exemplary overview of tomato varieties sorted by shape.
  • the term "food” is any substance consumed to provide nutritional support for the body. It is usually of plant or animal origin, and contains essential nutrients, such as carbohydrates, fats, proteins, vitamins, or minerals. The substance is ingested by an organism and assimilated by the organism's cells in an effort to produce energy, maintain life, or stimulate growth. The term food includes both substance consumed to provide nutritional support for the human and animal body.
  • shelf life or "post-harvest shelf life” designates the (average) length of time that a fruit is given before it is considered unsuitable for sale or consumption ('bad'). Shelf life is the period of time that products can be stored, during which the defined quality of a specified proportion of the goods remains acceptable under expected conditions of distribution, storage and display.
  • Shelf life is influenced by several factors: exposure to light and heat, transmission of gases (including humidity), mechanical stresses, and contamination by things such as micro-organisms .
  • Product quality is often mathematically modelled around the fruit firmness/softness parameter. Shelf-life may be defined as the (average) time it takes for fruits of a plant line to start to become bad and unsuitable for sale or consumption, starting for example from the first fruit of a plant entering breaker stage or turning stage or from the first fruit becoming fully red or from harvest.
  • the mutants according to the invention have a shelf life that is significantly longer than the shelf life of wild type plants, for example the number of days from the first fruit being in breaker stage (or turning stage, pink stage, red stage or from harvest) p to the first fruit starting to become 'bad' and unsuitable for sale or consumption is significantly longer, e.g. at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, days longer than fruits of control plants (such as wild type DHS plants), when plants are grown under the same conditions and fruits are treated the same way and kept under the same conditions.
  • control plants such as wild type DHS plants
  • the day when the first fruit of the wild type control plant (grown under the same conditions as the mutant plants and being at the same developmental stage) enters a certain stage can, for example, be taken as the starting point (day 1) from when on periodically (at certain time intervals, e.g. after 1, 2, 3, 4, 5 or 6 days) the fruits are observed until the day that the first fruit has passed the fully ripe stage and becomes 'bad' (as determinable visually and/or through assessing fruit softness).
  • the "ripening stage" of a tomato fruit can be divided as follows: (1) Mature green stage: surface is completely green; the shade of green may vary from light to dark. (2) Breaker stage: there is a definite break in color from green to tannish-yellow, pink or red on not more than 10% of the surface; (3) Turning stage: 10% to 30% of the surface is not green; in the aggregate, shows a definite change from green to tannish-yellow, pink, red, or a combination thereof. (4) Pink stage: 30% to 60 %> of the surface is not green; in the aggregate, shows pink or red color. (5) Light red stage: 60% to 90%>o of the surface is not green; in the aggregate, shows pinkish-red or red.
  • Mature Red stage More than 90% of the surface is not green; in the aggregate, shows red color. Colours may be measured with chromameters, such as the Minolta CR-400 Chromameter. Values between 10 and 20 for *a in CIE L*a*b color space measurements are indicative of a pink stage. Values greater than 20 for *a are indicative of the light red stage. Values may be determined by colour charts, see e.g. USDA, 1997. United States Standards for Grade of Fresh Tomatoes. US department of Agriculture Agricultural Marketing, Service, Washington, DC, or RH S Colour chart, Royal Horticultural Society, 2007.
  • Average refers herein to the arithmetic mean.
  • comparisons between different plant lines involves growing a number of plants of a line (e.g. at least 5 plants, preferably at least 10 plants per line) under the same conditions as the plants of one or more control plant lines (preferably wild type plants) and the determination of statistically significant differences between the plant lines when grown under the same environmental conditions.
  • the plants are of the same line or variety.
  • several fruits are harvested and measured for an average number. Each measurement is done on a fresh tomato, i.e. a tomato that has not been measured before. The measurement causes soft spots in the tomato and they may rot sooner. Further for the best comparison, the fruits are measured each time at the same spot on the tomato.
  • Softness or firmness may be measured by flat-plate compression, constant area compression or by puncture.
  • flat-plate compression tests whole tomato fruit are placed on a stationary plate. A specific force moves an upper plate to compress tomatoes at a certain deformation speed.
  • constant area compression tests pericarp samples are excised from the equatorial region of each tomato fruit. Thickness of tissue sections generally ranged from 6.5 to 7.5 mm. The excised pericarp Sections, with epidermal tissue intact, are placed exocarp down on a plate and compressed with a flat-ended cylindrical probe at a specific force and deformation speed. During puncture tests, whole tomato fruit are placed in a holder to prevent slipping.
  • Non-destructive firmness test are described in e.g. Panmanas Sirisomboon, Munehiro Tanaka. Takayuki Kojima (2012) Evaluation of tomato textural mechanical properties, J. Food Eng.; I l l (4):618— 624. and Cheng-chang Lien, Chyung Ay and Ching-Hua Ting (2009) Non-destructive impact test for assessment of tomato maturity, J. Food Eng. 91 : 402-407.
  • shelf-life the words “improved”, “increased”, “longer” and “extended” as used in conjunction with the word “shelf-life” are interchangeable and all mean that the fruits of a tomato plant according to the invention have on average, a longer shelf-life than the control fruits with wild type DHS.
  • SEQ ID NO: 1 shows the mutant dhs cDNA of the Solanum lycopersicum mutant having a mutation G429A.
  • SEQ I D NO: 2 shows the mutant dhs amino acid sequence of the Solanum lycopersicum mutant having a mutation V126I.
  • SEQ ID NO: 3 shows the Solanum lycopersicum wild type DHS cDNA based on NCBI Reference Sequence: AF296077; 1610 nucleotides
  • SEQ ID NO: 4 shows the Solanum lycopersicum wild type DHS protein sequence as derived from the mR A based on NCBI Reference Sequence: AF296077;.
  • SEQ ID NO: 5 shows the Solanum lycopersicum mutant DHS genomic DNA having a mutation G2882A when referred to wild type genomic sequence SL2.40ch02:39508000-39514899.
  • SEQ ID NO: 6 shows the Solanum lycopersicum wild type DHS genomic DNA as obtained from the solgenomics network sequence region SL2.40ch02:39508000-39514899). The position of the exons as described herein, is derived from this sequence.
  • SEQ I D NO: 7 shows the Forward primer to amplify DHS fragment.
  • SEQ I D NO: 8 shows the Reverse primer to amplify DHS fragment.
  • Figure 1 Measurement of firmness of tomato fruits 0-21 days after harvest for mutant plant of mutant 4282 (homozygous and heterozygous for the amino acid mutation 126V to 1261, and wild type DHS tomato plants.
  • Figure 2 Impact mutation 429GTT431 to 429ATT431 , 126V to 1261.
  • Figure 3 Alignment of SEQ I D NO: 2 (mutant 4282) with wild type amino acid sequence (SEQ I D NO: 4; AF296077).
  • Figure 4 Wild type genomic DNA (SEQ I D NO: 6) aligned with wild type cDNA (SEQ I D NO: 3) indicated in Figure 4 as "AF296077" showing intron and exon positions. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention relates to tomato plants and tomato fruits exhibiting an altered deoxyhypusine synthase (DHS) enzyme activity.
  • the altered DHS enzyme activity is obtained without the inclusion of foreign nucleic acids in the tomato plants' genomes.
  • the tomato plants of the present invention are non-transgenic.
  • the present invention further describes non-transgenic mutations in a deoxyhypusine synthase gene or allele of a tomato plant or a tomato fruit.
  • Deoxyhypusine synthase (EC 2.5.1.46) is an enzyme which is also known under its system name (eIF5A-precursor)-lysine:spermidine 4-aminobutyltransferase (propane- 1 ,3 -diamine-forming) DHS catalyzes the NAD-dependent oxidative cleavage of spermidine and the subsequent transfer of the butylamine moiety of spermidine to the epsilon-amino group of a specific lysine residue of the eIF-5A precursor protein to form the intermediate deoxyhypusine residue. DHS is also able to produce homospermidine from putrescine. The protein is found in all eukaryotic cells and is thought to be involved in senescence.
  • the altered DHS may be created and identified by a method known as TILLING.
  • TILLING Targeting Induced Local Lesions IN Genomes
  • TILLING is a general reverse genetic technique that uses traditional chemical mutagenesis methods to create libraries of mutagenized individuals that are later subjected to high throughput screens for the discovery of mutations.
  • TILLING combines chemical mutagenesis with mutation screens of pooled PCR products, resulting in the isolation of missense and non-sense mutant alleles of the targeted genes.
  • TILLING uses traditional chemical mutagenesis (e.g. EMS or MNU mutagenesis) or other mutagenesis methods (e.g.
  • SI nucleases such as CELl or ENDOl
  • electrophoresis such as a LI -COR gel analyzer system, see e.g. Henikoff et al. Plant Physiology 2004, 135: 630-636.
  • TILLING has been applied inmany plant species, such as tomato. (see world wide web tiling.ucdavis,edu/''index.php/Tomato__Tilling), rice (Till et al.
  • the invention and/or embodiments thereof is directed to a mutation in a polynucleotide encoding a mutant DHS protein.
  • the mutation may be located at position 429-430 i.e. nucleotides at position 429-43 1 do not encode a valine.
  • the isolated polynucleotide comprises a mutation at position 429 or 430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3.
  • the isolated polynucleotide comprises a mutation at position 429 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3.
  • the isolated polynucleotide comprises a mutation into an adenine (A).
  • Nucleotides 429-431 are GTT in wild type cDNA encoding wild type DHS protein.
  • GTT encodes a valine.
  • a mutation of the guanine (G) into an adenine (A) changes the GTT into ATT which encodes an isoleucine.
  • amino acid codons are degenerate and more than one three- nucleotide codon may code for an amino acid.
  • codons code for a valine GTT, GTC, GTA, GTG, and the following codons code for isoleucine ATT, ATC, ATA.
  • the present invention relates therefor to an isolated polynucleotide comprising a nucleic acid sequence comprising at least 70% sequence identity to SEQ ID NO:l and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e. nucleotides at position 429-431 do not encode a valine.
  • the present invention relates to an isolated polynucleotide comprising a nucleic acid sequence comprising at least 70% sequence identity to the nucleic acid sequence of the cDNA or mRNA encoding the S.
  • lycopersicum deoxyhypusme synthase ( DHS) protein as found in a plant deposited under number NCIMB 42270 and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e. nucleotides at position 429-43 1 do not encode a valine.
  • the present invention relates to an isolated polynucleotide comprising the nucleic acid sequence of the cDNA or mRNA encoding the S. lycopersicum DHS protein as found in a plant deposited under number NCIMB 42270.
  • the isolated polynucleotide is having at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% nucleic acid sequence identity to SEQ ID NO: l .
  • the isolated polynucleotide is having at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% nucleic acid sequence identity to the nucleic acid sequence of the cDNA or mRNA encoding the S. lycopersicum deoxyhypusine synthase ( DHS) protein as found in a plant deposited under number NCIMB 42270 .
  • DHS S. lycopersicum deoxyhypusine synthase
  • nucleotides 429-431 do not code for a valine and preferably code for an isoleucine.
  • the isolated polynucleotide comprises a mutation at position 429 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3.
  • the isolated polynucleotide comprises a mutation into an adenine (A) at position 429.
  • nucleotides 429-431 are ATT, ATC, or ATA.
  • the isolated polynucleotide may be DNA or RNA, e.g. cDNA or niR A. It is to be understood that for RNA a thymine (T) is to be changed for uracil (U) e.g. the T in SEQ ID NO: 1 is to be changed for U.
  • T thymine
  • U uracil
  • the present invention is directed to an isolated polynucleotide that hybridizes with SEQ I D NO: l , or a functional derivative of the isolated polynucleotide which hybridizes with SEQ I D N0: 1.
  • the polynucleotide hybridizes under stringent conditions, such as hybridization in a buffered solution of 0.9M NaCl at 55° C.
  • the isolated polynucleotide hybridizes with at least nucleotide 429-431 of SEQ ID NO: 1 and the nucleotides at position 429-43 1 do not encode a valine.
  • the isolated polynucleotide molecule hybridizes completely to the nucleotide sequence of SEQ I D NO: 1 and is 100% complementary (sequence identity) to SEQ I D NO: I .
  • the isolated polynucleotide that hybridizes to SEQ I D NO: 1 has a length that is 10%-200% of the length of SEQ I D NO: l , or in one embodiment, a length that is 20-180% of the length of SEQ I D NO: I . a length that is 30-170% of the length of SEQ I D NO: I .
  • a length that is 50-150% of the length of SEQ I D NO: l or in another embodiment a length that is 75-125% of the length of SEQ I D NO: l , in still another embodiment, a length that is 80-120% of the length of SEQ I D NO: 1 , preferably a length that is 90-1 10% of the length of SEQ I D NO: 1.
  • the present invention relates to an isolated polynucleotide comprising a genomic nucleic acid sequence comprising at least 70% (e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or even 99.7% ) sequence identity to SEQ I D NO:5 and comprising a mutation at any of position 2882-2883 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 6.
  • the present invention relates to an isolated polynucleotide comprising a nucleic acid sequence as defined by SEQ I D NO: 5.
  • the present invention relates to an isolated polynucleotide comprising a nucleic acid sequence comprising at least 70% (e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or even 99.7% ) sequence identity to the nucleic acid sequence of the genomic polynucleotide encoding the S.
  • 70% e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or even 99.7%
  • the present invention also relates to an isolated polynucleotide comprising a nucleic acid sequence of the genomic polynucleotide encoding the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCIMB 42270.
  • the isolated polynucleotide is having at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% sequence identity to SEQ ID NO:5, or to the nucleic acid sequence of the genomic polynucleotide encoding the S. lycopersicum deoxyhypusine synthase ( DHS ) protein as found in a plant deposited under number NC 1 MB 42270 .
  • DHS S. lycopersicum deoxyhypusine synthase
  • nucleotides 2882-2884 do not code for a valine and preferably code for a isoleucine.
  • the isolated polynucleotide comprises a mutation at position 2882 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 6.
  • the isolated polynucleotide comprises a mutation into an adenine (A).
  • nucleotides 2882-2884 are ATT, ATC, or ATA.
  • the isolated polynucleotide may be DNA or RNA, e.g. genomic DNA or premRNA. It is to be understood that for RNA the T in SEQ I D NO: 5 is to be changed for U.
  • DH S genes/proteins can be identified in silico, e.g. by identifying nucleic acid or protein sequences in existing nucleic acid or protein database (e.g. GENBANK, SWISSPROT, TrEMBL) and using standard sequence analysis software, such as sequence similarity search tools (BLASTN, BLASTP, BLASTX, T I. AST, FASTA, etc).
  • sequence similarity search tools BLASTN, BLASTP, BLASTX, T I. AST, FASTA, etc.
  • the present invention is directed to an isolated polynucleotide that hybridizes with SEQ I D NO:5, or a functional derivative of the isolated polynucleotide which hybridizes with SEQ I D N0:5.
  • the polynucleotide hybridizes under stringent conditions, such as hybridization in a buffered solution of 0.9M NaCl at 55° C.
  • the isolated polynucleotide hybridizes with at least nucleotide 2882-2884 of SEQ I D NO: 5.
  • the isolated polynucleotide molecule hybridizes completely to the nucleotide sequence of SEQ I D NO: 5 and is 100%o complementary (sequence identity) to SEQ I D NO:5.
  • the isolated polynucleotide that hybridizes to SEQ I D NO:5 has a length that is 10%-200% of the length of SEQ I D NO: l , preferably a length that is 20-180% of the length of SEQ I D NO: l , preferably a length that is 30-170% of the length of SEQ I D NO: 1 , preferably a length that is 50-150% of the length of SEQ I D NO: l , preferably a length that is 75- 125% of the length of SEQ I D NO: 1 , preferably a length that is 80-120% of the length of SEQ ID NO: l, preferably a length that is 90-1 10% of the length of SEQ I D NO: 1.
  • polypeptide encoded by a polynucleotide of the invention and/or embodiments, or a functional derivative thereof.
  • the polypeptide comprises the amino acid sequence comprising at least 70% (e.g.
  • the isolated polypeptide is having at least 72%, or 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or even 99.7%) amino acid sequence identity to SEQ I D NO:2, or the amino acid sequence of SEQ ID N0:2, or is a functional derivative thereof; and having a mutation at position 126 (or the corresponding position in a functional derivative thereof) when compared to wild type sequence SEQ ID NO:4.
  • said mutation is a change of an amino acid 126 into isoleucine.
  • the isolated polypeptide is having at least 72%, e.g.
  • the functional derivative of SEQ I D NO:2 has at least 70% amino acid sequence identity (e.g.
  • the isolated polypeptide has a mutation at position 126 when compared to wild type sequence SEQ I D NO:4.
  • said mutation is a change of an amino acid 126 into isoleucine.
  • the present invention relates in addition, to an isolated polypeptide comprising an amino acid sequence comprising at least 70% sequence identity to the amino acid sequence of the S. lycopersicum DHS protein as found in a plant deposited under number NCIMB 42270 and comprising an isoleucine at position 126 or a corresponding position in variants thereof, in one embodiments the isolated polypeptide is having at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or even 99.7% amino acid sequence identity to the amino acid sequence of the S.
  • the isolated polypeptide comprises the amino acid sequence of the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCIMB 42270.
  • the isolated polypeptide has a mutation at position 126 when compared to wild type sequence SEQ I D NO:4. Preferably said mutation is a change of an amino acid 126 into isoleucine.
  • the present invention relates further to a tomato plant, plant part, plant cell or a tomato fruit comprising any of the polynucleotides of the invention and/or embodiments thereof.
  • the present invention relates to a tomato plant, plant part, plant cell or a tomato fruit comprising any polypeptide of the invention and/or embodiments thereof.
  • the present invention relates in one aspect to a cultivated plant of the species Solarium lycopersicum, varieties, breeding lines or cultivars of the species Solatium lycopersicum, cultivated by humans and preferably having good agronomic characteristics.
  • the tomato fruits may have a variety of shapes such as selected from the group consisting of cherry, bell, blocky, currant, deep round oval or roma, flattened globe, grape, long blocky, long pointed, ox heart, pear, beefsteak, round, small pear, small pointed, staffer, and plum.
  • the tomato fruit has a shape selected from the group consisting of beefsteak, plum, round, or cherry.
  • the tomato fruit may have a variety of skin colours such as selected from the group consisting of dark, bi-color, deep pink, golden, green, orange, pink, red, white, and yellow.
  • the tomato fruit has colour selected from the group consisting of bicolor, red, black, deep pink, orange, yellow or pink.
  • the tomato fruit has a colour selected from the group consisting of red, yellow orange and pink.
  • the invention is also related to tomato plants having tomato fruit with different shapes and/or skin colours as defined above.
  • Varieties of Solarium lycopersicum may show various combinations of shapes, colours, number of fruits, Brix, etc.
  • Tables I and 2 show exemplary varieties with shape and colour.
  • the tomato plant may be determinate or indeterminate.
  • the tomato plant may be heirloom, open-pollinated or hybrid.
  • the tomato plant is a hybrid tomato plant.
  • the tomato plant may, thus, be any cultivated tomato, any commercial variety, any breeding line or other, it may be determinate or indeterminate, open pollinated or hybrid, producing fruits of any colour, shape and size.
  • the present invention and/or embodiments thereof relate to a tomato plant, tomato fruit, cell of a tomato plant and/or part of a tomato plant, wherein the polynucleotide of the invention and/or embodiments thereof is present in at least one allele (i.e. heterozygous for the mutant DHS allele), preferably in two alleles (i.e. homozygous for the mutant DHS allele).
  • the tomato plants of the present invention and/or embodiments thereof are homozygous for the mutant DHS gene. To generate plants comprising the mutant allele in homozygous form, selfing can be used.
  • the mutant DHS alleles according to the invention can be transferred to any other tomato plant by traditional breeding techniques, such as crossing, selfing, backcrossing, etc.
  • any type of tomato having reduced softening, increased firmness and/or longer shelf life due to the presence of at least one mutant DHS allele according to the invention may be generated.
  • Any tomato plant may be generated and/or identified having at least one mutant DHS allele in its genome and producing a DHS protein having reduced activity compared to wild type DHS protein.
  • the tomato plant may, thus, be any cultivated tomato, any commercial variety, any breeding line or other, it may be determinate or indeterminate, open pollinated or hybrid, producing fruits of any color, shape and size.
  • the mutant allele generated and/or identified in a particular tomato plant, or in a sexually compatible relative of tomato may be easily transferred into any other tomato plant by breeding (crossing with a plant comprising the mutant allele and then selecting progeny comprising the mutant allele). It is to be understood that in methods of the present invention tomato plants being heterozygous for the mutant DHS gene of the invention are also suitable e.g. to produce homozygous mutant DHS tomato plants.
  • the present invention relates to tomato fruits or tomato plants producing tomato fruits exhibiting delayed post-harvest softening of their tomato fruits due to altered DHS activity, preferably without the inclusion of foreign nucleic acids in the tomato plants' genomes.
  • the present invention describes tomato fruits and/or tomato plants producing tomato fruits exhibiting increased firmness of their tomato fruits post-harvest due to altered DHS activity, preferably without the inclusion of foreign nucleic acids in the tomato plants' genomes.
  • the present invention relates to tomato fruits and/or tomato plants producing tomato fruits exhibiting increased shelf life of their tomato fruits post-harvest due to altered DHS activity, preferably without the inclusion of foreign nucleic acids in the tomato plants' genomes.
  • a delayed softening, and/or increased firmness and/or extended shelf-life may have the advantage that more time is available for transport of picked fruits e.g. to retailers and supermarkets and/or that the consumer can keep the fruits longer.
  • Tomatoes may be harvested at mature green stage or at breaker stage, or at red or full red stage.
  • the present invention is especially useful for tomato fruits and tomato plants that are harvested at the red stage or at full red stage. Tomatoes at red or full red stage are normally much more vulnerable and sensitive then tomatoes harvested at mature green or breaker stage as they normally are already much softer.
  • the present invention provides an increased firmness for tomatoes that are at the red or full red stage and thereby increasing the shelf life for these tomatoes.
  • the present invention and/or embodiments thereof relate to a tomato fruit or a tomato plant producing a tomato fruit wherein the tomato fruit has slower softening and/or increased firmness and/or increased shelf life after harvest compared to a tomato fruit from a tomato plant homozygous for the wild type DHS polynucleotide as defined by SEQ I D NO 3 or 6; or comprising no other DHS polypeptide than the wild type DH S polypeptide as defined by SEQ I D NO: 4.
  • the invention relates to tomato fruit or a tomato plant of the invention having an increased post-harvest shelf life caused by a mutation in DH S allele wherein the longer post-harvest shelf life is at least 1 10% of the post-harvest shelf life of a tomato fruit from a tomato plant being homozygous for the wild type DHS polynucleotide as defined by SEQ I D NO 3 or 6; or being homozygous for a DHS allele encoding a wild type DHS polypeptide as defined by SEQ I D NO: 4.
  • the post-harvest shelf life is at least 1 15%>, e.g.
  • the post-harvest shelf life is at least 120%, or even at least 125%) of the post-harvest shelf life of a tomato fruit from a tomato plant being homozygous for the wild type DHS polynucleotide as defined by SEQ I D NO 3 or 6.
  • the post-harvest shelf life is at least 135%, or even at least 150%o, e.g. at least 165%o of the post-harvest shelflife of a tomato fruit from a tomato plant being homozygous for the wild type DHS polynucleotide as defined by SEQ I D NO 3 or 6.
  • the post-harvest shelf life is at least 180%, e.g.
  • the present invention and/or embodiments thereof relate to tomato fruit or a tomato plant producing a tomato fruit wherein the tomato fruit has a firmness of at least 5.5 N/mm at 2 weeks after harvest, preferably at least 6 N/mm, more preferably at least 6.5 N/mm.
  • the tomato fruit has a firmness of at least 4.5 N/mm at 4 weeks after harvest, e.g. at least 5 N/mm, or even at least 5.5 N/mm.
  • the tomato fruit has a firmness at 2-4 weeks after harvest that is at least 0.5 N/mm higher than a tomato fruit from a tomato plant homozygous for the wild type DHS polynucleotide as defined by SEQ ID NO 3 or 6, or homozygous for an DHS allele encoding the wild type DH S polypeptide as defined by SEQ ID NO: 4.
  • the tomato fruits are harvested at the red stage or at full red stage.
  • the present invention and/or embodiments thereof relate to a tomato plant according to the invention and/or any embodiment thereof comprising at least one desirable trait selected from the group consisting of total solids, pH, Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin colour, leaf length, internode length, fruit flesh colour, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • the tomato plant, tomato fruit, cell of a tomato plant and/or part of a tomato plant comprises a vector encoding for at least one desirable trait selected from the group consisting of total solids, pH, Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin colour, leaf length, internode length, fruit flesh colour, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • the plants according to the invention, which comprise mutant DHS do not produce fewer fruits than the wild type plants. Thus, fruit number per plant is preferably not reduced.
  • the plants according to the invention, which comprise mutant DHS show normal leaf morphology and do not show modified leaf morphology.
  • the plants according to the invention which comprise mutant DHS are not male sterile.
  • the invention further relates to tomato fruits produced by tomato plants of the invention and/or embodiments thereof.
  • the present invention relates to a tomato plant producing tomato fruits according to the invention and/or embodiments thereof.
  • the present invention also relate to pollen which are produced by a tomato plant according to the invention and/or any embodiment thereof.
  • the pollen comprise the mutant of the invention, i.e. a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 i.e. nucleotides at position 429-43 1 do not encode a valine ;or a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine.
  • the pollen comprise a mutation at position 429 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 or a mutation at position 2882 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6.
  • the mutation is a mutation of G to A.
  • the pollen comprises a polynucleotide according to the invention and/or any embodiment thereof or a polypeptide according to the invention and/or any embodiment.
  • the pollen comprises a polynucleotide comprising a nucleic acid sequence comprising at least 70% (e.g.
  • nucleic acid sequence identity to SEQ ID NO: I and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e. nucleotides at position 429-43 1 do not encode a val ine, more suitably the pollen comprise a polynucleotide comprising a nucleic acid sequence as defined by SEQ ID NO: 1.
  • the pollen comprises a polynucleotide comprising a nucleic acid sequence comprising a genomic nucleic acid sequence comprising at least 70% (e.g. at least 72%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even at least 99.1 % e.g. at least 99.2%, 99.4%, 99.5%, or even 99.7% or 99.8% or 99.9% ) nucleic acid sequence identity to SEQ I D NO:5 and comprising a mutation at any of position 2882-2883 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 6 i.e.
  • nucleotides at position 2882-2884 do not encode a valine.
  • the pollen comprise a polynucleotide comprising a nucleic acid sequence as defined by SEQ I D NO: 5.
  • the pollen comprises a polynucleotide comprising a nucleic acid sequence comprising at least 70% (e.g. at least 72%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even at least 99.1 % e.g.
  • nucleic acid sequence identity to the nucleic acid sequence of the cDNA or mR A encoding the S. lycopersicum deoxyhypusine synthase ( DHS ) protein as found in a plant deposited under number NCI MB 42270 and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 i.e.
  • nucleotides at position 429-43 I do not encode a val ine; or a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6 i.e. nucleotides at posit ion 2882- 2884 do not encode a valine
  • the pollen comprise a polynucleotide comprising a mutation at position 429 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 or a mutation at position 2882 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6.
  • the mutation is a mutation of G to A.
  • the pollen comprises a polynucleotide comprising the nucleic acid sequence of the cDNA or mRNA encoding the S. lycopersicum DH S protein as found in a plant deposited under number NCI MB 42270.
  • the present invention also relate to seed which are produced by a tomato plant according to the invention and/or any embodiment thereof.comprising the mutant of the invention, i.e. a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 i.e. nucleotides at position 429-43 1 do not encode a v al ine ;or a mutation at any of position 2882- 2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine.
  • the seed comprise a mutation at position 429 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 or a mutation at position 2882 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6.
  • the mutation is a mutation of ( i to A.
  • the seed comprises a polynucleotide according to the invention and/or any embodiment thereof or a polypeptide according to the invention and/or any embodiment.
  • the seed comprises a polynucleotide comprising a nucleic acid sequence comprising at least 70% (e.g.
  • nucleic acid sequence identity to SEQ I D NO: l and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3, i.e. nucleotides at position 429-43 1 do not encode a valine more suitably the seed comprise a polynucleotide comprising a nucleic acid sequence as defined by SEQ ID NO: 1.
  • the seed comprises a polynucleotide comprising a nucleic acid sequence comprising a genomic nucleic acid sequence comprising at least 70%(e.g. at least 72%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even at least 99.1 % e.g. at least 99.2%, 99.4%, 99.5%, or even 99.7% or 99.8% or 99.9% ) nucleic acid sequence identity to SEQ I D NO:5 and comprising a mutation at any of position 2882-2883 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 6 i.e.
  • nucleotides at position 2882-2884 do not encode a valine.
  • the seed comprise a polynucleotide comprising a nucleic acid sequence as defined by SEQ I D NO: 5.
  • the seed comprises a polynucleotide comprising a nucleic acid sequence comprising at least 70% (e.g. at least 72%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even at least 99.1% e.g. at least 99.2%, 99.4%, 99.5%, or even 99.7% or 99.8% or 99.9% ) nucleic acid sequence identity to the nucleic acid sequence of the cDNA or mRNA encoding the S.
  • DH S lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCI MB 42270 and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 i. e. nucleotides at position 429-43 1 do not encode a valine or a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6 i.e. nucleotides at position 2882-2884 do not encode a v al ine.
  • the seed comprise a polynucleotide comprising a mutation at position 429 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 or a mutation at position 2882 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6.
  • the mutation is a mutation of ( i to A. More suitably the seed comprises an isolated polynucleotide comprising the nucleic acid sequence of the cDNA or mRNA encoding the S. lycopersicum DHS protein as found in a plant deposited under number NCI MB 42270.
  • the present invention also relates to seed from which a plant according to the invention and/or any embodiment thereof can be grown, as well as a container comprising such seeds.
  • a seed-comprising container can be of various shapes (e.g. box, bag, can) and can comprise various numbers of seeds (e.g. up to 10, 100, 1000,or even more seeds such as at least 2000 e.g. at least 5000, 10.000 or even more seeds)
  • the tomato plant is grown from seeds according to the present invention and/or embodiments thereof. Seeds may be treated and / or primed.
  • the invention provides a cell culture or tissue culture of the mutant tomato plant of the invention.
  • the cell culture or tissue culture comprises regenerable cells.
  • Such cells can be derived from leaves, pollen, embryos, cotyledon, hypocotyls, meristematic cells, roots, root tips, anthers, flowers, seeds and stems.
  • vegetative propagations of plants according to the invention are an aspect encompassed herein. Likewise harvested fruits and fruit parts, either for fresh consumption or for processing or in processed form are encompassed. Fruits may be graded, sized and/or packaged. Fruits may be sliced or diced or further processed.
  • the present invention is directed to food and/or a food product comprising the tomato fruit according to the present invention and or embodiments thereof or parts thereof.
  • the food and/or a food product comprises the tomato fruit or parts thereof wherein the polynucleotide or polypeptide according to the present invention and/or embodiments thereof is present.
  • food and/or food products may be salads, sandwiches, tomato juice, tomato slices, tomato sauce, tomato paste, tomato soup, tomato ketchup and any other food or food product that comprises tomato such as pasta, pizza, salsa, and more.
  • the food product is a salad or sandwiches.
  • the present invention also is directed to parts of the tomato plant or tomato fruit of the invention.
  • the plant part is selected from the group consisting of a leaf, anther, pistil, stem, petiole, root, scion, rootstock, ovule, pollen, protoplast, tissue, seed, fruit, flower, cotyledon, hypocotyl, embryo and cell.
  • plant part comprises the mutation, polynucleotide and/or polypeptide according to the present invention and/or embodiments thereof. Definitions of the mutation, polynucleotide and/or polypeptide are amply described in the present application for example when referred to the pollen and seed.
  • a tomato plant comprising a scion according to the present invention and/or embodiments thereof is expressly encompassed in the present invention.
  • the present invention also relate to an isolated tomato plant cell comprising a polynucleotide, polypeptide and/or mutation according to the present invention and/or embodiments thereof the plant cell comprising the mutant of the invention, i.e. a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e. nucleotides at position 429-43 1 do not encode a valine or a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e. nucleotides at position 2882- 2884 do not encode a valine.
  • the plant cell comprises a mutation at position 429 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 or a mutation at position 2882 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6.
  • the mutation is a mutation of G to A.
  • the plant cell comprises a polynucleotide according to the invention and/or any embodiment thereof or a polypeptide according to the invention and/or any embodiment.
  • the plant cell comprises a polynucleotide comprising a nucleic acid sequence comprising at least 70% (e.g.
  • nucleic acid sequence identity to SEQ I D NO: I and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e. nucleotides at position 429-431 do not encode a valine, more suitably the plant cell comprise a polynucleotide comprising a nucleic acid sequence as defined by SEQ ID NO: 1.
  • the plant cell comprises a polynucleotide comprising a nucleic acid sequence comprising a genomic nucleic acid sequence comprising at least 70% (e.g. at least 72%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even at least 99.1% e.g. at least 99.2%, 99.4%, 99.5%, or even 99.7% or 99.8% or 99.9% ) nucleic acid sequence identity to SEQ I D NO: 5 and comprising a mutation at any of position 2882-2883 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 6.
  • a polynucleotide comprising a nucleic acid sequence comprising a genomic nucleic acid sequence comprising at least 70% (e.g. at least 72%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even at least 99.1% e.g. at least 99.2%, 99.4%
  • the plant cell comprise a polynucleotide comprising a nucleic acid sequence as defined by SEQ ID NO: 5.
  • the plant cell comprises a polynucleotide comprising a nucleic acid sequence comprising at least 70% (e.g. at least 72%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even at least 99.1% e.g. at least 99.2%, 99.4%, 99.5%, or even 99.7% or 99.8% or 99.9% ) nucleic acid sequence identity to the nucleic acid sequence of the cDNA or mRNA encoding the S.
  • lycopersicum deoxyhypusine synthase (DHS) protein as found in a plant deposited under number NCIMB 42270 and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 i.e. nucleotides at position 429-43 1 do not encode a valine or a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6 i.e. nucleotides at position 2882- 2884 do not encode a valine.
  • DHS lycopersicum deoxyhypusine synthase
  • the plant cell comprise a polynucleotide comprising a mutation at position 429 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 r a mutation at position 2882 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6.
  • the mutation is a mutation of G to A.
  • the plant cell comprises a polypeptide comprising an amino acid sequence comprising at least 70% sequence identity to SEQ I D NO: 2 and comprising a mutation at position 126 when compared to the wild type amino acid sequence defmed by SEQ ID NO: 4, more suitably the plant cell comprise a polypeptide comprising an amino acid sequence as defined by SEQ ID NO: 2.
  • the plant ceil comprises a polypeptide comprising an amino acid sequence comprising at least 70% (e.g. at least 72%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even at least 99.1% e.g. at least 99.2%, 99.4%, 99.5%, or even 99.7% or 99.8% or 99.9% ) amino acid sequence identity to the amino acid sequence of the S. lycopersicum deoxyhypusine synthase (DHS) protein as found in a plant deposited under number NCIMB 42270 and comprising a mutation at position 126 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 4.
  • DHS S. lycopersicum deoxyhypusine synthase
  • the mutation is a mutation of valine into isoleucine. More suitably the plant cell comprises an isolated polypeptide comprising the amino acid sequence of the S. lycopersicum DHS protein as found in a plant deposited under number NCIMB 42270.
  • the present invention is also directed to a tomato plant, tomato fruit, plant part and/or plant cell of the invention and/or embodiments thereof comprising a polynucleotide and/or polypeptide according to the invention and/or embodiments thereof and a vector encoding for at least one desirable trait, in suitable embodiments the desirable trait may be selected from the group consisting of total solids, pH, Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin colour, leaf length, internode length, fruit flesh colour, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • the tomato plants, tomato fruits, tomato plant parts and/or tomato plant cells of the present invention may be transgenic.
  • the present invention also describes transgenic mutations in a deoxyhypusine synthase gene or allele of a tomato plant or a tomato fruit, or tomato plant cell.
  • the transgenic plants may be made using the mutant DHS nucleotide sequences of the invention using known plant transformation and regeneration techniques in the art.
  • An "elite event" may be selected, which is a transformation event having the DHS gene (comprising a promoter operably linked to a nucleotide sequence encoding a mutant DHS protein) inserted in a particular location in the genome, which results in good expression of the desired phenotype.
  • the invention therefor also relates to a vector comprising a polynucleotide according to the present invention and/or embodiments thereof.
  • the vector comprises a polynucleotide comprising the nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of the cDNA or inR A encoding the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCIMB 42270.
  • the vector comprises a polynucleotide wherein the polynucleotide is having at least 72%>, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% nucleic acid sequence identity to SEQ ID NO:l, or to the nucleic acid sequence of the cDNA or mRNA encoding the S.
  • the vector comprises a polynucleotide comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i. e. nucleotides at position 429-43 1 do not encode a valine, or a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine.
  • the vector comprises a polynucleotide comprising a nucleic acid sequence comprising at least 70% sequence identity to SEQ I NO:l and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e. nucleotides at position 429-43 1 do not encode a val ine.
  • the vector comprise a polynucleotide comprising a mutation at position 429 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 or a mutation at position 2882 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6.
  • the mutation is a mutation of G to A.
  • the vector comprises a polynucleotide comprising a nucleic acid sequence as defined by SEQ I D NO: 1. . More suitably the vector comprises a polynucleotide comprising the nucleic acid sequence of the cDNA or mRNA encoding the S. lycopersicum DI IS protein as found in a plant deposited under number NCIMB 42270. Preferably the vector comprises a nucleotide sequence encoding a deoxyhypusine synthase protein according to the present invention and/or embodiments thereof.
  • a skilled person is well aware of methods and techniques to transform, plant cells and cells with vectors.
  • a skilled person is also well aware of techniques of creating a suitable vector comprising a polynucleotide according to the present invention.
  • the vector further comprises regulatory sequences operatively linked to the nucleic acid sequence such that the nucleic acid sequence is expressed in a plant cell into which it is transformed.
  • the regulatory sequences may include a promoter functional in the transformed plant cell, which promoter may be inducible or constitutive.
  • the regulatory sequences include a polyadenylation signal.
  • Promoter regulatory elements that are use ul in combination with the DH S gene include any plant promoter in general, and more particularly, a constitutive promoter such as the fig wart mosaic virus 3 S promoter, the cauliflower mosaic virus promoter, CaMV35S promoter, or the MAS promoter, or a tissue-specific or GST1 promoter or the Arabidopsis SAG12 promoter (See, for example, J.C. Palaqui et al, Plant Physiol.. 1 12: 1447-1456 (1996); Morton et al., Molecular Breeding, 1 : 1 23- 1 32 (1995); Fobert et al., Plant Journal, 6:567-577 (1994); and Gan et al.. Plant Physiol.. 1 13 :313 5 (1997), incorporated herein by reference).
  • the promoter is a constitutive promoter, most preferably a double 35 S promoter.
  • Vectors may be plasmids, preferably, or may be viral or other vectors known in the art to replicate and express genes encoded thereon in plant cells or bacterial cells.
  • the vector may become chromosomally integrated such that it can be transcribed to produce the desired DHS RNA.
  • Such plasmid or viral vectors can be constructed by recombinant DNA technology methods that are standard in the art.
  • the vector may be a plasmid vector containing a replication system functional in a prokaryotic host and an oligonucleotide or polynucleotide according to the invention.
  • the vector may be a plasmid containing a replication system functional in Agrobacterium and an oligonucleotide or polynucleotide according to the invention. Plasmids that are capable of replicating in Agrobacterium are well known in the art. See, Miki, et al.. Procedures for Introducing Foreign DNA Into Plants, Methods in Plant Molecular Biology and Biotechnology,, Eds. B.R. Glick and J.E. Thompson. CRC Press (1993), PP. 67-83. [0219] The invention also provides a plant cell transformed with a vector or combination of vectors as described above, a plantlet or mature plant generated from such a cell, or a plant part of such a plantlet or plant.
  • Transgenic plants made in accordance with the present invention may be prepared by DNA transformation using any method of plant transformation known in the art.
  • Plant transformation methods include direct co-cultivation of plants, tissues r cells with Agrobacterium tumefaciens or direct infection (Miki, et al., Meth. in Plant Mol. Biol, and Biotechnology, (1993), p. 67-88); direct gene transfer into protoplasts or protoplast uptake (Paszkowski, et al., EM BO J., 12:2717 (1984); electroporation (Fromm, et al..
  • the invention is directed to methods of producing a tomato plant, tomato fruit, tomato plant part, and/or tomato plant cell according to the invention and/or embodiments thereof.
  • the present invention relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato fruit, according to the present invention and/or embodiments thereof, said method comprising the steps of
  • the polynucleotide according to the invention is in one embodiment generated in a cultivated plant, but may also be generated in a wild plant or non-cultivated plant and then transferred into an cultivated plant using e.g. crossing and selection (optionally using interspecific crosses with e.g. embryo rescue to transfer the mutant allele).
  • a mutant DHS polynucleotide may be generated e.g. human induced mutation using mutagenesis techniques to mutagenize the target DHS gene or variant thereof in a tomato plant or in other Solanum species for example wild relatives of tomato, such as S. cheesmanii, S. chilense, S. habrochaites (L. hirsutum), S. chmielewskii, S.
  • lycopersicum x S. peruvianum, S. glandulosum, S. hirsutum, S. minutum, S. parviflorum, S. pennellii, S. peruvianum, S. peruvianum var. humifusum and S. pimpinellifolium and then transferred into a cultivated tomato plant, e.g. Solanum lycopersicum by traditional breeding techniques.
  • traditional breeding techniques encompasses herein crossing, selfing, selection, double haploid production, embryo rescue, protoplast fusion, transfer via bridge species, etc. as known to the breeder, i.e. methods other than genetic modification by which alleles can be transferred.
  • the introduction of a polynucleotide into a plant cell is by crossing two tomato plants whereinat least one tomato plant comprises a polynucleotide sequence according to the present invention and/or embodiments thereof.
  • at least one tomato plant comprises at least one other desirable trait.
  • said trait is selected from the group consisting of total solids, pH, Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin color, lea length, internode length, fruit flesh color, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • the introduction of a polynucleotide into a plant cell is by introducing a vector according to the present invention and/or embodiments thereof as defined above.
  • the vector comprises a polynucleotide according to the invention and/or embodiments thereof.
  • the introduction of a polynucleotide into a plant cell may be by introducing a mutation in a polynucleotide encoding a DHS protein in a tomato plant.
  • the mutation may be introduced by using a mutagen or radiation.
  • Suitable chemical mutagens include ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N-nitrosurea (ENU), trimethylamine (TEM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cy clopho sphamide , diethyl sulfate, acrylamide monomer, meiphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N' -nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7, 12-dimethylbenz(a)anthracene (DMBA), ethylene oxide, hexamethyiphosphoramide, bisulfan, diepoxyalkanes, diepoxyoctrane (DEO), diepoxybutane (DEB), 2- methoxy-6-choloro9[3-ethyl-2
  • the method of producing a tomato fruit, and/or tomato plant producing a tomato comprises
  • (iii) optionally cross plants grown from the seeds of (ii) with another tomato plant, preferably a second plant of (i).
  • the second tomato plant may also be an inbred line.
  • first and/or second tomato plant comprise a further desirable trait.
  • first and/or second tomato plant comprise a vector encoding a desirable trait.
  • Suitable traits may be selected from the group consisting of total solids, pH, Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin color, leaf length, internode length, fruit flesh color, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • a plant according to the invention is used as a parent plant to produce a hybrid plant and/or hybrid seeds.
  • Fl hybid tomato seeds i.e.
  • Fl hybrid seeds from which IT hybrid tomato plants can be grown are provided, comprising at least one mutated DHS allele (i.e. heterozygous) according to the invention.
  • Fl hybrid seeds may be harvested from a cross between two inbred tomato parent plants.
  • Such an Fl hybrid may comprise one (heterozygous) or two (homozygous) mutant DHS alleles according to the invention.
  • the fruit of said hybrid plant or plant grown from said seed have an increased firmness, delayed softening and/or increased shelf life after harvest compared to a tomato fruit from a tomato plant homozygous for the wild type DHS polynucleotide as defined by SEQ ID NO 3 or 6, or homozygous for an allele encoding the wild type DHS polypeptide as defined by SEQ ID NO: 4.
  • the method of producing a tomato fruit, and/or tomato plant producing a tomato according to the invention further comprises a step of selecting tomato plants.
  • Suitable tomato plants are selected when they produce a tomato fruit having an increased firmness, delayed softening and or increased shelf life after harvest than a tomato fruit from a tomato plant homozygous for the wild type DHS polynucleotide as defined by SEQ ID NO 3 or 6, or homozygous for an allele encoding the wild type DHS polypeptide as defined by SEQ ID NO: 4.
  • Suitable tomato plants are selected when they produce a tomato fruit having a firmness of at least 5.5 N/mm at 2 weeks after harvest, preferably at full red stage, preferably at least 6 N/mm, more preferably at least 6.5 N/mm.
  • tomato plants may be selected when they comprise a polynucleotide according to the invention using DNA or protein sequence analysis techniques known in the art.
  • tomato plants are selected when they comprise a nucleic acid sequence having at least 70% (e.g. at least 72%, 75%, 78%), 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%), or even 99.7%) nucleic acid sequence identity to the nucleic acid sequence of the cDNA or mRNA polynucleotide of SEQ ID NO:l comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 i.e.
  • nucleotides at position 429-43 I do not encode a valine; or a nucleic acid sequence having at least 70% (e.g. at least 72%o, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%), 99.5%), 99.6%, or even 99.7%>) nucleic acid sequence identity to the nucleic acid sequence of the genomic polynucleotide of SEQ ID NO: 5 comprising a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ I D NO: 6 i.e.
  • nucleotides at position 2882-2884 do not encode a valine, or a polynucleotide comprising the nucleic acid sequence having at least 70% (e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or even 99.7%) nucleic acid sequence identity to the nucleic acid sequence of the cDNA or mRNA encoding the S.
  • 70% e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or even 99.7%
  • lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NO MB 42270, and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 i.e. nucleotides at position 429-43 1 do not encode a valine.
  • Selection of tomato plants comprising the polynucleotide of the present invention may be carried by any method known to the skilled person.
  • Suitably polynucleotides are used in such selection methods that hybridize with SEQ ID NO:l and especially hybridize with the mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3.
  • tomato plants are selected comprising a polynucleotide hybridizing with SEQ ID NO: 1 and comprising a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e.
  • nucleotides at position 429-43 1 do not encode a val ine or a polynucleotide hybridizing with SEQ I NO: 5 and comprises a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine, or a polynucleotide hybridizing with the polynucleotide encoding the S. ly coper sicum deoxyhypusine synthase protein as found in a plant deposited under number NCIMB 42270.
  • tomato plants may be selected comprising a polypeptide comprising an amino acid sequence having at least 70% (e.g.
  • amino acid sequence identity to SEQ I D NO: 2 and comprising a mutation at position 126 when compared to the wild type amino acid sequence defined by SEQ I D NO: 4, preferably said mutation is a change of an amino acid 126 into isoleucine or a polypeptide comprising the amino acid sequence having at least 70% (e.g.
  • amino acid sequence identity to the amino acid sequence of the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCIMB 42270, preferably having a change of an amino acid 126 into isoleucine when compared to the wild type amino acid sequence defined by SEQ I D NO: 4.
  • the invention relates to a nucleotide sequence defined by SEQ I D NO: 1 wherein all nucleotides are identical to SEQ ID NO: l except for 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, provided the nucleotide at position 429 is identical to SEQ ID NO: 1.
  • the invention relates to a nucleotide sequence defined by SEQ I D NO: 5 wherein all nucleotides are identical to SEQ I D NO: 5 except for 1 , 2, 3, 4, 5, 6. 7, 8, 9, or 10 nucleotides, provided the nucleotide at position 2882 is identical to SEQ ID NO: 5.
  • the present invention relates to a method of producing a tomato plant, plant part and/or plant cell comprising a polynucleotide having a mutation in a nucleotide encoding for a deoxyhypusine synthase comprising the steps of:
  • mutagenized plant material (i) analyzing mutagenized plant material to identify a plant having at least one mutation in the nucleic acid sequence encoding for deoxyhypusine synthase, preferably said mutation is a mutation according to aspects of the invention and/or embodiments thereof.
  • Such mutagenized plant material may be obtained by treating plant material with a mutagen such as for example EMS, or other as listed herein.
  • the present invention and/or embodiments thereof relates to a method of producing a tomato plant, tomato plant part and/or tomato plant cell comprising a polynucleotide having a mutation in a nucleotide encoding for a deoxyhypusine synthase wherein the mutation is a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 i.e. nucleotides at position 429-43 1 do not encode a valine or a mutation at any of position 2882- 2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e.
  • nucleotides at position 2882-2884 do not encode a valine.
  • said mutation is a mutation at position 429 when compared to the wild type nucleic acid sequence defined by SEQ iD NO: 3 or a mutation at position 2882 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6.
  • the mutation is a mutation of G to A.
  • tomato seeds are mutagenized and then grown into Mi plants.
  • the Ml plants are then allowed to self-pollinate and seeds from the Mi plant are grown into M2 plants, which can then screened for mutations in their deoxyhypusine synthase genes.
  • An advantage of screening the M2 plants is that all somatic mutations correspond to the germiine mutations.
  • tomato plant materials including, but not limited to, seeds, pollen, plant tissue or plant cells, may be mutagenized in order to create a deoxyhypusine synthase-mutated tomato plants of the present invention.
  • the type of plant material mutagenized may affect when the plant DNA is screened for mutations.
  • the seeds resulting from that pollination are grown into Ml plants. Every cell of the Ml plants will contain mutations created in the pollen, thus these Mi plants may then be screened for deoxyhypusine synthase gene mutations instead of waiting until the M2 generation.
  • Mutagens that create primarily point mutations and short deletions, insertions, transversions, and/or transitions (about 1 to about 5 nucleotides), such as chemical mutagens or radiation, may be used to create the mutations of the present invention.
  • Mutagens conforming with the method of the present invention include, but are not limited to, ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl N-nitrosurea (ENU), triethylmelamine (TEM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N' -nitro Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7, 12 dimethyl benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO), diepoxybutane (BEB), and the like), 2
  • any method of plant DNA preparation known to those of skill in the art may be used to prepare the tomato plant DNA for deoxyhypusine synthase mutation screening. For example, see Chen & Ronald, Plant Molecular Biology Reporter, 17: 53-57, 1999; Stewart & Via, Bio Techniques, 14: 748-749, 1993. Additionally, several commercial kits are available, including kits from Qiagen (Valencia, CA) and Qbiogene (Carlsbad, CA). Prepared DNA from individual tomato plants may be pooled in order to expedite screening for mutations in the deoxyhypusine synthase genes of the entire population of plants originating from the mutagenized plant tissue. The size of the pooled group is dependent upon the sensitivity of the screening method used.
  • PCR Polymerase Chain Reaction
  • the primer is designed to amplify the regions of the deoxyhypusine synthase gene where useful mutations are most likely to arise. Most preferably, the primer is designed to detect mutations in the coding region of the deoxyhypusine synthase gene. Additionally, it is preferable for the primer to avoid known polymorphic sites in order to ease screening for point mutations. To facilitate detection of PCR products on a gel, the PCR primer may be labelled using any conventional labelling method.
  • the mutations are analyzed to determine its effect on the expression, translation, and/or activity of the deoxyhypusine synthase enzyme.
  • the PCR fragment containing the mutation is sequenced using standard sequencing techniques, in order to determine the exact location of the mutation within the deoxyhypusine synthase gene sequence.
  • Each mutation may be evaluated in order to predict its impact on protein function (i.e., completely tolerated to loss-of-function) using bioinformatics tools such as SIFT (Sorting Intolerant from Tolerant; Ng, P.C. and Henikoff, S.
  • the M2 or Ml plant may be backcrossed or outcrossed twice to create a BC 1 plant in order to eliminate background mutations. Then the backcrossed or outcrossed BC 1 plant may be self-pollinated in order to create a BC1F2 plant that is homozygous for the deoxyhypusine synthase mutation. Deoxyhypusine synthase mutant tomatoes remain firm longer post-harvest than tomatoes from wild type sibling lines or from parental lines.
  • the method of producing a tomato plant according to the invention preferably produces tomato plant producing a tomato fruit wherein the tomato fruit has a firmness of at least 5.5 N/mm at 2 weeks after harvest preferably at least 6 N/mm, more preferably at least 6.5 N/mm. Preferably the harvest is at full red stage.
  • the tomato fruit has slower softening and/or increased firmness compared to a tomato fruit from a tomato plant comprising the wild type DHS polynucleotide as defined by SEQ ID NO 3 or 6, or the wild type DHS polypeptide as defined by SEQ ID NO: 4.
  • the tomato fruit has a firmness of at least 4.5 N/mm at 4 weeks after harvest, preferably at least 5 N/mm, more preferably at least 5.5 N/mm.
  • the harvest is at full red stage.
  • the tomato fruit produced by methods of the invention has a firmness at 2-4 weeks after harvest that is at least 0.5 N/mm higher than a tomato fruit from a tomato plant homozygous for the wild type DHS polynucleotide as defined by SEQ ID NO 3 or 6, or homozygous for an allele encoding the wild type DHS polypeptide as defined by SEQ I D NO: 4.
  • the harvest is at full red stage.
  • the method of producing a tomato plant comprises at least one desirable trait selected from the group consisting of total solids, pH, Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin color, leaf length, internode length, fruit flesh color, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • the desirable trait is introduced into the tomato plant by transformation of a vector encoding for at least one desirable trait selected from the group consisting of total solids, pH, Brix, sugar content, uniformity of fruit size, fruit weight, fruit size, skin color, leaf length, internode length, fruit flesh color, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting, in a preferred embodiment, the method of producing a tomato plant, tomato plant produces a tomato fruit having a shape selected from the group consisting of cherry, bell, blocky, currant, deep round oval or roma, flattened globe, grape, long blocky, long pointed, oxheart, pear, beefsteak, round, small pear, small pointed, stuffer, and plum.
  • the method of producing a tomato plant tomato plant produces a tomato fruit having a skin colour selected from the group consisting of dark, bi-color, deep pink, golden, green, orange, pink, red, white, and yellow.
  • the method of producing a tomato plant the tomato plant is determinate or indeterminate.
  • the method of producing a tomato plant, the tomato plant is open-pollinated or hybrid.
  • the tomato plant is a hybrid tomato plant.
  • the invention relates to plants obtained in any of the methods described herein.
  • plants obtained from methods of the invention may be further selfed and/or crossed and progeny selected comprising the mutant allele and producing fruits with reduced softness, increased firmness and/or longer shelf-life due to the presence f the mutant allele compared to plants comprising the wild type DHS allele.
  • double haploid plants (and seeds from which double haploid plants can be grown), generated by chromosome doubling of haploid cells comprising an DHS mutant allele, and hybrid plants
  • a plant of the invention and/or embodiments thereof can be used in a conventional plant breeding scheme to produce more plants with the same characteristics or to introduce the mutated DHS allele into other plant lines or varieties of the same or related plant species.
  • the present invention further relates to a method of screening for a tomato plant which produces tomato fruits that have an increased firmness and/or decreased softening and/or increases shelf-life after harvest comprising
  • the method screens for the mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e. nucleotides at position 429-43 1 do not encode a valine; or a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine.
  • nucleic acid hybridization PCR technology direct, and gene sequencing of the DHS gene may be used to screen plant populations for mutant alleles.
  • Key Point screening is a sequence based method which can be used to identify plants comprising mutant DHS alleles (Rigola et al. PloS One, March 2009, Vol 4(3):e4761).
  • Suitable polynucleotides that hybridize with SEQ ID NO: 1 or SEQ ID NO: 5 as defined above may be used for screening and detecting mutation.
  • the present invention further relates to a method of screening tomato plants for the presence of a mutant DHS allele comprising the steps of:
  • (111) detect the presence of DNA having a sequence having at least 70% (e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5% ⁇ , 99.6%>, or even 99.7%>) nucleic acid sequence identity to a nucleic acid sequence as defined to SEQ ID NO 1 and having a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3 i.e.
  • nucleotides at position 429-430 do not encode a val i ne; or DNA having a sequence having at least 70% (e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or even 99.7%) nucleic acid sequence identity to a nucleic acid sequence as defined to SEQ ID NO 5 and having a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e.
  • nucleotides at position 2882-2884 do not encode a val ine; or having at least 70%o (e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or even 99.7%) nucleic acid sequence identity to a nucleic acid sequence of the cDNA or mRNA encoding the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCI MB 42270.
  • said DNA from plant material is pooled from more than one tomato plant.
  • the detection of a specific DNA sequence may be done by any method known to a skilled person such as nucleic acid hybridization, PG R technology direct, and gene sequencing of the DHS gene.
  • the present invention further relates to a method of screening tomato plants for the presence of a mutant DHS allele comprising the steps of
  • (ii) Optionally isolate protein from the plant material; (iii) detect the presence of a protein having a sequence having at least 70% (e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%), or even 99.7%>) amino acid sequence identity to the amino acid sequence as defined in SEQ I D NO: 2 and having a mutation at position 126 when compared to the wild type amino acid sequence as defined by SEQ I D NO: 4, or having at least 70%o (e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
  • amino acid sequence identity to an amino acid sequence of the S. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCIMB 42270.
  • the detection of a protein as defined by the invention may be done by any method known to a skilled person such as blotting, electrophoresis, mass spectroscopy, immuno staining, immunoprecipitation, NMR, microscopy and crystallography.
  • the present invention further relates to tomato plant, tomato seed, tomato fruit, tomato plant part, and/or tomato cell produced by any of the methods of aspect of the invention and/or embodiments thereof.
  • the present invention further relates to use of a tomato plant, tomato fruit, tomato plant part and/or tomato plan cell comprising a polynucleotide and/or polypeptide according to aspects of the invention and/or embodiments thereof.
  • the present invention further relates to use of polynucleotide and/or polypetide according to any aspect of the invention and/or embodiments thereof for producing a tomato plant, tomato fruit, tomato plant cell or tomato plant part.
  • the tomato plant produces tomato fruit that has an increased firmness, and/or delayed softening, and/or increased shelf-life when compared to a tomato fruit from a tomato plant homozygous for the wild type DHS polynucleotide as defined by SEQ I D NO 3 or 6, or homozygous for an allele encoding the wild type DHS polypeptide as defined by SEQ I D NO: 4.
  • the use of the polynucleotide and/or polypetide according to the invention for producing a tomato plant, tomato fruit, tomato plant cell or tomato plant part may comprise the following steps
  • the introduction of nucleic acids may be by introducing a vector comprising said nucleic acid sequence, by introducing a mutation into the nucleic acid encoding S. lycopersicum deoxyhypusine synthase protein by a mutagen or by crossing a tomato plant according to any aspect of the invention and/or embodiments thereof to a second tomato plant, as explained above.
  • the present invention further relates to use of polynucleotide and/or polypetide according to the invention for screening for mutation according to any aspect of the present invention and/or embodiments thereof.
  • the use of polynucleotide and/or polypetide according for screening for mutation comprises the steps
  • DNA and/or protein isolated from tomato plant material detect the presence of DNA having a sequence having at least 70% (e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or even 99.7%) nucleic acid sequence identity to a nucleic acid sequence as defined to SEQ I D NO 1 and having a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 i.e.
  • nucleotides at position 429-43 1 do not encode a valine; or DNA having a sequence having at least 70% (e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or even 99.7%) nucleic acid sequence identity to a nucleic acid sequence as defined to SEQ ID NO 5 and having a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e.
  • nucleotides at position 2882-2884 do not encode a valine; or having at least 70% (e.g. at least 72%, 75%, 78%, 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%o, 99.6%), or even 99.7%) nucleic acid sequence identity to a nucleic acid sequence of the cDNA or mRNA encoding the 5. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NCIMB 42270.; or detect the presence of a protein having a sequence having at least 70%o (e.g.
  • amino acid sequence identity to the amino acid sequence as defined in SEQ I D NO: 2 and having a mutation at position 126 when compared to the wild type amino acid sequence as defined by SEQ I D NO: 4; or having at least 70% (e.g.
  • nucleic acid and/or protein are explained above.
  • the present invention further relates to use of a polynucleotide for hybridizing to a polynucleotide according to the invention and/or embodiments thereof for screening and/or selecting tomato plants and tomato plant material.
  • the polynucleotide for hybridizing to a polynucleotide according to the invention may suitably be used in methods of screening according to the invention and/or embodiments thereof.
  • the polynucleotide for hybridizing to a polynucleotide according to the invention are described herein.
  • the present invention further relates to use of mutagen to produce a tomato plant, tomato seed, tomato plant part and/or tomato plant cell comprising a mutation in a nucleotide encoding for a S. lycopersicum deoxyhypusine synthase protein, preferably said mutation is a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 i.e. nucleotides at position 429-43 1 do not encode a valine; or a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6 i.e. nucleotides at position 2882-2884 do not encode a valine.
  • said use of mutagen comprises the steps of:
  • Analyzing mutagenized plant material to identify a plant having at least one mutation in the nucleic acid sequenc e encoding for deoxyhypusine synthase preferably said mutation is a mutation at any of position 429-430 when compared to the wild type nucleic acid sequence defined by SEQ I D NO: 3 or a mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6.
  • said plant material may be obtained from a tomato plant, tomato seed, tomato plant part and/or tomato plant cell; which may be treated with a mutagen.;
  • the present invention further relates to use of a tomato plant according to the present invention and/or embodiments thereof in a method to produce a transgenic tomato plant, preferably said transgenic tomato plant produces a tomato fruit that has an increased firmness, and/or delayed softening, and/or increased shelf life when compared to a tomato fruit from a tomato plant comprising the wild type DHS polynucleotide as defined by SEQ ID NO 3 or 6, or the wild type DHS polypeptide as defined by SEQ ID NO: 4.
  • the method to produce a transgenic tomato plant comprises introducing nucleic acids encoding for a second trait into said tomato plant according to the present invention and/or embodiments thereof.
  • the present invention and/or embodiments thereof relate to use of a tomato plant according to the present invention and/or embodiments thereof in a method to produce a transgenic tomato plant comprising introducing nucleic acids encoding for a second trait into said tomato plant according to the present invention and/or embodiments thereof, wherein the introduction is by introduction of a vector encoding for a second trait.
  • the present invention and/or embodiments thereof relate to use of a tomato plant according to the present invention and/or embodiments thereof in a method to produce a transgenic tomato plant comprising introducing nucleic acids encoding for a second trait into said tomato plan according to the present invention and/or embodiments thereof, wherein the introduction is by crossing said tomato plant according to the present invention and/or embodiments thereof with a second tomato plant comprising said second trait.
  • said second trait is selected from the group consisting of total solids, pH.
  • Brix sugar content, uniformity of fruit size, fruit weight, fruit size, skin color, leaf length, internode length, fruit flesh color, fruit per cluster, yield per plant, nutritional value of the fruit, pest resistance, disease resistance, speed of ripening, ease of harvesting.
  • the present invention further relates to tomato plant, tomato seed, tomato fruit, tomato plant part, and/or tomato cell produced by any of the use of the invention and/or embodiments thereof.
  • the invention relates to a tomato plant, or a part or cell thereof, comprising a mutation in an DHS allele wherein the DHS allele is as found in a plant of mutant 4282, a representative sample of seed of said mutant has been deposited under Accessi n Number NCI MB 42270.
  • Other embodiments of the invention relate to
  • a tomato plant cell of a plant of the invention i.e. comprising a mutation in an DHS allele.
  • a highly homozygous inbred line used in commercial processing tomato breeding was used for mutagenesis treatment with the following protocol. After seed germination on damp Whatman® paper for 24h, -20,000 seeds, divided in 8 batches of 2500 respectively, were soaked in 100 ml of ultrapure water and ethyl methanesulfonate (EMS) at a concentration of 1 % in conical flasks. The flasks were gently shaken for 16h at room temperature. Finally, EMS was rinsed out under flowing water. Following EMS treatment, seeds were directly sown in the greenhouse. Out of the 60% of the seeds that germinated, 10600 plantlets were transplanted in the field.
  • EMS ethyl methanesulfonate
  • M2 population is composed of 8810 seeds lots each representing one M2 family. O these, 585 families were excluded from the population due to low seed availability.
  • DNA was extracted from a pool of 10 seeds originating from each M2 seed lot. Per mutant line, 10 seeds were pooled in a Micronic® deepwell tube; http ://www.micronic. com from a 96 deep-well plate, 2 stainless balls were added to each tube.
  • the tubes and seeds were frozen in liquid nitrogen for 1 minute and seeds were immediately ground to a fine powder in a Deepwell shaker (Vaskon 96 grinder, Belgium; http ://www.vaskon. com) for 2 minutes at 16,8 Hz (80% of the maximum speed).
  • 300 ⁇ Agowa® Lysis buffer P from the AGOWA® Plant DNA Isolation Kit http://www.agowa.de was added to the sample plate and the powder was suspended in solution by shaking 1 minute at 16,8 Hz in the Deepwell shaker. Plates were centrifuged for 10 minutes at 4000 rpm.
  • 75 ⁇ of the supernatant was pipetted out to a 96 Kingfisher plate using a Janus MDT® ( Perk in Elmer, USA; http ://www.perkinelmer. com) platform (96 head).
  • the following steps were performed using a Perk in Elmer Janus® liquid handler robot and a 96 Kingfisher® (Thermo labsystems, Finland; world wide web thermo.com).
  • the supernatant containing the DNA was diluted with binding buffer (150 ⁇ ) and magnetic beads (20 ⁇ ).
  • the primers used were 5 ' -C AGCTCTC ATGTTTAGC ATTGG-3 ' (forward; SEQ I NO: 7) and 5 ' -C AGACAAGAGCGGATACCTCA-3 ' (reverse; SEQ ID NO: 8).
  • Hi h Resolution Melt curve analysis has been proven to be sensitive and high- throughput methods in human and plant genetics.
  • HRM is a non-enzymatic screening technique.
  • PCR amplification dye LCGreen+ dye, Idaho Technology Inc., UT, USA
  • LCGreen+ dye Idaho Technology Inc., UT, USA
  • the dye When captured in the molecule, the dye emits fluorescence at 510 nm after excitation at 470 nm.
  • a camera in a fluorescence detector (LightScanner, Idaho Technology Inc., UT, USA) records the fluorescence intensity while the DNA sample is progressively heated.
  • the double stranded PCR product starts to melt, releasing the dye.
  • the release of dye results in decreased fluorescence that is recorded as a melting curve by the fluorescence detector.
  • Pools containing a mutation form hetero duplexes in the post-PC R fragment mix. These are identified as differential melting temperature curves in comparison to homo duplexes.
  • Mutants showing a delayed ripening were selected and the type of mutation in the DHS gene was determined. The presence of the particular mutation in individual plants was confirmed repeating the HRM analysis on DNA from the individual M2 seed lots of the identified corresponding DNA pool.
  • the truss was harvested and stored in an open box in the greenhouse. Fruit condition of the fruits was recorded during the whole ripening period by making pictures from each truss. After harvest pictures were made per box containing all trusses from one genotype.
  • mutant 4282 The following mutant was identified: mutant 4282, and seeds were deposited at the NCI MB under the Accession numbers given above. Protocol measurement softening
  • Tomato ripening stage is determined by the tomato color (Royal Horticultural Society, 2007).
  • Figure 1 shows that tomato fruits from a tomato plant homozygous for the mutation have a delayed softening when compared to an azygous tomato plant. This will lead to an increased shelf life.
  • the firmness at harvest has been defined as 100% and firmness of the tomato fruits is expressed relative to this firmness at harvest.
  • Table 11 Shape of tomatoes: Bell, Blocky and Currant varieties
  • Table 12 Shape of tomatoes: Deep Round Oval, Oxheart, Long Blocky varieties
  • Ranger Table 13 Shape of tomatoes: Pear, small pear and small pointed varieties
  • Table 14 Shape of tomatoes: stuffer and undetermined varieties
  • Violaceum Krypni-Rozo Table 15 Shape of tomatoes: Plum varieties
  • Table 18 Shape of tomatoes: flattened globe varieties

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Abstract

L'invention concerne un polynucléotide isolé comprenant une séquence d'acide nucléique comprenant au moins 70 % d'identité de séquence avec SEQ ID NO: 1 et comprenant une mutation en l'une quelconque des positions 429-430 par rapport à la séquence d'acide nucléique de type sauvage définie par SEQ ID NO: 3 ; de préférence ladite mutation est un changement du nucléotide 429 en adénine.
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