Tomato plants having a non-transgenic alteration in the DHS gene FIELD OF THE INVENTION

[0001] 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. In addition, 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.

BACKGROUND

[0002] One of the main challenges facing today's tomato industry is how to deliver to a processing plant or to the marketplace tomato fruit that have been vine-ripened (and thus are desirable to consumers in terms of taste, texture, and colour), but that remain firm without the usual post-harvest ripening- related softening that reduces shelf life of harvested fruit. Researchers and breeders hope to develop new tomato plants that have the desirable qualities of vine-ripened fruit, but that are resistant to post-harvest softening and therefore display an extended shelf life. [0003] Fruit softening is one of the many ripening-related changes-including alterations in fruit texture, colour, aroma, and metabolism of sugars and organic acids -that occur as a result of a developmental program triggered by ethylene. The changes associated with ripening, in particular post- harvest softening, limit the shelf life of a fresh product, such as tomatoes. Recent data indicate deoxyhypusine synthase ( DHS ) and eukaryotic translation initiation factor 5 A (eIF-5A) are important regulators of senescence in plants and animals. DHS is an enzyme that converts the inactive form of elF- 5 A to its active form, hypusine-modified eIF-5A. By adding butylamine to a conserved lysine of eIF-5A, DHS catalyses the formation of deoxyhypusine which is subsequently converted to hypusine by the enzyme deoxyhypusine hydoxylase. 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).

[0004] 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.

[0005] Wang, et al ((2005). "Antisense Suppression of Deoxyhypusine Synthase in Tomato Delays Fruit Softening and Alters Growth and Development," Plant Physiology vol. 138, pp. 1372-82.) tried to alter the DHS gene by inserting it in an antisense orientation. Transgenic tomato plant lines with suppressed DHS expression were established and these plants exhibited delayed fruit softening. However half of the transformed plants were male sterile and did not produce fruit at all. Also significant morphological and physiological side effects were found such as deformed pollen, no fruit unless cross-pollinated with wild type pollen, larger and thicker leaves, more chlorophyll per unit surface area in the leaves, fewer internodes, in young leaves, 1.9 times higher photosynthesis activity than in wild tomatoes, corresponding with increased starch deposition in the leaves (up to 78% higher), increased size of the stem pith.

[0006] There is thus a need for tomato plants producing tomatoes with increased firmness, and/or delayed softening but wherein the tomato plant is male fertile. There is also a need for tomato plants producing tomatoes with increased firmness, and/or delayed softening but wherein the tomato plant does not show morphological side effects such as altered leaf morphology.

SUMMARY OF INVENTION

[0007] 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.

[0008] The present invention further relates to an isolated polynucleotide comprising a nucleic acid sequence as defined by SEQ I D NO: 1. [0009] 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.

[0010] In addition, 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.

[0011] Furthermore, 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.

[0012] 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.

[0013] The present invention relates also to an isolated polypeptide comprising an amino acid sequence as defined by SEQ ID NO: 2.

[0014] 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.

[0015] 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. [0016] Furthermore, 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.

[0017] In another aspect, 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.

[0018] In yet another aspect 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.

[0019] Moreover, the present invention relates to an isolated polynucleotide comprising a nucleic acid sequence as defined by SEQ ID NO: 5.

[0020] In addition, 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. [0021] 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.

[0022] Moreover, the present invention relates to an isolated polynucleotide wherein the polynucleotide hybridizes with SEQ I D NO: l or SEQ I D NO: 5. In a pre erred embodiment of the present invention and/or embodiments and or aspects thereof 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. In one embodiment 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. It is understood that in these embodiments 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.

[0023] 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. [0024] In addition, the present invention relates to a tomato plant or a tomato fruit comprising any polypeptide of the invention and/or embodiments thereof.

[0025] In one embodiment, 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.

[0026] In one embodiment, 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. [0027] In one embodiment, 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.

[0028] In one embodiment, 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.

[0029] In one embodiment, the present invention and/or embodiments thereof relate to a tomato plant that is detenriinate.

[0030] In one embodiment, 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.

[0032] In one embodiment, 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.

[0033] In another embodiment, 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. In yet another embodiment, 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.

[0034] In one embodiment, 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.

[0035] in one embodiment, 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.

[0036] In a preferred embodiment, 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. [0037] In a preferred embodiment, 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. [0038] In a preferred embodiment, 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.

[0039] In a preferred embodiment, 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. [0040] In one embodiment, the present invention and/or embodiments thereof relate to pollen produced by a tomato plant according to the invention and/or any embodiment thereof. In still another embodiment, 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. [0041] In a preferred embodiment, the present invention and/or embodiments thereof relate to seed produced by a tomato plant according to the invention and/or any embodiment thereof. In a preferred embodiment, 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. [0042] In one embodiment, 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.

[0044] In yet another embodiment, 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. In a preferred embodiment, 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. [0045] In still another embodiment, 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. In one embodiment, 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. In one embodiment, the present invention and/or embodiments thereof relate to a tomato plant comprising a scion according to the present invention and/or embodiments thereof.

[0046] In one embodiment, 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. In a preferred embodiment, 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. In another embodiment, 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. In yet another embodiment, 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.

[0047] In still another embodiment, 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. [0048] In one embodiment, the present invention and/or embodiments thereof relate to a vector comprising a polynucleotide according to the present invention and/or embodiments thereof. In one embodiment, 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. In one embodiment, 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. lycopersicum deoxyhypusine synthase protein as found in a plant deposited under number NC 1MB 42270, wherein the polynucleotide comprises a mutation at any of position 429 or 430 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3. In one embodiment, 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. In another embodiment, 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.

[0049] In one embodiment, 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. [0050] 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

(i) Introducing into a plant cell a nucleic acid sequence according to the present invention and/or embodiments thereof; (ii) Growing the plant preferably thereby expressing the nucleotide sequence;

(iii) Optionally allowing fruits to grow on the plant of b) and harvesting the tomato fruit.

[0051] In one embodiment, 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.

[0052] In yet another embodiment, 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.

[0053] In still another embodiment, 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. [0054] In one embodiment, 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.

[0055] in one embodiment, 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.

[0056] In another aspect, 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:

(i) Providing a first tomato plant wherein the first tomato plant is a tomato plant according to aspects and/or embodiments of the invention;

(ii) Providing a second tomato plant;

(iii) Crossing the first and second tomato plant

(iv) Harvest seeds from the crossing of (iii)

(v) Optionally cross plants grown from the seeds of (iv) with another tomato plant, preferably a second plant of (ii)

(vi) Optionally cross the plant grown from the seeds of (iv) with a first tomato plant.

[0057] In another aspect, 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:

(i) Providing a first tomato plant wherein the first tomato plant is a tomato plant according to aspects and/or embodiments of the invention;

(ii) Providing a second tomato plant;

(iii) Crossing the first and second tomato plant (iv) Harvest seeds from the crossing of (iii)

(v) Optionally cross plants grown from the seeds of (iv) with another tomato plant, preferably a second plant of (ii), wherein the first tomato plant is an inbred line.

[0058] In a preferred embodiment, 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.

[0059] In one embodiment, 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. [0060] In another embodiment, 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.

[0061] In one embodiment, 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.

[0062] In one embodiment, 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. the 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. [0063] In a preferred embodiment, 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. In one embodiment of this method, 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.

[0064] In still another embodiment, 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. nucleotides at position 429-43 1 do not encode a valine. [0065] In a preferred embodiment, 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.

[0066] In a preferred embodiment, 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. [0067] In another aspect, 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:

(i) obtaining plant material from a parent tomato plant; (ii) treating said plant material with a mutagen to create mutagenized plant material;

(iii) analysing said 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.

[0068] In one embodiment, the plant material in step (i) is a tomato seed. In another embodiment, the mutagen in step (ii) is EMS.

[0069] in one embodiment, 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.

[0070] In another aspect, 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:

(i) Providing a tomato plant wherein the first tomato plant is a tomato plant according to any aspect of the invention and/or embodiments thereof;

(ii) Introducing into said tomato plant nucleic acids for said second trait.

[0071] in one embodiment, 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.

[0072] In a preferred embodiment, 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:

(i) Providing a tomato plant wherein the first tomato plant is a tomato plant according to any aspect of the invention and/or embodiments thereof;

(ii) crossing the tomato plant according to (i) with a second tomato plant comprising said second trait;

(iii) harvest seeds from the crossing of (iii);

(iv) optionally selfing plants grown from seeds of (iii);

(v) optionally crossing plants grown from seeds of (iii) with the plant of (i) or (ii).

[0073] In one embodiment, 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.

[0074] In one embodiment, 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.

[0075] In one embodiment, 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.

[0076] In one embodiment, 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.

[0077] In one embodiment, 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.

[0078] In a preferred embodiment, 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.

[0079] In one 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 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. [0080] In one 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 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.

[0081] In one embodiment, 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) .

[0082] In another embodiment, 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. [0083] In still another 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 determinate or indeterminate.

[0084] In yet another 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 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.

[0085] In another aspect, 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:

(i) providing a population of tomato plants;

(ii) screening for a mutation as defined by the invention and embodiments thereof;

(iii) selecting a tomato plant carrying a mutation in the nucleic acid sequence encoding for a deoxyhypusine synthase, said mutation as by the invention and embodiments thereof.

[0086] In one embodiment, 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.

[0087] In another aspect, the present invention further relates to a method of screening tomato plants for the presence of a mutant DHS allele comprising the steps of

(i) providing material from a tomato plant

(ii) isolate DNA from the plant material;

(iii) detect the presence of DNA having a sequence having at least 70% sequence identity to a nucleic acid sequence as defined to SEQ I 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 v aline; 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%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.

[0088] In one embodiment, 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.

[0089] In another aspect, the present invention further relates to a method of screening tomato plants for the presence of a mutant DHS allele comprising the steps of

(i) providing material from a tomato plant ;

(ii) isolate protein from the plant material of (i);

(iii) detect the presence of 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 I D 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 I D NO: 4.

[0090] In another aspect, 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.

[0091] In another aspect, 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.

[0092] In a preferred embodiment, 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.

[0093] in another aspect, 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.

[0094] in one embodiment, 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.

[0095] In another aspect, 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. [0096] In one embodiment, 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. [0097] In one embodiment, 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;

(ii) Growing a tomato plant from said plant cell, preferably thereby expressing the nucleotide sequence;

(iii) Optionally harvesting the tomato fruit. [0098] In one embodiment, 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. [0099] In another embodiment, 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. [0100] in still another embodiment, 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. [0101] In another aspect, 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.

[0102] In one embodiment, 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;

(ii) isolate DNA and/or protein from said plant material;

(iii) detect the presence of DNA having a sequence having at least 70% 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 I 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%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.

[0103] In one embodiment, 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.

[0104] In another aspect, 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.

[0105] In another embodiment, 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.

[0106] In yet another embodiment, 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.

[0107] In a preferred embodiment, 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:

(i) obtaining plant material from a tomato plant, tomato seed, tomato plant part and/or tomato plant cell;

(ii) treating said plant material, tomato seed, tomato plant part and/or tomato plant cell with a mutagen to create mutagenized plant material;

(iii) analysing said 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 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.

[0108] In another aspect, 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.

[0109] In one embodiment, 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. [0110] In another embodiment, 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. [0111] In still another embodiment, 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. [0112] In one embodiment, 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.

[0113] In another aspect, 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.

DETAILED DESCRIPTION General definitions

[0114] The term "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. An "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.

[0115] 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).

[0116] "Expression of a gene" refers to the process wherein a DNA region, which is operabiy linked to appropriate regulatory regions, particularly a promoter, is transcribed into an 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.

[0117] The terms "protein", "peptide sequence", "amino acid sequence" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 -dimensional structure or origin. A "fragment" or "portion" of DHS protein may thus still be referred to as a "protein". An "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. [0118] An "active protein" or "functional protein" is a protein which has protein activity as measurable in vitro, e.g. by an in vitro activity assay, and/or in vivo, e.g. by the phenotype conferred by the protein. 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.

[0119] "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. [0120] A fragment of the mutant DHS protein refers to any subset of the molecule.

[0121] Variant peptides may be made by direct chemical synthesis, for example, using methods well known in the art.

[0122] An analogue of the mutant DHS refers to a non-natural protein substantially similar to either the entire protein or a fragment thereof. [0123] 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. Alternatively, the protein can be produced in a cell-free system. Ranu, et al., Meth. Enzymol, 60:459-484, (1979).

[0124] 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.

[0125] 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.

[0126] As used herein, the term "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. For instance, 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.

[0127] 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. Generally, the default parameters are used, with a gap creation penalty = 10 and gap extension penalty = 0.5 (both for nucleotide and protein alignments). For nucleotides 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

(http://www.ebi.ac.uk/rools/psa/emboss_needle/). Alternatively 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. gap creation penalty = 10, gap extension penalty = 0.5, using scoring matrix DNAFULL for nucleic acids an Blosum62 for proteins). Such sequences are also referred to as 'variants' herein, e.g. other variants of mutant DHS alleles and mutant DHS proteins than the specific nucleic acid and amino acid sequences disclosed herein can be identified, which have the same effect on increased firmness and/or slower softening, and/or increased longer shelf- life of the fruits comprising such variants. [0128] The term "hybridisation" as used herein 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. For typical high stringency conditions, 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. For lower stringencies the temperature of hybridisation is reduced to about 42°C below the melting temperature (TM) of the duplex. 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.

[0129] As used herein, the phrase "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. For example, 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. It is to be understood that 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.

[0130] As used herein, the phrase "a 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.

[0131] The term "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. In a diploid cell of an organism, alleles of a given gene are located at a specific location, or locus (loci plural) 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). [0132] The term "locus" (plural loci) 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.

[0133] "Wild type allele" (WT) refers herein to a version of a gene encoding a fully functional protein (wild type protein). Such a sequence encoding 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.

[0134] "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)) may lead to the encoded protein having reduced in vitro and/or in vivo functionality (reduced function) or no in vitro and/or in vivo functionality (loss-of-function), e.g. due to the protein e.g. being truncated or having an amino acid sequence wherein one o more amino acids are deleted, inserted or replaced. 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.

[0135] "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). Such plants are for example suitable controls in phenotypic assays. Preferably wild type and/or mutant plants are "cultivated tomato plants". For example the cuitivar Moneymaker is a wild type plant, as is cuitivar Ailsa Craig, cuitivar Tapa and many others. [0136] "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.

[0137] Other fully functional mutant DHS protein-encoding alleles (i.e. alleles which confer firmness to the same extent as the protein of mutant sequence SEQ ID NO 4) 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. Likewise the 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.

[0138] It is noted that nucleotide sequences referred to herein ( SEQ I D NO: 1 and SEQ I D NO: 3) are cDNA, i.e. coding DNA sequences, encoding the proteins of SEQ I D NO: 2 and SEQ I D NO: 4. Obviously, when reference is made to these cDNA nucleotide sequences, it is understood that the 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. Thus, when reference is made to 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. Further, when reference is made to a tomato plant comprising a nucleotide sequence encoding a protein according to the invention (such as a mutant protein of SEQ I D No: 2, or a different mutant), this encompasses different nucleotide sequences, due to the degeneracy of the genetic code. In one embodiment the plant comprises the genomic DHS sequence depicted in SEQ I D NO: 5 or a genomic DHS sequence substantially identical thereto (e.g. 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: 5), but with one or more mutations in said sequence, especially in the exons of said genomic sequence, causing a change in the activity of the encoded DHS protein.

[0139] The following 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. [0140] 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.

[0141] The term "functional derivative" of a nucleic acid (or poly- or oligonucleotide) 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. Preferably the functional derivative comprises the mutation of the invention.

[0142] 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.

[0143] 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. Preferably the variant comprises the mutation as defined by the invention. [0144] A "homologue" refers to a fragment or variant sequence from a different plant genus or species. Preferably the homologue comprises the mutation as defined by the invention.

[0145] 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. Preferably the analogue comprises the mutation as defined by the invention.

[0146] As used herein, 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. Also any developmental stage is included, such as seedlings, immature and mature, etc. Preferably the plant part or derivative comprises the mutation as defined by the current invention. [0147] A "plant line" or "breeding line" refers to a plant and its progeny. As used herein, the term "inbred line" refers to a plant line which has been repeatedly selfed.

[0148] "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. Therefore, the term "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.

[0149] "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). [0150] "Tomato plants" or "cultivated tomato plants" are plants of the Solanum lycopersicum, i.e. varieties, breeding lines or cultivars of the species Solanum lycopersicum, cultivated by humans and having good agronomic characteristics; preferably such plants are not "wild plants", i.e. plants which generally have much poorer yields and poorer agronomic characteristics than cultivated plants and e.g. grow naturally in wild populations. "Wild plants" include for example ecotypes, PI (Plant Introduction) lines, landraces or wild accessions or wild relatives of a species. The so-called 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. [0151] Wild relatives of tomato include S. arcanum, S. chmielewskii, S. neorickii ( = L. parviflorum), S. cheesmaniae, S. galapagense, S. pimpinellifolium, S. chilense, S. corneliomulleri, S. habrochaites ( = L. hirsutum), S. huaylasense, S. sisymbriifolium, S. peruvianum, S. hirsutum or S. pennellii.

[0152] 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.

[0153] 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. [0154] 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. [0155] The term "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. I n one embodiment 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. Thus, to determine the number of days required from a certain stage (e.g. from breaker stage or a later stage) to 'bad' stage, 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 (e.g. breaker stage or a later 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).

[0156] 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. (6) 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.

[0157] "Average" refers herein to the arithmetic mean.

[0158] It is understood that 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. Preferably the plants are of the same line or variety. In general 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.

[0159] Softness or firmness may be measured by flat-plate compression, constant area compression or by puncture. For 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. For 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. A flat- ended cylindrical probe is used with a specific force and deformation speed. 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.

[0160] In this application 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.

[0161] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". It is further understood that, when referring to "sequences" herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids or nucleic acids) are referred to. "Position 429-430" of a sequence refers to position429 or 430 or both, in the sequence. Nucleotides are numbered counting the "A" (adenine) in the ATG start codon as number 1.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING [0162] SEQ ID NO: 1 : shows the mutant dhs cDNA of the Solanum lycopersicum mutant having a mutation G429A.

[0163] SEQ I D NO: 2: shows the mutant dhs amino acid sequence of the Solanum lycopersicum mutant having a mutation V126I. [0164] SEQ ID NO: 3:shows the Solanum lycopersicum wild type DHS cDNA based on NCBI Reference Sequence: AF296077; 1610 nucleotides

[0165] 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;.

[0166] 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.

[0167] 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.

[0168] SEQ I D NO: 7 shows the Forward primer to amplify DHS fragment. [0169] SEQ I D NO: 8 shows the Reverse primer to amplify DHS fragment.

BRIEF DESCRIPTION OF THE FIGURES

[0170] 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. [0171] Figure 2: Impact mutation 429GTT431 to 429ATT431 , 126V to 1261.

[0172] Figure 3: Alignment of SEQ I D NO: 2 (mutant 4282) with wild type amino acid sequence (SEQ I D NO: 4; AF296077).

[0173] 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

[0174] The present invention relates to tomato plants and tomato fruits exhibiting an altered deoxyhypusine synthase (DHS) enzyme activity. Preferably, the altered DHS enzyme activity is obtained without the inclusion of foreign nucleic acids in the tomato plants' genomes. In a one embodiment 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. [0175] 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.

[0176] The altered DHS may be created and identified by a method known as TILLING. TILLING (Targeting Induced Local Lesions IN Genomes) 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. Thus, TILLING uses traditional chemical mutagenesis (e.g. EMS or MNU mutagenesis) or other mutagenesis methods (e.g. radiation such as UV) followed by high-throughput screening for mutations in specific target genes, such as DHS according to the invention. SI nucleases, such as CELl or ENDOl , are used to cleave heteroduplexes of mutant and wild type target DNA and detection of cleavage products using e.g. 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. 2007, BMC Plant Biol 7: 19), Arabidopsis (Till et al. 2006, Methods Mol Biol 323: 127-35),-Brassica, maize (Till et al. 2004, BMC Plant Biol 4: 12), etc. Also EcoTILLING, whereby mutants in natural populations are detected, has been widely used, see Till et al. 2006 (Nat Protoc 1 : 2465-77) and Comai et al. 2004 (Plant .1 37: 778-86).

[0177] In one aspect 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. In another embodiments 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. In another embodiment 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. In still another embodiment, the isolated polynucleotide comprises a mutation into an adenine (A). [0178] 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. As is well known, amino acid codons are degenerate and more than one three- nucleotide codon may code for an amino acid. For example the following codons code for a valine: GTT, GTC, GTA, GTG, and the following codons code for isoleucine ATT, ATC, ATA. It can be seen that for three three-nucleotide codons for valine, GTT, GTC, GTA, the change of the first guanine into an adenine into ATT, ATC, ATA respectively, will lead to a isoleucine in the translated protein.

[0179] 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. In addition, 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.

[0180] Furthermore, 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. [0181] In one embodiments 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 . In another embodiment 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 . Because of the degenerate coding of DNA/RNA for amino acids, polynucleotides having alternative amino acid coding may still express DHS and are expressly encompassed in the present invention. The important part is that the nucleotides 429-431 do not code for a valine and preferably code for an isoleucine. In one embodiment, the isolated polynucleotide comprises a mutation at position 429 when compared to the wild type nucleic acid sequence defined by SEQ ID NO: 3. In one embodiment the isolated polynucleotide comprises a mutation into an adenine (A) at position 429. In still another embodiment, 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.

[0182] In one aspect, 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. Preferably, the polynucleotide hybridizes under stringent conditions, such as hybridization in a buffered solution of 0.9M NaCl at 55° C. Preferably 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. In one embodiment of this aspect of the invention, 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 . or even 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.

[0183] Furthermore, 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.

[0184] Moreover, the present invention relates to an isolated polynucleotide comprising a nucleic acid sequence as defined by SEQ I D NO: 5.

[0185] In addition, 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. lycopersicum DI I S protein as found in a plant deposited under number NCIMB 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. [0186] 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. [0187] In one embodiment 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 . Because of the degenerate coding of DNA/RNA for amino acids, polynucleotides having alternative amino acid coding may still express DH S and are expressly encompassed in the present invention. The important part is that the nucleotides 2882-2884 do not code for a valine and preferably code for a isoleucine. Preferably, 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. Preferably, the isolated polynucleotide comprises a mutation into an adenine (A). Preferably, 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.

[0188] Other putative 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).

[0189] In one aspect, 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. Preferably, the polynucleotide hybridizes under stringent conditions, such as hybridization in a buffered solution of 0.9M NaCl at 55° C. Preferably the isolated polynucleotide hybridizes with at least nucleotide 2882-2884 of SEQ I D NO: 5. In one embodiment of this aspect of the invention, 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. [0190] In another embodiment of the invention, there is provided an isolated polypeptide encoded by a polynucleotide of the invention and/or embodiments, or a functional derivative thereof. In one embodiment the polypeptide comprises the amino acid sequence comprising at least 70% (e.g. 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. Preferably said mutation is a change of an amino acid 126 into isoleucine. . In one embodiments the isolated polypeptide is having at least 72%, e.g. at least 75%, 78%, 80%>, 83 %o, 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 NO:2. In one embodiment, the functional derivative of SEQ I D NO:2 has at least 70% amino acid sequence identity (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% ) to SEQ ID NO:2 and provides an altered DHS activity comparable to a polypeptide with sequence SEQ ID NO: 2. Preferably 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.

[0191] 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. 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 embodiment 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. In one embodiment 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.

[0192] 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. In addition, 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. [0193] 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. Preferably the tomato fruit has a shape selected from the group consisting of beefsteak, plum, round, or cherry. In addition, 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. Preferably the tomato fruit has colour selected from the group consisting of bicolor, red, black, deep pink, orange, yellow or pink. Most preferably 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. Preferably 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.

[0194] In one embodiment, 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). In one embodiment 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. Thus 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.

[0195] Furthermore in another aspect, 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. [0196] In another aspect, 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.

[0197] In yet another aspect, 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.

[0198] 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. [0199] In one embodiment, 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. [0200] In another aspect and/or embodiments thereof, 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. In one embodiment, the post-harvest shelf life is at least 1 15%>, e.g. 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. In another aspect and/or embodiments thereof 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. In another aspect and/or embodiments thereof the post-harvest shelf life is at least 180%, e.g. at least 200%o or even at least 250 % 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. [0201] In still another embodiment, 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. In one embodiment, 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. Preferably, 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. Preferably the tomato fruits are harvested at the red stage or at full red stage. [0202] in one embodiment, 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. Preferably 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. [0203] Preferably 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. In one embodiment the plants according to the invention, which comprise mutant DHS show normal leaf morphology and do not show modified leaf morphology. In another embodiment the plants according to the invention, which comprise mutant DHS are not male sterile. [0204] The invention further relates to tomato fruits produced by tomato plants of the invention and/or embodiments thereof. In addition, the present invention relates to a tomato plant producing tomato fruits according to the invention and/or embodiments thereof.

[0205] The present invention also relate to pollen which are produced by a tomato plant according to the invention and/or any embodiment thereof. Preferably, 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. Preferably 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. Preferably the mutation is a mutation of G to A. Preferably 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. Suitably, 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. at least 99.2%, 99.4%, 99.5%, or even 99.7% or 99.8% or 99.9% ) 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. Suitably, 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. More suitably the pollen comprise a polynucleotide comprising a nucleic acid sequence as defined by SEQ I D NO: 5. Suitably, 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. 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 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, in one embodiment 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. Preferably the mutation is a mutation of G to A. More suitably 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.

[0206] 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. Preferably 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. Preferably the mutation is a mutation of ( i to A. Preferably 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. Suitably, 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 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. Suitably, 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. More suitably the seed comprise a polynucleotide comprising a nucleic acid sequence as defined by SEQ I D NO: 5. Suitably, 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. lycopersicum deoxyhypusine synthase ( DH S) 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. Preferably 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. Preferably 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. [0207] 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. It is understood that such 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) In a preferred embodiment, the tomato plant is grown from seeds according to the present invention and/or embodiments thereof. Seeds may be treated and / or primed.

[0208] According to a further aspect 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.

[0209] Also 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.

[0210] In addition, 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. Preferably 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. Suitably 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. In one embodiment the food product is a salad or sandwiches.

[0211] The present invention also is directed to parts of the tomato plant or tomato fruit of the invention. Suitably 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. Preferably 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.

[0212] 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. In one embodiment 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. Preferably the mutation is a mutation of G to A. Preferably 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. Suitably, 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 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. Suitably, 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. More suitably the plant cell comprise a polynucleotide comprising a nucleic acid sequence as defined by SEQ ID NO: 5. Suitably, 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. Preferably 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. Preferably the mutation is a mutation of G to A. Suitably, 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. Suitably, 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. In one embodiment 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.

[0213] 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.

[0214] 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. Suitably 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. In preferred embodiments 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. lycopersicum deoxyhypusine synthase ( DHS) protein as found in a plant deposited under number NCIMB 42270 Preferably 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. Preferably, 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. Preferably 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. Preferably the mutation is a mutation of G to A. More preferably 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.

[0215] 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.

[0216] Suitably, 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. Optionally, the regulatory sequences include a polyadenylation signal.

[0217] 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). Preferably, the promoter is a constitutive promoter, most preferably a double 35 S promoter.

[0218] 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. For example, 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. Alternatively, 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.

[0220] 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.. Nature, 319:719 (1986); particle bombardment (Klein et al., 30 Bio Technology, 6:559-563 (1988); injection into meristematic tissues of seedlings and plants (De LaPena, et al., Nature, 325:274-276 (1987); injection into protoplasts of cultured cells and tissues (Reich, et al., BioTechnology, 4:1001-1004 (1986)). Generally a complete plant is obtained from the transformation process. Plants are regenerated from protoplasts, callus, tissue parts or explants, etc. Plant parts obtained from the regenerated plants in which the mutated DHS is expressed such as leaves, flowers, fruit, seeds and the like are included in the definition of "plant parts" as used herein. Progeny, variants and mutants of the regenerated plants are also included in the definition of plant."

[0221] In another aspect 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.

[0222] 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

(i) Introducing into a plant cell a polynucleotide according to the present invention and/or embodiments thereof as defined above; (ii) Optionally growing a plant from said plant cell preferably thereby expressing the nucleotide sequence;

(iii) optionally harvesting the tomato fruit.

[0223] 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). Thus, 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. The term "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. In a one embodiment 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. In one embodiments, at least one tomato plant comprises at least one other desirable trait. Preferably 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. Alternatively 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. Preferably the vector comprises a polynucleotide according to the invention and/or embodiments thereof. In addition, 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. Suitably 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-chloro-ethyl]aminopropylamine]a cridine dihydro chloride (ICR- 170), and formaldehyde. Suitable radiation is UV radiation or radioactive radiation. [0224] The present invention further relates to a method of producing a tomato fruit, and/or tomato plant producing a tomato comprising

(i) Crossing a first tomato plant wherein the first tomato plant is a tomato plant according to the invention with a second tomato plant; (ii) Optionally harvest seeds from the crossing of (i)

(iii) optionally cross plants grown from the seeds of (ii) with another tomato plant, preferably a second plant of (i)

(iv) optionally cross the plant grown from the seeds of (ii) with the first tomato plant.

[0225] In a one embodiment the method of producing a tomato fruit, and/or tomato plant producing a tomato comprises

(i) Crossing a first tomato plant wherein the first tomato plant is a tomato plant according to the invention with a second tomato plant;

(ii) Optionally harvest seeds from the crossing of (i);

(iii) optionally cross plants grown from the seeds of (ii) with another tomato plant, preferably a second plant of (i).

[0226] The second tomato plant may also be an inbred line.

[0227] The methods described above provide hybrid plants and hybrid seeds. In circumstances, the first and/or second tomato plant comprise a further desirable trait. Optionally, the 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. Thus, in one embodiment a plant according to the invention is used as a parent plant to produce a hybrid plant and/or hybrid seeds. In one embodiment Fl hybid tomato seeds (i.e. 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. In one embodiment 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. In one embodiment 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. In addition, 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. Suitably 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. 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. or a polynucleotide hybridizing with SEQ I D NO: 5 and especially hybridize with the mutation at any of position 2882-2883 when compared to the wild type nucleotide acid sequence defined by SEQ ID NO: 6. In a preferred embodiment 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. Furthermore tomato plants may be selected comprising a polypeptide comprising an amino 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%) 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. 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. 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. In still another aspect 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. In yet another aspect 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.

[0228] In another aspect, 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:

(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.

[0229] In one embodiment, 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. Preferably 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. Preferably the mutation is a mutation of G to A.

[0230] In one embodiment of the present invention, 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. One of skill in the art would understand that a variety of 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. However, the type of plant material mutagenized may affect when the plant DNA is screened for mutations. For example, when pollen is subjected to mutagenesis prior to pollination of a non-mutagenized plant, 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 methoxy-6-chloro-9[3-(e thyl-2-chloro-ethyl)aminopropylamino] acridine dihydro chloride (ICR- 170), and formaldehyde. 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. Preferably, groups of four or more individuals are pooled. After the DNA samples are pooled, the pools are subjected to deoxyhypusine synthase gene-specific amplification techniques, such as Polymerase Chain Reaction (PCR). For a general overview of PG R. see PCR Protocols: A Guide to Methods and Applications (Inns, M., Gelfand, D., Sninsky, J., and White, T., eds.), Academic Press, San Diego, 1990. Any primer specific to the deoxyhypusine synthase genes or the sequences immediately adjacent to the deoxyhypusine synthase genes may be utilized to amplify the deoxyhypusine synthase genes within the pooled DNA sample. Preferably, 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.

[0231] Once an M2 or Ml plant having a mutated deoxyhypusine synthase gene is identified, the mutations are analyzed to determine its effect on the expression, translation, and/or activity of the deoxyhypusine synthase enzyme. First, 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. Nuc Acids Res 31 :3812-3814, 2003), PSSM (Position-Specific Scoring Matrix; Henikoff, J.G. and Henikoff, S. Comput Appl Biosci, 12: 135- 143, 1996) and PA SESNP 5 (TaylorN.E. and Greene, E.A. Nuc Acids Res, 31 :3808-381 1 , 2003). For example, a SIFT score that is less than 0.05 and a large change in PSSM score (roughly 10 or above) indicate a mutation that is likely to have a deleterious effect on protein function. I f the initial assessment of the mutation in the M2 plant indicated it to be of a useful nature and in a useful position within a deoxyhypusine synthase gene, then further phenotypic analysis of the tomato plant containing that mutation is pursued. First, 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.

[0232] 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. In a preferred embodiment, 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. Preferably, 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. Preferably the harvest is at full red stage. Preferably 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. Preferably the harvest is at full red stage. In one embodimentembodiment, the method of producing a tomato plant, the 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. Preferably 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. In a preferred embodiment, the method of producing a tomato plant, tomato plant produces a tomato fruit hav ing a skin colour selected from the group consisting of dark, bi-color, deep pink, golden, green, orange, pink, red, white, and yellow. In a preferred embodiment, the method of producing a tomato plant, the tomato plant is determinate or indeterminate. In one embodiment, the method of producing a tomato plant, the tomato plant is open-pollinated or hybrid. Preferably the tomato plant is a hybrid tomato plant.

[0233] In one aspect the invention relates to plants obtained in any of the methods described herein.

[0234] Optionally 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.

[0235] Also 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

(and seeds from which hybrid plants can be grown) comprising a mutant DHS allele in their genome are en compassed herein, whereby the double haploid plants and hybrid plants produce tomato fruits having reduced softness, increased firmness and/or longer shelf life fruits according to the invention.

[0236] 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.

[0237] In another aspect, 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

(i) screening a population of one or more tomato plants for a mutation as defined by the invention and embodiments thereof;

(ii) Optionally selecting a tomato plant carrying a mutation in the nucleic acid sequence encoding for a deoxyhypusine synthase, said mutation as by the invention and embodiments thereof.

[0238] Preferably 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. There are many methods that may be used to screen for a certain mutation. Screening may be done on nucleic acid material or on protein material. For example nucleic acid hybridization, PCR technology direct, and gene sequencing of the DHS gene may be used to screen plant populations for mutant alleles. For example 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.

[0239] I another aspect, the present invention further relates to a method of screening tomato plants for the presence of a mutant DHS allele comprising the steps of:

(i) Optionally providing a tomato plant material ;

(11) Optionally isolate DNA from the plant material;

(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%ο, 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. In one embodiment 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.

[0240] In another aspect, the present invention further relates to a method of screening tomato plants for the presence of a mutant DHS allele comprising the steps of

(i) Optionally providing a tomato plant material ;

(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%,

99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or even 99.7%) 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.

[0241] In another aspect, 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. [0242] In another aspect, 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.

[0243] In another aspect, 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. Preferably 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

(i) Introducing into a plant c ell a nucleic acid sequence according to aspects of the invention and/or embodiments thereof; (ii) Optionally Growing a tomato plant from said plant cell, preferably thereby expressing the nucleotide sequence;

(iii) optionally harvesting the tomato fruit.

[0244] 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.

[0245] In another aspect, 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. Suitably the use of polynucleotide and/or polypetide according for screening for mutation comprises the steps

(i) in 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. 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% (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%o) 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. Optionally said DNA or protein is from plant material from more than one tomato plant. Methods to detect the presence of a nucleic acid and/or protein are explained above.

[0246] In another aspect, 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.

[0247] In another aspect, 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. Suitably 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. Optionally 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.;

[0248] In another aspect, 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. Preferably, 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.

[0249] In a preferred embodiment, 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.

[0250] in a preferred embodiment, 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. Preferably 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.

[0251] In another aspect, 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.

[0252] In one aspect 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

1. A tomato plant cell of a plant of the invention, i.e. comprising a mutation in an DHS allele.

2. The plant cell of embodiment 1 , wherein the plant cell is a seed cell.

3. A plant cell from a tomato plant, or a plant cell from a part of the tomato plant, wherein the tomato plant is produced by growing seed of tomato mutant 4282, and wherein a representative sample of said seed has been deposited under Accession Number NCI MB 42270.

4. The plant cell of embodiment 4 wherein the plant cell is from a part of the tomato plant and wherein the plant cell is a cell from a cutting, a cell culture or a tissue culture.

5. A plant cell from i) a tomato plant or ii) a tomato seed wherein the plant or seed is a descendant of tomato mutant 4282, wherein a representative sample of seed of mutant 4282 has been deposited under Accession Number NCI MB 42270, wherein the descendant expresses the mutant DHS protein as mutant 4282 when grown under substantially similar environmental conditions, wherein the descendent is produced by self-pollinating mutant 4282 or crossing mutant 4282 with another tomato plant. 6. Use of tomato mutant 4282, wherein a representative sample of seed of mutant 4282 has been deposited under Accession Number NC 1MB 42270, to cross with another tomato plant and optionally select progeny from said crossing.

7. Use of tomato mutant 4282, wherein a representative sample of seed of mutant 4282 has been deposited under Accession Number NCI M B 42270, to produce a genetic marker profile. 8. Use of tomato plant of the invention i.e. comprising a mutant DHS allele in homozygous or heterozygous form, for tomato fruit production.

9. Use of tomato plant of the invention i.e. comprising a mutant DHS allele in homozygous or heterozygous form, in a method of producing a tomato plant having a desired trait.

10. Use of tomato plant of the invention i.e. comprising a mutant DHS allele in homozygous or heterozygous form, in a tomato breeding program or in a tomato seed production program. Seed Deposits

[0253] A representative sample of seeds of tomato mutants according to Example 1, were deposited by Nunhems B.V. and accepted for deposit on July 25, 2014 at the NCI MB Ltd. (Ferguson Building, Craib stone Estate, Bucksburn Aberdeen, Scotland AB21 9YA, UK) according to the Budapest Treaty, under the Expert Solution (EPC 2000, Rule 32(1)). Seeds were given the following deposit number: NC I MB 42270 (mutant 4282).

[0254] The Applicant requests that samples of the biological material and any material derived therefrom be only released to a designated Expert in accordance with Rule 32(1) EPC or related legislation of countries or treaties having similar rules and regulation, until the mention of the grant of the patent, or for 20 years from the date of filing if the application is refused, withdrawn or deemed to be withdrawn.

[0255] Access to the deposit will be available during the pendency of this application to persons determined by the Director of the U.S. Patent Office to be entitled thereto upon request. Subject to 37 C.F.R. § 1.808(b), all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent. The deposit will be maintained for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent whichever is longer, and will be replaced if it ever becomes nonviable during that period. Applicant does not waive any rights granted under this patent on this application or under the Plant Variety Protection Act (7 USC 2321 et seq.).

EXAMPLES General methods Mutagenesis

[0256] 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. From these 10600 plantlets, 1790 were either sterile or died before producing fruit. For each remaining Ml mutant plant one fruits was harvested and its seeds isolated. The obtained population, named 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. [0257] 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 μΐ 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 μΐ 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 μΐ) and magnetic beads (20 μΐ). Once DNA was bound to the beads, two successive washing steps were carried out (Wash buffer 1 : Agowa wash buffer 1 1/3, ethanoi 1/3, isopropanol 1/3; Wash buffer 2: 70% ethanol, 30% Agowa wash buffer 2) and finally eluted in elution buffer (100 μΐ MQ, 0,025 μΐ Tween). [0258] Grinding ten S. lycopersicum seeds produced enough DNA to saturate the magnetic beads, thus hi hly homogenous and comparable DNA concentrations of all samples were obtained. Comparing with lambda DNA references, a concentration of 30 ng/μΐ for each sample was estimated. Two times diluted DNA was 4 fold flat pooled. 2 μΐ pooled DNA was used in multiplex PCRs for mutation detection analysis.

[0259] Primers used to amplify gene fragments for High Resolution Melt (HRM) curve analysis were designed using a computer program (Primer3, http://primer3.sourceforge.net/). The length of the amplification product was limited between 200 and 400 base pairs. Quality of the primers was determined by a test PGR reaction that should yield a single product.

[0260] Polymerase Chain Reaction (PGR) to amplify gene fragments. lOng of genomic DNA was mixed with 4μ1 reaction buffer (5x Reaction Buffer), 2μ1 lOxLC dye ((LCGreen+ dye, Idaho Technology Inc., UT, USA), Spmole of forward and reverse primers each, 4nmole dNTPs (Life Technologies, NY, USA) and 1 unit DNA polymerase (Hot Start 11 DNA Polymerase) in a total volume of ΪΟμί. Reaction conditions were: 30s 98°C, then 40 cycles of 10s. 98°C, 15s 60°C, 25s of 72°C and finally 60s at 72°C. 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).

[0261] Hi h Resolution Melt curve analysis (HRM) has been proven to be sensitive and high- throughput methods in human and plant genetics. HRM is a non-enzymatic screening technique. During the PCR amplification dye (LCGreen+ dye, Idaho Technology Inc., UT, USA) molecules intercalate between each annealed base pair of the double stranded DNA molecule. 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. At a temperature dependent on the sequence specific stability of the DNA helices, 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. [0262] 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. When the presence of the mutation, based on the HRM profile, was confirmed in one of the four individual M2 family DNA samples, the PCR fragments were sequenced to identify the mutation in the gene. Once the mutation was known, the effect of such an mutation was predicted using a computer program CODDLe (for Choosing codons to Optimize Discovery of Deleterious Lesions, http://www.proweb.org/coddle/) that identifies the region(s) of a user-selected gene and of its coding sequence where the anticipated point mutations are most likely to result in deleterious effects on the gene's function. [0263] Seeds from M2 families that contain mutations with predicted effect on protein activity were sown for phenotypic analysis of the plants. Homozygous mutants were selected or obtained after so! ting and subsequent selection. The effect of the mutation on the corresponding protein and phenotype of the plant was determined. [0264] Seeds containing the identified mutations were germinated and plants were grown in pots with soil the greenhouse with 16/8 light dark regime and 18°C night and 22-25°C day temperature. For each genotype 5 plants were raised. The second, third and fourth inflorescence were used for the analysis. The inflorescences were pruned remaining six flowers per inflorescence that were allowed to set fruit by self-pollination. The dates of fruit set of the first and sixth flower was recorded as was the date of breaker and red stage of the first and sixth fruit. At red stage of the fourth fruit 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.

[0265] At later stages fruit condition was determined based on visual assessment of the fruits and the date when the oldest fruit became 'bad' was recorded and further fruit deterioration was recorded (indicated by further fruit softness assessed by pinching the fruits, and visual assessment of dehydration/water loss, breaking of the skin and fungal growth).

[0266] The following mutant was identified: mutant 4282, and seeds were deposited at the NCI MB under the Accession numbers given above. Protocol measurement softening

[0267] Seeds from wild type cv. Tapa and mutant 4282 in the same genetic background were sown and the seedlings were grown under standard growing conditions in the greenhouse. Ten plants were grown for each genotype; wild type, mutant 4282 with the mutation homozygously or heterozygously present and plants without the mutation, but from the same M2 mutant family (azygous). [0268] Three clusters of tomatoes were selected at each plant. Each cluster was labelled with a colour label that indicated the cluster age; the oldest cluster (#1) was marked with a yellow label, the second oldest cluster (#2) was marked with a blue label and the third oldest cluster (#3) was marked with a red label. The 3 ^rd and 4 ^th tomato of each cluster was selected for measurement of firmness. By this, six tomatoes where followed from each plant. For every tomato the date of the breaker stage (breakdown of chlorophyll), the yellow/pink stage and full red were recorded, classified according to USDA (1997). The colour of the tomato represents the stage of development and therefore a colour chart (Royal Horticultural Society, 2007) was used to determine (the range of) colour(s) that belongs to a ripening stage (Table 1). Tomatoes that entered the breaker stage were marked with a white label at the joint. Every 2 days the population was checked for fruit entering the breaker stage. The fruits were collected for measurement at the day they were just full red.

Table 1 : Tomato ripening stage is determined by the tomato color (Royal Horticultural Society, 2007).

[0269] Harvested tomatoes were stored at room temperature (22-24°C) until measurement after either 0, 7, 14 or 21 days. Firmness was measured using a Single Column Tabletop Testing Systems (Instron, System ID: 3342L2018; Force Transducer model 2519-104) and Bluehill Software (Instron, 825 University Ave, Norwood, MA 02062-2643, USA). Each measurement was carried out only once per fruit and in such a way that the fruits were not damaged. A tomato fruit was compressed between two flat steel plates with an incremental force from 0.1 Newton (N) to 4 (N). The firmness was calculated as the distance of compression relative to the fruit size (as described by Sirisomboon et al 2012, Journal of food engineering; Volume 1 11 , Issue 4, Pages 618-624, which is enclosed by reference, (Figure 1). [0270] 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 2: Tomato colors-Bicolor varieties

Bi-color

Ananas Noir Mr. Strip ey Indian Stripe Bi-color

Armenian Northern Lights Isis Candy

Bi -Color Cherry Oaxacan Jewel Lucky Cross

Big Rainbow Old German Lyn's Mahogany Garnet

Patty's Yellow Striped

Big Tiger Beefsteak Magiia Rosa

Big Zebra Peppermint Marizoi Magic

Blush Pineapple Marvel Stripe

Mortgage Lifter, bi-color

Cacady's Folly Piriform strain

Copia Pixie Stripe Tiger Like

Csiko Botermo Rambling Red Stripe Tiger Tom

Dagma's Perfection Red Lightning Tigerella

Elberta Girl Red Zebra Tye-Dye

Georgia Streak Schimmeig Striped Hollow Vintage Wine

German Johnson Pink Speckled Roman Virginia Sweets

Gold Medal Striped Cavern Zebra Cherry

Green Zebra Cherry Striped German Hillbilly

Sunset Falls Hugh's

Table 3: Tomato colors-Black varieties

Black

Black Nyagous Brown Berry Black

Black Cherry Paul Robeson Carbon

Black Krini Purple Russian Cherokee Chocolate

Black Mauri Sleeping Lady Chocolate Cherry

Black Pear Southern Night indigo Rose

Black Pearl Sunchocola Japanese Trifle Black

Black Plum Black Sea Man Black Zebra

Black Prince Black Velvet Brandywine Black

Table 4: Tomato colors-Deep pinkvarieties

Deep Pink

Boondocks Pruden's Purple Early Wonder

Cherokee Purple Purple Smudge Eva Purple Ball

Chianti Rose Razzle Dazzle German Giant

Chiquita Rosalita German Head

Clear Pink Early Rose De Berne Giant Tree

Coustralee Sugary Gulf State Market

Earl's Faux Sweet Quartz Wins All

Honey Hybrid Violaceum Krypni-Rozo Zapotec Pink Ribbed

Lisa King Watermelon Beefsteak Pink Wonder Deep Pink

New Big Dwarf Porter

Table 5: Tomato colors-Bicolor varieties

Golden Green White Undetermined

Aunt Ruby's German Cosmonaut Volkov

Amarillo Cherry Coyote Red

Aunt Ruby's German

Carolina Gold Green Cream Sausage Grandma's Pick

Cerise Orange Cherokee Green Great White Seminis 0172-1432

Golden Gem Dorothy's Green Italian Ice

Golden Girl Fried GreenTomato Old Ivory Egg

Golden Queen, Super Snow

USDA Strain Granny Smith White

Goldie Green Bell Pepper White Beauty

Husky Gold Green Doctors White Currant

Lime Green Salad Green Envy

Mountain Gold Green Giant

Nugget Green Grape

Sunny Goliath Green Sausage

Sunray Green Zebra

Sweet Gold Sungreen Garden Tasty Evergreen Table 6: Tomato colors-Orange varieties

Tab!e 7: Tomato colors-Pink varieties

Pink

Amish Paste German Pink Micado Violettor

Aunt Ginny's German Queen Momotaro Pink

Big Pink Giant Belgium Mortgage Lifter

Brandymaster Pink Giant Syrian Pearly Pink

Brandywine Gum Drop Pink Peach

Caspian Pink Husky Pink Pink Ping Pong

Kalman's Hungarian

Crnkovic Yugoslavian Pink Pink Pounder

Fenda Kolb Pink Stuffer

Torbay Mexico Red Rose

Tough Boy Redfield Beauty Sheboygan

Trucker's Favorite Rose Summer Pink

Ultra Pink Sakura Honey Sunpeach

Thai Pink Egg Tidweli German Sweet Treats

Table 8: Tomato colors-yellow varieties

Ye!Iow

Aunt Gertie's Gold Lemon Boy Wapsipinicon Peach

Aunt Molly's Ground Cherry Lemon Cherry White Cherry

Azoychaka Lemon Drop Wonder Light

Banana Legs Lemon Tree Yellow Bell

Beam's Yellow Pear Limmony Yellow Brandywine

BHN 901 Lollipop Yellow Magic Yellow

BHN YC ! Many el Yellow Peach

Chello Morning Light Yellow Pear

Dr. Carolyn Plum Lemon Yellow Perfection

El Dorado Pork Chop Yellow Staffer

Galina Snow White Solid Gold

Garden Peach Snowberry Sunny Boy

Golden Mama Solar Power Sunshine Heirloom

Golden San Marzano Ildi Taxi

Hartman's Yellow Gooseberry Jubilee Topaz r Huan u

Table 9: Tomato colors-Red varieties

Red

Burgess Stuffing

506 Bush BHN 189 Tomato Skorospelka

Amai BUN 268 Burpee's Big Boy Slava

Amelia BHN 543 Bush Big Boy Small Fry

Andrew Rahart's

Jumbo Red BHN 589 Bush Goliath Smarty

Andrina BHN 762 Cabernet Snacker's Delight

Anna Aasa BHN 826 Camelia Solar Fire

Applause BHN 961 Campbell's 1327 Sophya Red

Arkansas Traveler Big Beef Capaya Spitfire

Austin's Red Pear Big Boy Carmelita Sprite

Big

Baby Cakes Raspberry Carmello St. Nick

Bloody

Basket Vee Butcher Casa del Sol St. Pierre

Beefmaster Bolseno Celebration Steak Sandwich

Box Car

Believe It Or Not Willie Celebrity Stone

Bella Rosa Bradley Celebrity Supreme Sugar Lump

Brandywine

Bellestar OTV Ceylon Sugar Plum

Brandywine

Better Boy Red Chalk's Early Jewel Sugar Snack

Bulgarian #

BHN 1021 7 Champion Sun Cherry

Bulgarian

Christmas Grapes Triumph Charger Sun King

Church Early Doll Cherries Jubilee Sunleaper

Classica Early Girl Cherry Blossom Sunrise

Early

Cluster Grande Goliath Cheny Brandywine Sunstart

Early

Colonial Harvest Cherry Buzz Super Bush Red

Conestoga Elfin Cherry Roma Super Fantastic

Cordova Empire Cherry Sweetie Super Marmande

Corona Enchantment Frazier's Gem Super Sweet 100

Costoluto Genovese Fabulous Fresh Salsa SuperSauce

Crista Fantastic Fruity Cherry Supersonic

Cupid (S 2036) Favorita Gabriel le Supersteak

Dacquiri Ferline Gardener's Delight SVR 1400

Dafel Finishline German Red Strawberry Sweet 100

Debaro First Light Gill's All Purpose Sweet Baby Girl

Debut First Prize Glacier Sweet Chelsea

Five Star

Defiant PhR Grape Glamour Sweet Cluster

Deli/ia FLA 47R Grandero Plum Sweet Elite

Dona FLA 7514 Grandeur Sweet Hearts

Double Rich Floradade Grandma's Pick Sweet Million

Druzba Floralina Greater Baltimore Sweet Olive

Earliana Florida 47 Gremlin Sweet Seedless

Hard Rock Florida 91 Haliey 3155 Sweethearts

Harlequin Fox Mighty Sweet Talladega

Health Kick La Roma I I I Mini Charm Tami G

High Carotene Lady Finger Miroma Tamina Red

Holland Ladybug Montesino Tasti-Lee

Homestead Legend Moreton Tinkerbeil

Homesweet Lunch Box Mosaico Tip-Top

Honey Bunch Malinowski Moskvich Tocan

Mama

Hy-Brix Leone Mountain Fresh Tolstoi

Italian Heirloom Manitoba Mountain Magic Tomatoberry Garden

Jasper Margo Mountain Merit Tommy Toe

Jazzy Mariana Mr. Ugly Tribeca

Jersey Devil Marmande Napa Grape Tribute

Jet Star Marmara Napoli Trophy

Martian

Jetsonic Giant Nature Bites Ultimate Opener

Jolly Elf Martin Nectar Ultra Sweet

Martino's

Juliet Roma Neves Azorean Red Valley Girl

New Hampshire Red

KC 146 Matchless Pickling Velvet Red

Mater

Kimberly Sandwich Old Brooks Viva Italia

Koralik Matina Oiivade Wes

Red House

Pony Express Oroshan Wild Cherry

Free Red

Standing

Porterhouse Red Pear Out Damn Spot Seattle's Best of All

Praire Fire Red Pearl Pamella Seminis 1236

Premio Red Robin Pantano Romanesco Sapho

Primo Red Red Rocket Park's Early Challenge Sara's Galapagos

Pritchard Red Star Peacevine Scarlet Red

Queens Riesentraube Pellicore Serrat

Quick Pick Roma Peron Shady Lady

Rosso

Quimbaya Sicilian Phoenix Shasta

Ramapo Rostova Picus Red Grape

Ranger Rowdy Red Plum Crimson Silvery Fir Tree

Royal

Ravello Mountie Plum Regal Sioux

Red Candy Royesta Poibig Santa Clara Canner

Red Cherry Large

Fruited Rutgers Salt Spring Sunrise Santiam

Rutgers

Red Cup improved PS Sanibel Seminis Grape 9137

Red Defender S 151496 Red Fig

Table 1Θ: Shape of tomatoes: Beefsteak varieties Beefsteak

Amana Orange German Head Kellogg's Breakfast

Ananas Noir German Johnson Pink Kolb

Armenian German Pink Limmony

Aunt Ginny's German Queen Lisa King

Aunt Ruby's German Green Giant Belgium Lucky Cross

Big Rainbow Giant Syrian Persimmon

Big Raspberry Giant Tree Pineapple

Big Zebra Gold Medal Pork Chop

Black Grandma's Pick Porterhouse

Boondocks Grandma's Pick Rose

Brandymaster Pink Great White Rose De Berne

Brandywine Hawaiian Pineapple Southern Night

Brandywine Black Heirloom Orange Striped German

Brandywine OTV Hillbilly Supersteak

Brandywine Red Hugh's Tidwell German

Burpee's Big Boy Italian Heirloom Trucker's Favorite

Cherokee Chocolate Mexico Vintage Wine

Chianti Rose Mortgage Lifter Virginia Sweets

Church Mortgage Lifter, bi-coior strain Watermelon Beefsteak

Coustralee Mr. Ugly White Queen Crnkovic Yugoslavian Neves Azorean Red White Wonder

Dagma's Perfection Oaxacan Jewel Wins All

Earl's Faux Old German Yellow Brandywine

Georgia Streak Orange Slice Martian Giant

German Giant Orange Wellington Marvel Stripe

Patty's Yellow Striped Beefsteak Mater Sandwich

Table 11: Shape of tomatoes: Bell, Blocky and Currant varieties

Table 12: Shape of tomatoes: Deep Round Oval, Oxheart, Long Blocky varieties

Deep Round Ova! Oxheart Long Blocky

Roma German Red Strawberry Capaya

Orange Strawberry Dacquiri

Rostova Kalman's Hungarian Pink

Wes La Roma I I I

Quimbaya

Ranger Table 13: Shape of tomatoes: Pear, small pear and small pointed varieties

Table 14: Shape of tomatoes: stuffer and undetermined varieties

Stuffer Undetermined

Burgess Stuffing Tomato Aunt Molly's Ground Cherry

Green Bell Pepper Cosmonaut Volkov Red

Schimmeig Striped Hollow Purple Smudge

Striped Cavern Red Lightning

Yellow Stuffer SVR 1400

Violaceum Krypni-Rozo Table 15: Shape of tomatoes: Plum varieties

Table 16: Shape of tomatoes: round varieties

Round

506 Bush Early Girl Razzle Dazzle

Arkansas Traveler Early Goliath Red Cup

Aunt Gertie's Gold Glacier Red House Free Standing Round

Believe It Or Not Golden Delight Red Rocket

Bella Rosa Grandeur Red Rose

BHN 961 Green Giant Red Zebra

Big Pink Green Zebra Rowdy Red

Big Tiger Gulf State Market Royesta

Black Prince High Carotene Salt Spring Sunrise

Black Sea Man Holland Seattle's Best of All

Black Velvet Homestead Shady Lady

Black Zebra Honey Hybrid Shasta

Bloody Butcher Husky Gold Sioux

Bradley Husky Pink Skorospelka

Bulgarian # 7 Indian Stripe Slava

Bulgarian Triumph Jaune Flamme Solar Power

Bush Goliath Jetsonic St. Pierre

Cabernet Jubilee Summer Pink

Campbell's 1327 KC 146 Sunray

Caro Rich Kimberly Sunset Falls

Chalk's Early Jewel Legend Sunstart

Cherokee Green Lemon Boy Super Fantastic

Clear Pink Early Lime Green Salad Sweet Cluster Round

Csiko Botermo Manyel Sweet Seedless

Dads Sunset Matina Sweet Tangerine

Dafel Momotaro Tamina

Djena Lee's Golden Girl Moonglow Tangella

Dona Mountain Magic Tangerine Mama

Dorothy's Green Mr. Stripey Taxi

Double Rich Nectarine Tiger Like

Eariiana New Big Dwarf Tiger Tom

Early Doll Nyagous Tigerelia

Elberta Girl Orange Blossom Tolstoi

Empire Oroshan Topaz or Hiian u

Eva Purple Ball Out Damn Spot Torbay

Ferline Pamella Trophy

First Light Park's Early Challenge Ultimate Opener

First Prize Phoenix Ultra Pink

Frazier's Gem Pink Peach Valencia

Fried GreenTomato Pink Staffer Wapsipinicon Peach

Garden Peach Praire Fire Woodle Orange

Gill's All Purpose Premio Yellow Magic

Rambling Red Stripe Prize of the Trials Yellow Peach Round

Pruden's Purple Yellow Perfection

Table 17: Shape of tomatoes: cherry varieties

Cherry

Amarillo Golden Gem Sapho

Andrina Golden Girl Small Fry

Anna Aasa Goldie Snow White

Aunt Ruby's German Cherry Green Doctors Snowberry

Baby Cakes Green Zebra Cherry Sugar Lump

BHN 268 Hartman's Yellow Gooseberry Sugar Snack

BHN 762 Honey Bunch Sun Cherry

Bi-Color Cherry Indigo Rose Sun Gold

Black Cherry Isis Candy Sunchocola

Black Pearl Italian Ice SunSugar

Brown Berry Jasper Super Snow White

Camelia Jazzy Super Sweet 100

Cerise Orange Koraiik Sweet 100

Chello Ladybug Sweet Baby Girl

Cherries Jubilee Lemon Cherry Sweet Chelsea

Cherry Blossom Lemon Drop Sweet Gold Cherry

Cherry Brandywine Lollipop Sweet Million

Cherry Buzz Mosaico Sweet Orange

Cherry Sweetie Nature Bites Sweet Quartz

Chiquita Nectar Sweet Treats

Chocolate Cherry Orange Panuche Tomatoberry Garden

Christmas Grapes Peacevine Tommy Toe

Coyote Pearly Pink Toronjina

Dr. Carolyn Pink Ping Pong Velvet Red

Favorita Power Pops White Cherry

Fox Red Cherry Large Fruited White Currant

Fruity Cherry Red Robin Wild Cherry

Galina Red Star Zebra Cherry

Gardener's Delight S 151496

Table 18: Shape of tomatoes: flattened globe varieties

Flattened Globe

Amelia Fantastic Pritchard

Andrew Rahart's Jumbo Red Fenda Queens

Applause Finishiine Quick Pick

A/oychaka FLA 47R Ramapo Flattened Globe

Basket Vee FLA 7514 Red Defender

Beefmaster Floradade Rosso Sicilian

Better Boy Floralina Royal Mountie

BHN 1021 Florida 47 Russian Persimon

BU 189 Florida 91 Rutgers

BHN 543 Glamour Rutgers Improved PS

BHN 589 Golden Queen, USDA Strain Sanibel

BHN 826 Granny Smith Santa Clara Canner

Big Beef Greater Baltimore Santiam

Big Boy Homesweet Scarlet Red

Black Krini Jet Star Seminis 0172-1432

Bolseno Lyn's Mahogany Garnet Seminis 1236

Box Car Willie Malinowski Silvery Fir Tree

Bush Big Boy Manitoba Sleeping Lady

Carbon Margo Solar Fire

Carmelita Marizol Magic Spitfire

Carmello Marmande Steak Sandwich

Carolina Gold Marmara Stone

Caspian Pink Martin Sun King

Celebration Matchless Sun 1 caper Flattened Globe

Celebrity Micado Violettor Sunny Boy

Celebrity Supreme Moreton Sunny Goliath

Ceylon Moskvich Sunrise

Champion Mountain Fresh Sunshine Heirloom

Charger Mountain Gold Super Bush

Cherokee Purpl Mountain Merit Super Marmande

Cluster Grande Nebraska Wedding Supersonic

Colonial Northern Lights Talladega

Conestoga Old Brooks Tasii-I.ee

Copia Orange King Tasty Evergreen

Corona Pantano Romanesco To can

Costoluto Genovese Paul Robeson Tough Boy

Crista Pellicore Tribeca

Debut Peppermint Tribute

Defiant PliR Peron Tye-Dye

Delizia Pink Pounder Ultra Sweet

Dru/ba Pink Wonder Valley Girl

Early Harvest Pixie Stripe White Beauty

Early Wonder Poibig

Fabulous Primo Red Table 19: Shape of tomatoes: grapevaneties

Grape

Amai Maglia Rosa Seminis Grape 9137

BHN YC1 Mighty Sweet Smarty

Black Mauri Mini Charm Snaeker's Delight

Cherry Roma Montesino Solid Gold

Cupid (S 2036) Morning Light Sprite

Elfin Napa Grape St. Nick

Five Star Grape Nugget Sugar Plum

Gabrielle Orange Fizz Sugary

Golden Sweet Orange Santa Sungreen Garden

Green Envy Ravel lo Sunpeach

Green Grape Red Candy Sweet Elite

Gum Drop Red Grape Sweet Hearts

Harlequin Red Pearl Sweet Olive

Hy-Brix Riesentraube Sweethearts

Ildi Rosalita Tami G

Jolly Elf Sakura Honey Thai Pink Egg

Juliet Tinkerbell