Patent Description:
In recent years, consumer convenience as well as the development of new products has contributed to the increasing choice of processed vegetables and fruits available on the market. Ready-to-eat products, i.e. in a cut, washed and packaged form, may include lettuce (Lactuca sativa) and other leafy vegetables such as chicory (Cichorium intybus) and endive (Cichorium endivia), either individually processed or in mixed compositions. One of the most important and frequently encountered problems during harvesting, processing and storage of vegetables is the development of wound-induced surface discoloration visible by a pink discoloration at the wound surface of the plants or parts thereof which gradually turns brown after prolonged storage. Other crop plants such as potato (Solanum tuberosum), onion (Allium cepa), artichocke (Cynara cardunculus var. Scolymus), rice (Oryza sativa) , corn (Zea mays), peach (Prunus persica), eggplant (Solanum melongen), celery and celeriac (Apium graveolens), apple (Malus domestica), banana (Musa acuminate), soy (Glycine max), pear (Pyrus x bretschneideri), wheat (Triticum aestivum), radish (Raphanus sativus), cabbage and cauliflower (Brassica oleracea) etc. may also be subject to the wound-induced surface discoloration visible on the plant or parts thereof, such as leaves, whole plant heads, fruits, inflorescences, seeds, curds, stems, tubers, bulbs and roots etc..

Wound-induced surface discoloration or wound-induced discoloration is caused by a strong wound response at and around the wound and leads to a rapid deterioration of the harvested and optionally processed product. Consumers consider discoloration of vegetables and fruits to be unattractive and to compromise the product quality, thus reducing the product's marketability and/or leading to a waste of harvested and optionally processed products.

The wound response is a means of a plant or part thereof to heal the wound and defend itself against pathogens by creating a new insulation barrier. The response is a complex biological response of a plant to physical injury such as cutting or bruising, and implies the activity of numerous proteins. The local response is mainly aimed at closing the wound surface which is effectuated by the local death of cells at or just behind the wound surface. In addition to these visible effects, other responses like increased respiration or ethylene production are known to be induced.

At the biochemical level, studies have shown that wounding can lead to the induction of the phenylpropanoid pathway (PP pathway) which is required for inter alia the production of polyphenols and other compounds important for the plant.

The first step of the PP pathway is the conversion of the amino acid phenylalanine into cinnamic acid by the phenylalanine ammonia-lyase (PAL). PAL is enhanced upon wounding by the induction of gene expression of at least one of its isoforms. This response leads to the formation of polyphenols which are oxidized by the polyphenol oxidase (PPO). PPO is residing in plastids and is released and activated upon wounding. Oxidation of polyphenols lead to the formation of highly reactive quinones, that can react with amino acids or proteins which leads to pink, brown or black discoloration.

In order to reduce the wound-induced surface discoloration in vegetables such as lettuce, many post-harvest and post-processing treatments have been developed and applied. Examples of chemical or physical treatments are the packaging of fresh cut leafy vegetables under a modified atmosphere, the application of edible coatings, heat-shock treatment and the addition of chemicals.

Although these treatments prevent the appearance of the wound-induced discoloration, the harvested and eventually processed product is still susceptible to discoloration if the package is damaged or shortly after opening the package. In addition, the use of chemicals and the need for specialized equipment for such treatments significantly increases costs. For these reasons, a more viable genetically-based solution which works to reduce wound-induced surface discoloration in plants is preferred.

In the research leading to the present invention, it was surprisingly found that modifications to a F5H gene homolog in plants lead to a reduction of wound-induced surface discoloration, as compared to plants or parts thereof not comprising such modifications in their corresponding wild type F5H gene homologs. The F5H gene homologs code for Ferulate <NUM>-hydroxylase (F5H) protein homologs. In Arabidopsis thaliana two F5H gene homologs are described and called F5H1 and F5H2. The F5H enzyme is part of a of the PP pathway where it is responsible for the hydroxylation of coniferaldehyde and coniferyl alcohol. The F5H protein belongs to a new family of plant cytochrome P450-dependent mono-oxygenase called CYP84. However, the implication of F5H in the wound-induced surface discoloration has not yet been described.

It is an object of the present invention to provide a plant that shows reduced wound-induced surface discoloration.

The present invention thus provides a Lactuca sativa plant comprising two modified F5H gene homologs, wherein said gene homolog comprises a modification as compared to its corresponding wild type F5H gene homolog, wherein the modification leads to a reduction or absence of the protein expression and/or activity of the F5H protein homolog as compared to the expression and/or activity of the protein produced by the corresponding wild type F5H gene homolog, wherein the presence of the modified F5H gene homolog in the plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homolog, and wherein the plant comprises a first modified F5H gene homolog called F5H1, the wild type of which has SEQ ID No. <NUM>, and a second modified F5H gene homolog called F5H2, the wild type of which has SEQ ID No. <NUM>.

The term "reduced" is always measured in relation to the wound-induced surface discoloration of a control plant or part thereof that has no such modifications to its F5H gene homologs and is therefore a wild type plant comprising wild type F5H gene homologs and does not show reduced wound-induced surface discoloration. As used herein, a plant showing a "reduced wound-induced surface discoloration" or a "reduction of wound-induced surface discoloration" is a plant having a reduced wound-induced surface discoloration as compared to the wound induced surface discoloration of a wild type plant. Therefore, an improvement of the reduced wound-induced surface discoloration is defined by a delayed appearance and/or reduced intensity of the discoloration as compared to a plant not comprising a modified F5H gene homolog. A reduced intensity of discoloration is visible by a less intense discoloration of the wound surface and/or the discolored surface is smaller as compared to the discoloration and surface of the wound induced surface discoloration of a wild type plant. Ultimately, the wound-induced discoloration is completely absent. A delayed appearance of the discoloration means that the onset of discoloration occurs later in time. The plant thus maintains its fresh appearance longer, which is in fact an increase of shelf life.

Disclosed herein are the sequences of modified F5H gene homologs in order to identify plants comprising said modifications that lead to the trait of the invention.

The number of F5H gene homologs within one specific species differs among different plant species. According to the definition of gene homolog described in this application, it was found, that the Lactuca sativa plant genome comprises two F5H gene homologs. One homolog called herein F5H1, is located on chromosome <NUM> and has the wild type DNA coding sequence (CDS) represented in SEQ ID No: <NUM> and encodes the wild type F5H1 protein having SEQ ID No: <NUM>. The other lettuce homolog herein called F5H2, is located on chromosome <NUM> and has the wild type DNA coding sequence represented in SEQ ID No: <NUM> and encodes the wild type F5H2 protein having SEQ ID No: <NUM>.

It was also found that the genomes of artichocke (Cynara cardunculus var. Scolymus), rice (Oryza sativa), corn (Zea mays), peach (Prunus persica) and eggplant (Solanum melongena) also comprise two F5H gene homologs the wild type SEQ ID numbers of which are listed in Table <NUM>. The species chicory (Cichorium intybus), endive (Cichorium endivia), celery and celeriac (Apium graveolens) and apple (Malus domestica) comprise three F5H gene homologs the wild type SEQ ID numbers of which are listed in Table <NUM>. The genome of banana (Musa acuminata) comprises four F5H homologs the wild type SEQ ID numbers of which are listed in Table <NUM>. The genomes of soy (Glycine max), pear (Pyrus x bretschneideri), wheat (Triticum aestivum), radish (Raphanus sativus) and cabbage and cauliflower (Brassica oleracea) comprise five F5H homologs the wild type SEQ ID numbers of which are listed in Table <NUM>, whereas the genomes of potato (Solanum tuberosum) and onion (Allium cepa) comprise one F5H gene homolog in their genomes, the homolog the wild type SEQ ID numbers of which are listed in Table <NUM>.

Thus, the invention relates to a Lactuca sativa plant comprising two F5H gene homologs that are modified as compared to the nucleotide sequence of the wild type genes (SEQ ID No: <NUM> and <NUM>), encoding the wild type proteins (SEQ ID No: <NUM> and <NUM>). Additionally, disclosed herein are other plants comprising a modified F5H gene homolog in their genome such as Solanum tuberosum, Allium cepa, Cynara cardunculus var. Scolymus, Oryza sativa, Zea mays, Prunus persica, Solanum melongena, Cichorium intybus, Cichorium endivia, Apium graveolens, Malus domestica, Musa acuminate, Glycine max, Pyrus x bretschneideri, Triticum aestivum, Raphanus sativus and Brassica oleracea.

Further disclosed herein is a modified F5H gene homolog of a plant belonging to the species Solanum tuberosum, Allium cepa, Lactuca sativa, Cynara cardunculus var. Scolymus, Oryza sativa, Zea mays, Prunus persica, Solanum melongena, Cichorium intybus, Cichorium endivia, Apium graveolens, Malus domestica, Musa acuminate, Glycine max, Pyrus x bretschneideri, Triticum aestivum, Raphanus sativus or Brassica oleracea, wherein the modified F5H gene homolog comprises at least one modification as compared to its wild type sequence and wherein the modification leads to reduced wound-induced surface discoloration to the plant. It is not intended to claim a modified F5H gene of Arabidopsis thaliana or the sequence of the NCBI database with the accession no. XP_011028697 (Predicted: cytochrome P450 84A1-like [Populus euphratica]), in this application.

Also disclosed herein is a modified F5H1 gene homolog having the sequence represented by SEQ ID No: <NUM>, that leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H1 gene homolog.

Furthermore, disclosed herein is a modified F5H1 gene homolog having the sequence represented by SEQ ID No: <NUM> and a modified F5H2 gene homolog having the sequence represented by SEQ ID No: <NUM>, <NUM>, <NUM>, <NUM> or <NUM>, leading to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homologs.

Disclosed herein is a plant comprising a modified F5H gene homolog, wherein said gene homolog comprises a modification as compared to its corresponding wild type F5H gene homolog, wherein the presence of the modified F5H gene homolog in a plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homolog.

Additionally, disclosed herein is a plant comprising a modified F5H gene homolog, wherein said gene homolog comprises a modification as compared to its corresponding wild type F5H gene homolog, wherein the presence of the modified F5H gene homolog in a plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homolog and wherein the wild type F5H gene sequence is represented by any one of SEQ ID Nos: <NUM> to <NUM>.

A plant comprising a modified F5H gene homolog, wherein the plant is selected from the group consisting of Solanum tuberosum, Allium cepa, Lactuca sativa, Cynara cardunculus var. Scolymus, Oryza sativa, Zea mays, Prunus persica, Solanum melongena, Cichorium intybus, Cichorium endivia, Apium graveolens, Malus domestica, Musa acuminate, Glycine max, Pyrus x bretschneideri, Triticum aestivum, Raphanus sativus and Brassica oleracea, the wild type F5H gene sequence SEQ ID numbers of which are listed in Table <NUM> is also disclosed herein. It is not intended to claim a Arabidopsis thaliana plant comprising a modified F5H gene, in particular the one disclosed in the publication of <NPL>) or in <NPL>), or a plant comprising the sequence of the NCBI database with the accession no. XP_011028697 (Predicted: cytochrome P450 84A1-like [Populus euphratica]), in this application.

Further disclosed herein is a plant comprising two modified F5H gene homologs, wherein the presence of the modified F5H gene homologs in a plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homologs.

A plant comprising two modified F5H gene homologs of the invention, wherein the plant is selected from the group consisting of Lactuca sativa, Cynara cardunculus var. Scolymus, Oryza sativa Japonica, Zea mays, Prunus persica, Solanum melongena, Cichorium intybus, Cichorium endivia, Apium graveolens, Malus domestica, Musa acuminate, Glycine max, Pyrus x bretschneideri, Triticum aestivum, Raphanus sativus and Brassica oleracea, the wild type F5H gene sequence SEQ ID numbers of which are listed in Table <NUM> is also disclosed herein.

Furthermore, disclosed herein is a plant comprising three modified F5H genes homolog, wherein the presence of the modified F5H gene homologs in a plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homologs.

A plant comprising three modified F5H gene homologs of the invention, wherein the plant is selected from the group consisting of Cichorium intybus, Cichorium endivia, Apium graveolens, Malus domestica, Musa acuminate, Glycine max, Pyrus x bretschneideri, Triticum aestivum, Raphanus sativus and Brassica oleracea, the wild type F5H gene sequence SEQ ID numbers of which are listed in Table <NUM> is also disclosed herein.

Also, disclosed herein is a plant comprising four modified F5H gene homologs, wherein the presence of the modified F5H gene homologs in a plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homologs.

A plant comprising four modified F5H gene homologs of the invention, wherein the plant is selected from the group consisting of Musa acuminate, Glycine max, Pyrus x bretschneideri, Triticum aestivum, Raphanus sativus and Brassica oleracea, the wild type F5H gene sequence SEQ ID numbers of which are listed in Table <NUM> is also disclosed herein.

Further disclosed herein is a plant comprising five or more modified F5H gene homologs, wherein the presence of the modified F5H gene homologs in a plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homologs.

A plant comprising five or more modified F5H gene homologs of the invention, wherein the plant is selected from the group consisting of Glycine max, Pyrus x bretschneideri, Triticum aestivum, Raphanus sativus and Brassica oleracea, the wild type F5H gene sequence SEQ ID numbers of which are listed in Table <NUM> is also disclosed herein.

In addition, disclosed herein are edible plants such as vegetables, fruits and cereals comprising a modified F5H gene homolog, wherein said gene homolog comprises a modification as compared to its corresponding wild type F5H gene homolog and wherein the presence of the modified F5H gene homolog in the plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homolog.

A "plant of the invention" as used herein, is a Lactuca sativa plant comprising two modified F5H gene homologs, wherein said gene homolog comprises a modification as compared to its corresponding wild type F5H gene homolog, wherein the modification leads to a reduction or absence of the protein expression and/or activity of the F5H protein homolog as compared to the expression and/or activity of the protein produced by the corresponding wild type F5H gene homolog, wherein the presence of the modified F5H gene homolog in the plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homolog, and wherein the plant comprises a first modified F5H gene homolog called F5H1, the wild type of which has SEQ ID No. <NUM>, and a second modified F5H gene homolog called F5H2, the wild type of which has SEQ ID No. <NUM>.

The relationship between genes is defined as homologous. In this application "homologous genes" refers to two related genes originating from a common ancestral gene. Homologous sequences are termed "homologs" and this term may be applied to both genes and proteins. The terms "homologous" or "homologs" may be used interchangeably. Homologous genes encode homologous proteins. According to our definition, the wild type sequences of the F5H protein homologs as disclosed herein are listed in Table <NUM>. Moreover, all the F5H protein homologs that were identified during the research leading to the invention, have the five motifs the consensus sequence of which is represented in Table <NUM> and provided by MAST (Motif Alignment & Search Tool) and MEME (Multiple Em for Motif Elicitation) in their amino acid sequences with a certain degree of variation.

Preferably the F5H protein homologs as disclosed herein comprise the motifs represented in Table <NUM> with an identity percentage of at least <NUM>%, more preferably with an identity percentage of at least <NUM>%, even more preferably with an identity percentage of at least <NUM>%, even more preferably with an identity percentage of at least <NUM>%, even more preferably with an identity percentage of at least <NUM>%, even more preferably with an identity percentage of at least <NUM>%, even more preferably with an identity percentage of at least <NUM>%, even more preferably with an identity percentage of at least <NUM>%, even more preferably with an identity percentage of at least <NUM>% and most preferably with an identity percentage of <NUM>%. The consensus sequences of the five motifs are listed in the Table <NUM> and are highlighted in the alignment of the F5H protein orthologs in <FIG>.

"Orthologous genes" are homologous genes present in different species and originated from a common ancestral gene and separated by a speciation event. The terms "orthologous genes" or "orthologs" may be used interchangeably. Disclosed herein are thus modifications to F5H gene homologs within a species and F5H gene orthologs of different species, all leading to a reduced wound-induced surface discoloration in said species. The SEQ ID numbers of the wild type sequences of the F5H protein and gene orthologs are listed in Table <NUM>.

In this application a "gene" comprises exonic sequences and regulatory sequences such as a promoter sequence, UTR and polyadenylation signals and if present it also comprises intronic sequences. Modification to a F5H gene homolog creates a "modified gene homolog" by at least one change in the nucleotide sequence of the gene. The terms "modification" and "mutation" may be used interchangeably. Generally, modifications change the expression of the gene and/or the activity of the protein encoded by the gene that comprises the modification. Modifications to the gene sequence may inhibit gene transcription such that the expression of the modified gene is prevented or reduced or may lead to unstable mRNA. Modifications may also be changes to the sequence of the F5H gene that lead to a reduced level, reduced activity or a complete absence of the encoded protein activity. In some cases, modifications can also lead to an overexpression of the protein that may be responsible for the modified phenotype. A non-limitative list of examples of modifications and techniques in order to modify the genes is described in this application.

As used herein, "wild type" or "WT" refers to the form of an organism as it would occur in nature, in this case a plant not showing a reduction in wound-induced surface discoloration.

As used herein, a wild type gene or gene homolog refers to an unmodified F5H gene as it would occur in a plant not showing a reduction in wound-induced surface discoloration. The wild type plant is used as control plant that does not carry a modified F5H gene homolog and therefore does not show the reduced wound-induced surface discoloration. To be comparable, the plant that comprises a modified F5H gene homolog and the wild type plant should be selected from the same type, preferably the same variety, at the same age and be grown under the same conditions.

As used herein, the term "a F5H gene" or "a modified F5H gene" means one or more modified F5H genes. A "plant comprising a modified F5H gene homolog" comprises one or more modified F5H gene homologs.

In this application, the word "trait" refers to the phenotype of the plant. "Trait of the invention", "trait", or "phenotypic trait", "phenotype", "characteristic" may be used interchangeably. The trait of the invention as used herein is the reduced wound-induced surface discoloration as a result of the presence of a modified F5H gene homolog and its corresponding F5H protein.

As used herein the "modified F5H gene homolog of the invention" refers to a combination of two modified F5H gene homologs, wherein said gene homolog comprises a modification as compared to its corresponding wild type F5H gene homolog, wherein the modification leads to a reduction or absence of the protein expression and/or activity of the F5H protein homolog as compared to the expression and/or activity of the protein produced by the corresponding wild type F5H gene homolog, wherein the presence of the modified F5H gene homolog in the plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homolog, and wherein a first modified F5H gene homolog is called F5H1, the wild type of which has SEQ ID No. <NUM>, and a second modified F5H gene homolog is called F5H2, the wild type of which has SEQ ID No. <NUM>. "Modified F5H gene homolog of the invention", "gene of the invention", "F5H gene of the invention", "F5H gene homolog of the invention" be used interchangeably.

The invention also relates to a plant of the invention, wherein the modification leads to a premature stop codon.

The invention relates to a method for producing a plant exhibition reduced wound-induced surface discoloration, comprising reducing the endogenous level of F5H1 protein in the plant, wherein the endogenous level of F5H1 protein in the plant is reduced by mutating a wild type F5H1 gene homolog, and/or wherein reducing the endogenous level of F5H1 protein in the plant is accomplished by reducing the expression of a F5H1 gene homolog of the plant by gene silencing or RNAi.

The invention further relates to a method for producing a plant exhibition reduced wound-induced surface discoloration, comprising reducing the endogenous level of F5H1 protein in the plant, wherein the mutation is effected by CRISPR, by a chemical agent, radiation, or a combination thereof.

The modification of a F5H gene can be introduced by means of mutagenesis. Several chemical or physical treatments are known to the person skilled in the art which can be used to induce genetic mutations in plant species like lettuce. Mutagenesis comprises the random introduction of at least one modification by means of one or more chemical compounds, such as ethyl methanesulphonate (EMS), nitrosomethylurea, hydroxylamine, proflavine, N-methyl-N-nitrosoguanidine, N-ethyl-N-nitrosourea, N-methyl-N-nitro-nitrosoguanidine, diethyl sulphate, ethylene imine, sodium azide, formaline, urethane, phenol and ethylene oxide, and/or by physical means, such as UV-irradiation, fast-neutron exposure, X-rays, gamma irradiation, and/or by insertion of genetic elements, such as transposons, T-DNA, retroviral elements. Mutagenesis also comprises the more specific, targeted introduction of at least one modification by means of homologous recombination, oligonucleotide-based mutation induction, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems.

Seeds of the plant to be modified may be treated with a solution containing different concentrations of a mutagen like EMS. EMS alkylates primarily Guanine (G) residues of a DNA strand, which during DNA replication causes pairing with Thymine (T) instead of Cytosine (C). Therefore, GC base pairs change to AT base pairs at a frequency which is determined by the effective dose of EMS and the activity of the mismatch repair system of the plant. The effective dose of EMS depends on the concentration used, the seed size and other physical properties and the time of incubation of the seeds in the EMS solution. The seeds, which have been treated with EMS are typically called M1 seeds. As a consequence of the treatment, the tissues of the M1 seeds contain random point mutations in the genomes of their cells and those present in the subpopulation of cells, which will form the germline tissue (germinal cells) will be transferred to the next generation, which is called M2. Mutations or combinations thereof which are haplo-insufficient thereby causing sterility or which induce embryo lethality will not be transferred to the M2 generation. It should be noted that although most EMS induced mutations and the resulting trait are recessive, there is a possibility that dominant mutations leading to a semi-dominant or dominant trait can occur.

A plant comprising a modified F5H gene homolog, wherein said gene homolog is mutated as compared to its corresponding wild type F5H gene homolog, wherein the presence of the modified F5H gene homolog in the plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homolog is disclosed herein.

In particular, a plant comprising a modified F5H gene homolog, wherein said gene homolog comprises a man-made mutation as compared to its corresponding wild type F5H gene homolog, wherein the presence of the modified F5H gene homolog in the plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homolog is also disclosed herein.

In one embodiment, the invention relates to a plant comprising a reduced F5H1 and F5H2 expression, wherein the reduction is caused by any of the methods of the invention.

When the expression of a modified F5H gene is absent or reduced in the context of this invention, this means that the gene expression leading to the synthesis of a functional protein is prevented and thus absent or that the expression of the modified F5H gene is less than the expression of the wild type F5H gene leading to a lower level of the protein. The said prevention or reduction of gene expression is herein directly or indirectly responsible for the trait of reduced wound-induced surface discoloration.

Gene expression may also be prevented or reduced by preventing the transcription of the gene with for example RNA oligonucleotides or DNA oligonucleotides, or preferably by the expression of a negatively acting transcription factor acting on a F5H gene promoter. Other examples of methods to prevent or reduce the gene expression are the destabilization of the F5H mRNA or transcript, preferably by means of nucleic acid molecules that are complementary to the F5H mRNA or transcript selected from the group consisting of antisense RNA, RNAi molecules, Virus-Induced Gene Silencing (VIGS) molecules, co-suppressor molecules, RNA oligonucleotides or DNA oligonucleotides. Such methods for destabilizing mRNA or transcripts are well known to the person skilled in the art.

Examples of modifications leading to the reduction or absence of the F5H activity are modifications leading to premature stop codons, frame shifts or amino acid substitutions in the encoded protein. The said reduction or absence of the F5H protein activity is herein directly or indirectly responsible for the trait of reduced wound-induced surface discoloration. A reduced activity of the F5H protein may occur for example by introducing one or more mutations into the coding sequence of a F5H gene. Mutation(s) to the F5H gene may affect the biological function of the encoded protein, as compared to F5H protein encoded by a wild type F5H gene where no such mutation(s) is present.

The plants of the invention were created by using the mutagenic agent EMS, as described in Example <NUM>. Plants grown from seeds treated one time with the mutagenic agent and selected for their ability to show reduced wound-induced surface discoloration, comprise at least one mutation in one F5H gene homolog of their genome. In order to introduce modifications to the other F5H gene homologs of the plant, the seeds already carrying one or more modified F5H gene homologs may be treated for additional rounds with the mutagenic agent and selected after each round of treatment with the mutagenic agent for their ability to show reduced wound-induced surface discoloration. For example, a Lactuca sativa plant having two F5H gene homologs in their genome was treated two times with the mutagenic agent EMS in order to introduce a modification in the two F5H gene homologs of this plant.

Modifications to the gene may be recessive, dominant or intermediate. The terms "intermediate and "semi-dominant" may be used interchangeably. In case of a recessive trait, the modification of the gene needs to be present in homozygous state for the trait to be completely visible. Some of the modifications described herein are recessive and thus only confer the reduced wound-induced surface discoloration if both alleles of the gene have the modification. Modifications that are dominant or intermediate can also be visible in heterozygous state. The heterozygous phenotype of an intermediate trait lies between the phenotypes of the homozygous dominant and the homozygous recessive genotypes.

Modification in the F5H1 or F5H2 gene homolog may be present in a heterozygous or in a homozygous state. Preferably the modification in the F5H1 gene homolog is present in a homozygous state. The genotype of the plant can be confirmed by using molecular markers. Preferably, the genotype of the plants is confirmed by using the molecular markers described in Example <NUM>.

Disclosed herein is a plant comprising a F5H gene homolog that comprises a modification as compared to the corresponding wild type sequence, wherein the modification results in a different phenotype displaying reduced wound-induced surface discoloration.

Also disclosed herein is a plant comprising a F5H gene homolog that comprises a modification as compared to the corresponding wild type sequence, wherein the modification results in a different phenotype displaying reduced wound-induced surface discoloration as compared to a plant not comprising the modified F5H gene homolog.

In the context of this application, the reduced wound-induced discoloration is preferably caused by a modification that is present in the genome of lettuce seeds which were deposited with the NCIMB under accession number NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> and NCIMB <NUM>. The mutations are described in Table <NUM>.

Seed of seed lot 16E. 607_B01 was deposited with the NCIMB under accession number NCIMB <NUM>. The deposited seeds comprise mutation <NUM> in the F5H1 gene homolog.

Seed of seed lot 16E. 607_B02 was deposited with the NCIMB under accession number NCIMB <NUM>. The deposited seeds comprise mutation <NUM> in the F5H1 gene homolog and mutation <NUM> in the F5H2 gene homolog.

Seed of seed lot 16E. 607_B03 was deposited with the NCIMB under accession number NCIMB <NUM>. The deposited seeds comprise mutation <NUM> in the F5H1 gene homolog and mutation <NUM> in the F5H2 gene homolog.

Seed of seed lot 16E. 607_B04 was deposited with the NCIMB under accession number NCIMB <NUM>. The deposited seeds comprise mutation <NUM> in the F5H1 gene homolog and mutation <NUM> in the F5H2 gene homolog.

Seed of seed lot 16E. 607_B05 was deposited with the NCIMB under accession number NCIMB <NUM>. The deposited seeds comprise mutation <NUM> in the F5H1 gene homolog and mutation <NUM> in the F5H2 gene homolog.

Seed of seed lot 16E. 607_B06 was deposited with the NCIMB under accession number NCIMB <NUM>. The deposited seeds comprise mutation <NUM> in the F5H1 gene homolog and mutation <NUM> in the F5H2 gene homolog.

The lettuce F5H1 gene homolog comprising mutation <NUM> is represented by SEQ ID No: <NUM>.

The lettuce F5H2 gene homolog comprising mutation <NUM> is represented by SEQ ID No: <NUM>.

Modifications to a gene may lead to premature stop codons, frame shifts, amino acid substitutions or splice variants in the corresponding protein sequence. The modifications in the protein sequence are the results of substitution, deletions and changes of base pairs in the DNA sequences coding for proteins.

When the modification to the DNA sequence leads to a premature stop codon, the transcription results in a truncated version of the encoded protein. The modification may occur in a region of the protein sequence that contains one or more domains or active sites essential to perform its function and/or to interact with its substrate or other proteins and/or to fold properly into a functional protein.

When the modification to the DNA sequence leads to a frame shift mutation, the protein translation will result in an entirely different amino acid sequence as the WT sequence and often results in a premature stop codon. The translated protein will usually have a different biological function than the WT protein. These modifications are due to the insertion or deletion of a number of base pairs that is not a multiple of three, leading to the shift of the triplet codon encoding the individual amino acid of the protein, relative to the original open-reading frame changing thereby the amino acid sequence of the protein. If the insertion or deletion is a multiple of three it may also lead to a different amino acid sequence as the wild type sequence.

The modification of one or more base pairs in the coding sequence of a DNA sequence can lead to an amino acid change in the encoded protein sequence. Due to the redundancy of the genetic code some mutations lead to the same amino acid, these mutations are called "silent mutations". Moreover, some amino acid changes are "conservative", i.e. they lead to the replacement of one amino acid by another amino acid with comparable properties, for example similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity or the amphipathic nature of the residues, such that the mutation is unlikely to dramatically influence function of the mature protein and/or change its folding. Other amino acid changes are non-silent, non-conservative amino acid changes and are replacements of an amino acid by another amino acid with different chemical properties in domains that play a role in substrate-recognition, the active site of enzymes, interaction domains or in major structural domains and such amino acid changes may partly or completely destroy the functionality of the encoded protein, without necessarily affecting the expression level of the encoding gene. These types of mutations may lead to detrimental stability, functionality and/or structural effects of the encoded protein. Non-silent and non-conservative amino acid changes can also lead to an over-expression of the encoded protein.

Mutations in the promoter sequence of the F5H gene may also perturb the biological function of the encoded F5H protein, as such mutations may lead to a complete lack of transcription of the gene (e.g. subsequently resulting in a complete absence of the F5H protein), or to a significantly decreased and biologically inadequate level of transcription (e.g. subsequently resulting in a reduced level of the F5H protein) or to an overexpression of the F5H protein (e.g. subsequently resulting in a higher level of the F5H protein).

In the present invention gene expression analysis was performed by measuring the RNA of the F5H1 gene homolog and the F5H2 gene homolog. The analysis showed that the expression of F5H1 and F5H2 is induced upon wounding. The expression of F5H1 seems to start earlier than the expression of F5H2 and the expression of F5H1 seems to be reduced in plants having mutation <NUM> and optionally a mutation in the F5H2 gene homolog such as mutation <NUM> or mutation <NUM>.

Further disclosed herein is a plant that comprises a modified F5H gene homolog wherein said gene homolog comprises a modification as compared to its corresponding wild type F5H gene homolog, wherein the presence of the modified F5H gene homolog in a plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homolog.

The invention relates to a Lactuca sativa plant comprising a first modified F5H gene homolog called F5H1, the wild type of which has SEQ ID No. <NUM>, and optionally a second modified F5H gene homolog called F5H2, the wild type of which has SEQ ID No. <NUM>.

The invention also relates to a Lactuca sativa plant, wherein the modified F5H1 gene homolog is homozygously present and the modified F5H2 gene homolog is either heterozygously or homozygously present.

The invention further relates to a Lactuca sativa plant, wherein the modified F5H1 gene comprises a premature stop codon.

The invention further relates to a Lactuca sativa plant, wherein the modified F5H1 gene comprises a premature stop codon that is caused by a mutation C > T at position <NUM> of SEQ ID No: <NUM>.

Disclosed herein is another plant of the invention listed in <FIG> and the premature stop codon is caused by a mutation at a position that corresponds to position <NUM> of SEQ ID No:<NUM> in Lactuca sativa.

The invention further relates to a Lactuca sativa plant, comprising a first modified F5H gene homolog called F5H1, the wild type of which has SEQ ID No. <NUM>, and a second modified F5H gene homolog called F5H2, the wild type of which has SEQ ID No. <NUM>, wherein the modified F5H2 gene encodes a protein having an amino acid substitution of Threonine to Isoleucine at position <NUM> of the encoded F5H2 protein of SEQ ID No: <NUM>, and/or an amino acid substitution of Glycine to Glutamic acid at position <NUM> of the encoded F5H2 protein of SEQ ID No: <NUM>, and/or an amino acid substitution of Serine to Phenylalanine at position <NUM> of the encoded F5H2 protein of SEQ ID No: <NUM>, and/or an amino acid substitution of Glycine to Glutamic acid at position <NUM> of the encoded F5H2 protein of SEQ ID No: <NUM>, and/or an amino acid substitution of Glycine to Glutamic acid at position <NUM> of the encoded F5H2 protein of SEQ ID No: <NUM>.

The invention further relates to a Lactuca sativa plant, comprising a first modified F5H gene homolog called F5H1, the wild type of which has SEQ ID No. <NUM>, and optionally a second modified F5H gene homolog called F5H2, the wild type of which has SEQ ID No. <NUM>, wherein the amino acid substitution of Threonine to Isoleucine at position <NUM> of the encoded F5H2 protein is the result of a nucleotide change C > T at position <NUM> of SEQ ID No: <NUM>, the amino acid substitution of Glycine to Glutamic acid at position <NUM> of the encoded F5H2 protein is the result of a nucleotide change G > A at position <NUM> of SEQ ID No: <NUM>, the amino acid substitution of Serine to Phenylalanine at position <NUM> of the encoded F5H2 protein is the result of a nucleotide change C > T at position <NUM> of SEQ ID No: <NUM>, the amino acid substitution of Glycine to Glutamic acid at position <NUM> of the encoded F5H2 protein is the result of a nucleotide change G > A at position <NUM> of SEQ ID No: <NUM> and the amino acid substitution of Glycine to Glutamic acid at position <NUM> of the encoded F5H2 protein is the result of a nucleotide change G > A at position <NUM> of SEQ ID No: <NUM>.

Disclosed herein is another plant of the invention listed in <FIG> and the amino acid substitution is at a position that corresponds to the position in Lactuca sativa.

The invention relates to a Lactuca sativa plant, comprising a modified F5H1 gene, which gene comprises a premature stop codon and a F5H2 gene comprising an amino acid substitution, wherein the premature stop codon in the F5H1 gene is caused by a mutation C > T at position <NUM> of SEQ ID No: <NUM> and the amino acid substitution in the F5H2 gene selected from Threonine to Isoleucine at position <NUM> of the encoded F5H2 protein is the result of a nucleotide change C > T at position <NUM> of SEQ ID No: <NUM>, the amino acid substitution of Glycine to Glutamic acid at position <NUM> of the encoded F5H2 protein is the result of a nucleotide change G > A at position <NUM> of SEQ ID No: <NUM>, the amino acid substitution of Serine to Phenylalanine at position <NUM> of the encoded F5H2 protein is the result of a nucleotide change C > T at position <NUM> of SEQ ID No: <NUM>, the amino acid substitution of Glycine to Glutamic acid at position <NUM> of the encoded F5H2 protein is the result of a nucleotide change G > A at position <NUM> of SEQ ID No: <NUM> or the amino acid substitution of Glycine to Glutamic acid at position <NUM> of the encoded F5H2 protein is the result of a nucleotide change G > A at position <NUM> of SEQ ID No: <NUM>.

In one embodiment, the invention relates to a Lactuca sativa plant of the invention, comprising a modified F5H1 gene, which gene comprises a premature stop codon and a F5H2 gene comprising an amino acid substitution, wherein the premature stop codon in the F5H1 gene is caused by a mutation C > T at position <NUM> of SEQ ID No: <NUM> and the amino acid substitution in the F5H2 gene Glycine to Serine at position <NUM> of the encoded F5H2 protein is the result of a nucleotide change G > A at position <NUM> of SEQ ID No: <NUM>.

In particular, the invention relates to a Lactuca sativa plant that comprises one modification to the F5H1 gene homolog wherein the modification leads to a premature stop in the coding sequence of the lettuce F5H1 gene in combination with a modification to the F5H2 gene wherein the modification leads to an amino acid substitution. When both mutations, the mutation leading to a premature stop codon in the F5H1 gene homolog and the amino acid substitution in the F5H2 protein sequence, are carried by a plant the effect is enhanced and the plant comprising these mutations shows an delayed wound-induced surface discoloration. Therefore, a plant comprising a modified F5H1 gene homolog and a modified F5H2 gene homolog shows delayed wound-induced surface discoloration, as compared to a plant comprising only a modified F5H1 gene homolog.

A segregation analysis is performed with the F2 plants resulting from a cross of a plant showing reduced wound induced surface discoloration and comprising a mutation in the F5H1 gene homolog and a mutation in the F5H2 gene homolog in a homozygous state with a wild type plant that does not comprise a modified F5H gene homolog. The resulting F1 plants are selfed and the phenotype of the F2 plants grown from the obtained seeds is analyzed. The F2- plants comprise the mutation(s) in a homozygous state, heterozygous state or do not comprises any mutation. The results of the segregation analysis of the trait of the invention shows that in order to show reduced wound induced surface discoloration the plant needs to comprise the mutation <NUM> on the F5H1 homolog (C<NUM> >T<NUM> in the F5H1 gene homolog) preferably in a homozygous state. Plants that comprise the mutation <NUM> in the F5H1 homolog in a homozygous state and a mutation in the F5H2 gene homolog such as the mutation <NUM> in a homozygous or heterozygous state show a reduced wound-induced surface discoloration as compared to plants comprising only mutation <NUM> in the F5H1 gene homolog.

In addition, disclosed herein is a plant comprising a modification to the F5H1 gene homolog of a Lactuca sativa plant as indicated in Table <NUM>.

Also disclosed herein is a combination of mutation <NUM> of the F5H1 gene homolog and one or more of mutation <NUM>, <NUM>, <NUM>, <NUM> and <NUM> of the F5H2 gene homolog of a Lactuca sativa plant as indicated in Table <NUM>.

The invention further relates to a modified F5H gene homolog of the invention that confers reduced wound-induced surface discoloration to a plant.

The invention further relates to the use of a modified F5H gene homolog of the invention for the development of a plant exhibiting reduced wound-induced surface discoloration.

The modified F5H gene homologs that have been identified in the course of this research and described in this application, are certainly not the only modifications to the F5H gene homologs that would lead to the trait of the invention. By using methods described herein or known in the art, the skilled person is very well capable of introducing the described or other mutations that have the same or a similar effect in lettuce or in every other plant comprising a F5H gene homolog.

By using a phenotypic screening test as described herein it can be established whether the wound-induced surface discoloration is reduced compared to the discoloration of a WT plant. The phenotypic test can be used to detect reduced wound-induced surface discoloration in lettuce and in other crops that have a F5H gene homolog. The modification of a gene homolog leading to reduced wound-induced surface discoloration can be used for any plant that may be subject to discoloration, but it is in particular useful for vegetables or fruits.

Moreover, the skilled person is also capable of detecting other F5H gene homologs other than the homologs characterized in this application. The skilled person may detect other gene homologs in the crops as described herein or in other crops that are not described in this application. After detecting these other homologs, the skilled person is capable of modifying their sequences by using methods described in this application or known in the art. By modifying other F5H gene homologs the reduction of the wound-induced surface discoloration may be enhanced.

Amino acid substitutions may occur in regions of the protein that do not significantly affect the protein structure, function and stability. However, amino acid substitutions that occur at a position within a well conserved domain may affect the expression level or activity level of the protein. Multiple sequence alignments between F5H protein orthologs reveals highly conserved positions that may be relevant to the stability, function and/or structure of the F5H protein. It was found that F5H protein homologs comprises five conserved motifs as listed in Table <NUM> and highlighted in the protein alignment (<FIG>), were identified in all the species. Non-conservative amino acid changes within these conserved regions may disrupt the stability, functionality, and/or structure of the encoded F5H protein. However, modifications outside these motifs may also have an effect the stability, functionality, and/or structure of the encoded F5H protein.

More in particular, the substitution of a highly conserved Glycine residue at position <NUM> in the lettuce F5H2 protein (SEQ ID No: <NUM>), which is encoded by G<NUM>G<NUM>A<NUM> of the lettuce F5H2 DNA sequence (SEQ ID No: <NUM>), with a Glutamic acid residue, which is encoded by G<NUM>A<NUM>A<NUM>, leads in combination with mutation <NUM> to reduced wound-induced surface discoloration to a plant as compared to a wild type plant and to a reduced wound-induced surface discoloration as compared to a plant comprising only mutation <NUM>.

More in particular, the substitution of a highly conserved Serine residue at position <NUM> in the lettuce F5H2 protein (SEQ ID No: <NUM>), which is encoded by T<NUM>C<NUM>T<NUM> of the lettuce F5H2 DNA sequence (SEQ ID No: <NUM>), with a Phenylalanine residue, which is encoded by T<NUM>T<NUM>T<NUM>, leads in combination with mutation <NUM> to reduced wound-induced surface discoloration to a plant as compared to a wild type plant and to a reduced wound-induced surface discoloration as compared to a plant comprising only mutation <NUM>.

More in particular, the substitution of a highly conserved Glycine residue at position in the lettuce F5H2 protein (SEQ ID No: <NUM>), which is encoded by G<NUM>G<NUM>A<NUM> of the lettuce F5H2 DNA sequence (SEQ ID No: <NUM>), with a Glutamic acid residue, which is encoded by G<NUM>A<NUM>A<NUM>, leads in combination with mutation <NUM> to reduced wound-induced surface discoloration to a plant as compared to a wild type plant and to a reduced wound-induced surface discoloration as compared to a plant comprising only mutation <NUM>.

The present disclosure is broadly applicable to all plant species and crops that carry at least one functional F5H gene homolog in their genome. The F5H genes present in other plant species are called "gene orthologs" and are coding for F5H proteins having the same or a similar function. Identification of F5H orthologues, i.e. F5H genes in other species, can be performed in many crops, methods for which are known in the art. In the present research, orthologs of the F5H gene were identified in other crops by comparing the lettuce F5H protein sequences (SEQ ID No: <NUM> and <NUM>) against sequences of other plant genomes using a Basic Local Alignment Search Tool (BLAST) program. The best hits per species were identified as candidate F5H orthologous genes, listed in Table <NUM>. Multiple sequence alignments of the protein sequences using CLUSTAL confirmed that the candidate genes were orthologous F5H genes (<FIG>). Once the DNA sequences of orthologous F5H genes and their encoded F5H proteins are known, this information may be used to modulate or modify the proteins encoded by said genes using the methods described herein or known by the person skilled in the art.

Thus, also disclosed herein is a plant of the species Solanum tuberosum, Allium cepa, Cynara cardunculus var. Scolymus, Oryza sativa, Zea mays, Prunus persica, Solanum melongena, Cichorium intybus, Cichorium endivia, Apium graveolens, Malus domestica, Musa acuminate, Glycine max, Pyrus x bretschneideri, Triticum aestivum, Raphanus sativus and Brassica oleracea, comprising one or more modified F5H gene homologs in its genome which modified homologs lead to reduced wound-induced surface discoloration.

The invention relates to a method for selecting a plant showing reduced wound-induced surface discoloration, wherein the method comprises screening a plant or a population of plants for the presence of a modified F5H gene homolog of the invention leading to reduced wound-induced surface discoloration in a plant, optionally applying a phenotypic test to identify plants showing reduced wound-induced surface discoloration, and selecting a plant showing reduced wound-induced surface discoloration.

Methods used to detect and select plants that show a reduction of the wound-induced discoloration are for example a phenotypic test such as the test described and illustrated in Example <NUM> and/or the use of molecular markers as characterized in Example <NUM>. Both methods may be used to directly or indirectly detect and select the plants exhibiting a reduction of the wound-induced surface discoloration and comprising a modified F5H gene in the F1 or in any further generation resulting from a cross with a parent plant showing the reduced wound-induced discoloration and comprising a modified F5H gene homolog.

The trait of the invention can phenotypically be determined in a leaf disc test as described in Example <NUM> comprises the step of inducing a wound by taking for example a leaf disc of the plant. The shape of the sample is not limited to discs, but rather a piece of the leaves with a wound, regardless of the shape. The sample of the leaf is incubated between wetted filter papers moistened with buffer MES and after an incubation of three, five and ten days at <NUM>, it is compared to a leaf sample taken from a control plant that does not carry a modified F5H gene homolog in its genome, i.e. a wild type plant, that was incubated for the same time, under the same conditions.

The presence and the intensity of wound-induced surface discoloration on the different leaf disc samples can be evaluated on an appropriate scale in order to compare them. The skilled person can use a scale with any subdivision. In the phenotypic analysis described in Example <NUM> the wound-induced surface discoloration appears as a pink colored ring around the edges of the leaf disc. When the color is saturated and the wound-induced surface discoloration is very strong, the discoloration may appear red to very dark red. An example of scale is from <NUM> to <NUM>, wherein <NUM> means that no discoloration on the edges is visible, score <NUM> means that the leaf disc has a very slight pink discoloration around the edges, score <NUM> means that the leaf disc has a thin ring of red/pink discoloration around the edges, score <NUM> means that the leaf disc has a darker and thicker ring of red/pink discoloration around the edges as compared to a leaf disc having score <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> and score <NUM> means that the leaf disc has a very dark red and thick ring of discoloration around the edges. An example of each score is represented in <FIG>. The scale described in this application is an example of a scale that could be used to attribute a score to a leaf disc of a plant to test and to another plant in order to compare them and to identify the reduced wound-induced surface discoloration. The scoring of the leaf discs should be preferably done by the one person.

In order to identify the reduced wound-induced surface discoloration, the score of the plant to test should be compared to the score of the wild type plant at for example <NUM>, <NUM> and <NUM> days after sampling. Leaf discs taken from plants that show a reduced wound-induced surface discoloration have a score that is higher than the score of leaf discs of a wild type plant at the same day of incubation. Preferably the leaf discs taken from a plant showing a reduced wound-induced surface discoloration have score <NUM> or score <NUM>. A plant comprising a modified F5H1 gene and a modified F5H2 gene has a higher score than a plant comprising a modified F5H1 gene homolog alone at the same day of incubation. To be comparable the leaf disc test of the plants should be performed under the same conditions with plants grown under the same conditions.

Alternatively, the wound-induced surface discoloration can also be determined by cutting the plant or a part thereof and storing this cut part until it shows wound-induced surface discoloration. The evaluation of the wound-induced surface discoloration can be performed by reproducing the usual storage conditions of the plants. The plants at mature stage are harvested and cut into pieces. For lettuce plants the cutting method may depend on the lettuce variety used to perform the test. The plant pieces are washed and stored at <NUM>-<NUM> in a cool cell in plastic bags. After <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> days after washing the presence and the intensity of the wound-induced surface discoloration are evaluated. In order to detect the discoloration, the cut leaf pieces were compared to the cut leaf pieces of the WT plant. The presence and the intensity of the wound-induced surface discoloration is detectable by the presence and intensity of a pink discoloration on the cutting edges that can be evaluated by using a scale from <NUM> to <NUM>, wherein <NUM> means that no signs of wound-induced surface discoloration are visible, <NUM> means that first little traces of pink discoloration (nearly not visible, only a glow) are visible, <NUM> means the pink discoloration is appearing on some edges, <NUM> means that the pink discoloration is visible on all cutting edges, <NUM> means that a strong pink discoloration is visible on all cutting edges and <NUM> means that a dark pink discoloration is visible on all cutting edges. A plant showing reduced wound- induced surface discoloration has a higher score than a wild type plant. To be comparable the phenotypical analysis of whole lettuce heads should be performed under the same conditions with plants grown under the same conditions.

Disclosed herein is a molecular marker for detecting in the genome of a plant a mutation causative of reduced wound-induced surface discoloration in said plant or part thereof, wherein the marker is a mutation in any of the wild type sequences, the SEQ ID numbers of which are shown in Table <NUM>.

The invention however relates to a molecular marker for detecting in the genome of a plant a mutation causative of reduced wound-induced surface discoloration in said plant or part thereof, wherein the mutation is a nucleotide change of C > T at position <NUM> of SEQ ID No: <NUM>.

The invention relates to a molecular marker for detecting in the genome of a plant a mutation causative of reduced wound-induced surface discoloration in said plant or part thereof, wherein the mutation is an amino acid substitution from Threonine to Isoleucine at position <NUM> of the encoded protein as a result of a change C > T at position <NUM> of SEQ ID No: <NUM>, and/or an amino acid substitution of Glycine to Glutamic acid at position <NUM> of the encoded protein as a result of a change G > A at position <NUM> of SEQ ID No: <NUM> and/or an amino acid substitution of Serine to Phenylalanine at position <NUM> of the encoded protein as a result of a change C > T at position <NUM> of SEQ ID No: <NUM>, and/or an amino acid substitution of Glycine to Glutamic acid at position <NUM> of the encoded protein as a result of a change G > A at position <NUM> of SEQ ID No: <NUM>, and/or an amino acid substitution Glycine to Glutamic acid at position <NUM> of the encoded protein as a result of a change G > A SNP at position <NUM> of SEQ ID No: <NUM>.

The invention further relates to the use of a molecular marker as described herein, to identify or develop a plant showing reduced wound-induced surface discoloration, or develop other markers linked to a modified F5H gene homolog of the invention.

The invention also relates to a method for identifying molecular markers linked to the reduced wound-induced surface discoloration of a plant, comprising:.

Also disclosed herein is the use of a molecular marker to identify a modification in a F5H gene homolog that leads to a reduced wound-induced surface discoloration, or to develop other markers linked to a modified F5H gene homolog of the invention. A molecular marker is based upon the modification to the F5H gene homologs that underlies the trait. A non-exclusive list of suitable molecular markers is provided in this application. The person skilled in the art is familiar with creating and using them for detecting and selecting plants with a modified F5H gene homolog causative of reduced wound-induced surface discoloration during breeding.

During the research that led to the present invention a number of EMS induced SNP mutations were identified in the two F5H gene homologs of Lactuca sativa. One of the identified SNPs in F5H1 resulted in a stop codon in the protein and five of the identified SNPs in F5H2 resulted in an amino acid change in the protein sequence. The SNPs can be used as markers for detecting the presence of a modified F5H gene homolog in the genome of a plant.

In a particular embodiment, one suitable molecular marker is the C<NUM> >T<NUM> SNP in the F5H1 gene of lettuce (Lactuca sativa) represented in Table <NUM> (Example <NUM>).

In a particular embodiment, one suitable molecular marker is the C<NUM> > T<NUM> SNP in the F5H2 gene of lettuce (Lactuca sativa) represented in Table <NUM> (Example <NUM>).

In a particular embodiment, one suitable molecular marker is the G<NUM> > A<NUM> SNP in the F5H2 gene of lettuce (Lactuca sativa) represented in Table <NUM> (Example <NUM>).

The invention relates to the molecular markers and the use of these markers to identify the modified F5H gene homologs leading to reduced wound-induced surface discoloration in all the plants comprising a F5H gene homolog. The SNP markers mentioned above are particularly suitable for use in Lactuca sativa.

The invention relates to a method of determining the presence of a modified F5H gene homolog in a plant of the invention, comprising the steps of obtaining a sample of nucleic acids from said plant, comparing said nucleic acids to a sample of nucleic acids obtained from a reference plant comprising the wild type F5H gene homolog, and detecting a polymorphism between the two nucleic acid samples, wherein the detected polymorphism is indicative of the presence of said modified homolog.

Preferably, the wild type F5H gene homolog is any one of the sequences of which the SEQ ID numbers are listed in Table <NUM>.

A modified F5H gene may be introduced into any other genetic background of the same or a different species. The plant lacking the modification may have other desired traits. For sexually compatible plants the introgression can be achieved through crossing and/or backcrossing and selecting in the first generation in which the reduced wound-induced surface discoloration is detectable. Crossing can optionally be followed by embryo rescue techniques or other techniques that result in a successful combination and introgression, which techniques are known to the person skilled in the art. The parent plants may be plants grown directly from the deposited seeds or progeny plants from the seed or a progeny plant from seeds that are identified to have the trait of the invention by other means.

When a trait is dominant monogenic, it can be introgressed into another plant in only one generation (F1). When a trait is recessive and/or involves more than one gene, introgression may encompass a breeding process that takes multiple generations. Introgression is used herein to describe the entire process. For a dominant trait, the selection of the plants carrying the modification can start in the F1 or any further generation resulting from a cross between a plant with the desired trait and a plant without this trait. For a recessive trait, the selection with a phenotypic test and/or with the use of molecular markers is started in the F2 or any further generation resulting from a cross or alternatively from a backcross.

In a particular embodiment, one or more modified lettuce F5H gene homologs can be introgressed from a Lactuca sativa plant carrying the modified lettuce F5H gene homolog into a Lactuca sativa plant lacking modified lettuce F5H gene homologs using standard breeding techniques.

The invention further relates to propagation material suitable for producing a plant that comprises one or more modified F5H genes in its genome and exhibits reduced wound-induced discoloration. In one embodiment, the propagation material is formed by parts of the plant that are suitable for sexual reproduction, in particular a microspore, pollen, ovary, ovule, embryo sac and egg cell. In another embodiment, the propagation material is formed by parts suitable for vegetative reproduction, in a particular a microspore, pollen, ovary, ovule, embryo, embryo sac, egg cell, cutting, root, root tip, hypocotyl, cotyledon, stem, leaf, flower, anther, seed, meristematic cell, protoplast and a cell, or a tissue culture thereof.

A plant grown or regenerated from the said propagation material, which plant comprises in its genome one or more modified F5H genes as defined herein, providing the plant with reduced wound-induced surface discoloration is disclosed herein.

In particular, the plant produced from the propagation material comprises the modified F5H gene homolog as found in lettuce plants grown from seeds, and of which representative seed was deposited with the NCIMB under accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> and NCIMB <NUM>.

Additionally, disclosed herein is the use of a plant of the invention that comprises a modified F5H gene, which provides to a plant the reduction of wound-induced surface discoloration, in plant breeding to confer this trait.

Also disclosed herein is a method for producing a plant showing reduced wound-induced surface discoloration comprising:.

In one embodiment the plant is phenotypically selected and/or selected by use of molecular markers.

Furthermore, disclosed herein is a method for production of a plant that shows reduced wound-induced surface discoloration, comprising.

In addition, disclosed herein is a method for production of a plant that shows reduced wound-induced surface discoloration, comprising.

Additionally disclosed herein is a method of introducing another desired trait into a plant which has the trait of the invention, comprising:.

In one embodiment the selection for plants that show reduced wound-induced surface discoloration is done in the F1 or any further generation by using the markers described in Example <NUM>. In another aspect selection for the trait is started in the F2 of a cross or alternatively of a backcross. Selection of plants in the F2 can be done phenotypically as well as by using the said marker(s) which directly or indirectly detect the modified F5H gene underlying the trait.

Selection for plants that show reduced wound-induced surface discoloration may be started in the F3 or a later generation.

The plant comprising a F5H gene homolog of the invention may be a plant of an inbred line, a hybrid, a doubled haploid, or of a segregating population.

Disclosed herein is a method for the production of a plant that shows reduced wound-induced surface discoloration by using a doubled haploid generation technique to generate a doubled haploid line comprising the said trait.

Also disclosed herein is hybrid seed that can be grown into a plant that shows reduced wound-induced surface discoloration and to a method for producing such hybrid seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed, wherein said first parent plant and/or said second parent plant is the plant as claimed.

Further disclosed herein is a method for producing a hybrid plant that that shows reduced wound-induced surface discoloration, comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed, of which the first parent plant and/or the second parent plant is a plant that shows reduced wound-induced surface discoloration, and growing said hybrid seeds into hybrid plants that show reduced wound-induced surface discoloration.

A method of producing a hybrid plant seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant plant seed, wherein said first parent plant and/or said second parent plant comprises a modified F5H gene homolog of the invention is also disclosed herein.

Furthermore, disclosed herein is a method for the production of a plant that shows reduced wound-induced surface discoloration, comprising growing the plant from a seed that comprises a modified F5H gene homolog in its genome that leads to the trait of reduced wound-induced surface discoloration. The seeds are suitably seeds of which a representative sample was deposited with the NCIMB under accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> and NCIMB <NUM>.

A method for seed production comprising growing plants from seeds of which a representative sample was deposited with the NCIMB under accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> and NCIMB <NUM>, allowing the plants to produce seeds, and harvesting those seeds is additionally disclosed herein. Production of the seeds is suitably done by crossing or selfing.

Also disclosed herein is a method for the production of a plant that shows reduced wound-induced surface discoloration by tissue culture using a plant as described herein as the source of the tissue.

Furthermore, disclosed herein is a method for the production of a plant that shows reduced wound-induced surface discoloration by vegetative reproduction of parts of a plant as described herein.

Additionally, a method for the production of a plant that shows reduced wound-induced surface discoloration by using a method for genetic modification to introgress the said trait into the plant from a plant of the invention is disclosed herein. Genetic modification comprises transgenic modification or transgenesis, using a gene from a non-crossable species or a synthetic gene, and cisgenic modification or cisgenesis, using a natural gene, coding for an (agricultural) trait, from the crop plant itself or from a sexually compatible donor plant.

A breeding method for the development of plants that show reduced wound-induced surface discoloration wherein germplasm comprising said trait is used is further disclosed herein. Representative seed of said plant comprising the modified F5H gene homolog and being representative for the germplasm was deposited with the NCIMB under accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> and NCIMB <NUM>.

In addition, disclosed herein is a method for the production of a plant that shows reduced wound-induced surface discoloration wherein progeny or propagation material of a plant comprising the modified F5H gene homolog conferring said trait is used as a source to introgress the said trait into another plant. Representative seed of said plant comprising the modified F5H gene homolog was deposited with the NCIMB under accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> and NCIMB <NUM>.

Furthermore, disclosed herein is a plant showing reduced wound-induced surface discoloration, which plant is obtainable by any of the methods herein described and/or familiar to the skilled person.

In the course of breeding a new plant carrying a modified F5H gene homolog, desirable agronomic traits may be introduced into plant independently of the modified F5H gene. As used herein, "desirable traits" include but are not limited to e.g. improved yield, leaf shape, leaf size, leaf number, leaf color, seed number, seed size, plant vigor, plant height, bolting, and resistance to one or more diseases or disease causing organisms. Any one of these desirable traits may be combined with a modified F5H gene homolog.

Also disclosed herein is a method for producing an agronomically elite plant that shows the reduced wound-induced surface discoloration of the invention, comprising introgressing a modified F5H gene homolog into a agronomically elite plant. This can be achieved by methods described in this application or known in the art.

In yet a further embodiment the agronomically elite plant is an inbred line or a hybrid.

As used herein, a plant of an inbred line is a plant of a population of plants that is the result of three or more rounds of selfing, or backcrossing; or which plant is a double haploid. An inbred line may e.g. be a parent line used for the production of a commercial hybrid.

As used herein, a hybrid plant is a plant which is the result of a cross between two different plants having different genotypes. More in particular, a hybrid plant is the result of a cross between plants of two different inbred lines, such a hybrid plant may e.g. be a plant of an F<NUM> hybrid variety.

A plant comprising the modified F5H gene of the invention may be an agronomically elite plant.

In the context of this invention an agronomically elite plant is a plant having a genotype that as a result of directed crossing and selection by human intervention results into an accumulation of distinguishable and desirable agronomic traits which allow a producer to harvest a product of commercial significance.

The invention also relates to a food product comprising a part of a plant of the invention. The food product may comprise one or more harvested parts of plants of the invention, to food products which may comprise harvested leaves of plants of the invention, either in natural or in processed form, and to a container which may comprise one or more plants of the invention in a growth substrate for harvest of leaves from the lettuce plant in a domestic environment. The harvested part or food product may be, or comprise the head and/or part of a plant such as leaves of a plant of the invention. The food product or harvested part, may have undergone one or more processing steps. Such a processing step might comprise but is not limited to any one of the following treatments or combinations thereof: cutting, washing, or a salad mixture which may comprise parts of the plant of the invention such as leaves. The processed form that is obtained is also part of the invention. All the food products and harvested parts carry in their genome modified F5H gene homolog of the invention.

The invention relates to a part of a plant of the invention, wherein the part is a leaf, a whole head of a plant, a fruit, an inflorescence, a seeds, a curd, a stem, a tuber, a bulb or a root, optionally in processed form.

The invention further relates to a seed capable of developing into a plant of the invention.

The invention also relates to a seed of a plant of the invention, wherein the seed comprises a modified F5H gene homolog in its genome.

Disclosed herein is a cell of a plant of the invention, which cell comprises a modified F5H gene in its genome and provides the plant with reduced wound-induced discoloration. Such cell may be either isolated from or may be part of a plant or parts thereof.

Also disclosed herein is a cell of a lettuce plant (Lactuca sativa), which lettuce plant shows reduced wound-induced surface discoloration as found in a lettuce plant grown from seed as deposited with the NCIMB on <NUM> February <NUM> under NCIMB and having one of the accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, or NCIMB <NUM>.

Additionally, disclosed herein is a cell of a plant, which plant comprises a modified F5H gene and shows reduced wound-induced surface discoloration and a cell of a plant that comprises a modified F5H gene and that shows reduced wound-induced surface discoloration, which plant is obtainable by crossing a plant comprising a modified F5H gene and selecting for a plant that shows a reduced wound-induced surface discoloration.

Further disclosed herein is a cell of a lettuce plant (Lactuca sativa), which lettuce plant shows reduced wound-induced surface discoloration as found in a lettuce plant grown from seed as deposited with the NCIMB on <NUM> February 2016under NCIMB and having one of the accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, or NCIMB <NUM>, which lettuce plant is obtainable by crossing a lettuce plant with a lettuce plant grown from seed as deposited with the NCIMB on <NUM> February 2016under NCIMB and having one of the accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, or NCIMB <NUM>, and selecting for a lettuce plant that shows a reduced wound-induced surface discoloration.

In addition, disclosed herein is the use of seeds that were deposited under NCIMB under accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> and NCIMB <NUM>, for developing plants showing reduced wound-induced surface discoloration into another lettuce plant (Lactuca sativa).

Also disclosed herein is the use of the seeds of which a representative sample was deposited under NCIMB under accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> and NCIMB <NUM> for transferring the reduced wound-induced surface discoloration trait into another agronomically valuable lettuce plant.

The use of a plant that comprises a modified F5H gene homolog of the invention and shows reduced wound-induced surface discoloration, as a crop is also disclosed herein.

In particular, disclosed herein is the use of a lettuce plant (Lactuca sativa) that exhibits reduced wound-induced surface discoloration, as found in a lettuce plant grown from seed as deposited with the NCIMB on <NUM> February <NUM> and having one of the accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> or NCIMB <NUM>, as a crop.

Also disclosed is the use of a plant that comprises a modified F5H gene homolog of the invention and shows reduced wound-induced surface discoloration, as a source of seed.

The use of a lettuce plant (Lactuca sativa) that exhibits reduced wound-induced surface discoloration, as found in a lettuce plant grown from seed as deposited with the NCIMB on <NUM> February <NUM> and having one of the accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> or NCIMB <NUM>, as a source of seed is also disclosed herein.

Further disclosed herein is the use of a plant that comprises a modified F5H gene homolog of the invention and shows reduced wound-induced surface discoloration, as a source of propagation.

The use of a lettuce plant (Lactuca sativa) that exhibits reduced wound-induced surface discoloration, as found in a lettuce plant grown from seed as deposited with the NCIMB on <NUM> February <NUM> and having one of the accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> or NCIMB <NUM>, as a source of propagating material is also disclosed herein.

Also disclosed herein is the use of a plant that comprises a modified F5H gene homolog of the invention and shows reduced wound-induced surface discoloration, for consumption.

Additionally, disclosed herein is the use of a lettuce plant (Lactuca sativa) that exhibits reduced wound-induced surface discoloration, as found in a lettuce plant grown from seed as deposited with the NCIMB on <NUM> February <NUM> and having one of the accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, <NUM> or NCIMB <NUM>, for consumption and the use of a modified F5H gene homolog of the invention, for conferring reduced wound-induced surface discoloration to a plant.

Disclosed herein is the use of a F5H gene homolog as found in seeds that were deposited with the NCIMB on <NUM> February <NUM> and having one of the accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> or NCIMB <NUM>, for conferring reduced wound-induced surface discoloration to a lettuce plant (Lactuca sativa).

The use of a plant as a recipient of modified F5H gene homologs of the invention is also disclosed herein.

Also disclosed herein is the use of a lettuce plant (Lactuca sativa) as found in seeds that were deposited with the NCIMB on found in seeds that were deposited with the NCIMB on <NUM> February <NUM> and having one of the accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> or NCIMB <NUM>.

The use of modified F5H gene of the invention for conferring reduced wound-induced surface discoloration to a plant is also disclosed herein.

In particular, disclosed herein is the use of a modified F5H gene homolog as found in seeds that were deposited with the NCIMB on <NUM> February <NUM> and having one of the accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, or NCIMB <NUM>, conferring reduced wound-induced surface discoloration to a lettuce plant (Lactuca sativa).

In addition, disclosed herein is the use of seeds that were deposited with the NCIMB on <NUM> February <NUM> and having one of the accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, or NCIMB <NUM>, for transferring reduced wound-induced surface discoloration into another lettuce plant (Lactuca sativa).

Seeds of lettuce plants (Lactuca sativa) of the invention that comprise a modified F5H gene homolog which lead to reduction of wound-induced surface discoloration, were deposited with the NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK on February <NUM>, <NUM> under deposit accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> and NCIMB <NUM>.

The deposited seeds do not meet the DUS criteria which are required for obtaining plant variety protection, and can therefore not be considered to be plant varieties.

In the examples reference is made to the following figures:.

Table <NUM> shows the F5H1 and F5H2 mutations and the effect on the encoded F5H1 and F5H2 protein sequences. Positions are as in the sequences of Lactuca sativa, SEQ ID No: <NUM> (F5H1 CDS sequence wild type), SEQ ID No: <NUM> (F5H2 CDS sequence wild type), SEQ ID No: <NUM> (F5H1 protein sequence wild type) and SEQ ID No: <NUM> (F5H2 protein sequence wild type).

In order to create Lactuca sativa plant mutants, approximately <NUM> seeds of the lettuce varieties Troubadour, Apache, Yorvik and Roderick were treated with EMS by submergence of the seeds in an aerated solution of either <NUM>% (w/v) or <NUM>% (w/v) EMS for <NUM> hours at room temperature. Following EMS treatment, the M1 seeds were rinsed with water, germinated and grown in a greenhouse at <NUM> at <NUM> hours light, <NUM> hours dark regime in order to produce M2 seeds that were harvested and bulked. The resulting population of M2 plants was screened for lettuce plants that showed reduced wound-induced discoloration by using the phenotypic test described in Example <NUM>.

A lettuce plant which was a mutant of the variety Troubadour and did exhibit a reduced wound-induced surface discoloration compared to the WT following <NUM> to <NUM> days incubation of a leaf disc sample in the phenotypic test described in Example <NUM> was selected and crossed with a second lettuce plant from the variety Troubadour. An F6 line, 11K200310, was produced from this cross after repeated cycles of inbreeding in combination with plant and line selection. Selection was performed to maintain the reduced wound-induced surface discoloration and to reduce the effect of undesirable background mutations.

A second round of EMS treatment was then performed on <NUM> seeds of the line 11K200310, in the same manner as previously described in the first EMS treatment, but using a concentration of <NUM>% EMS instead. EMS treated seeds were then sown in a glasshouse and the resulting population of plants were then screened again for lettuce plants with wound-induced surface discoloration using the phenotypic test as described in Example <NUM>.

Plants from Example <NUM> that were grown from seeds that were either treated with one round or two rounds of EMS, were screened for their potential to show reduced wound-induced surface discoloration. Leaf disc samples were taken from the plants when these had developed approximately <NUM> true leaves being of the size to take a sample without cutting the middle vein. For this experiment the plants were at mature stage (approximately <NUM> months old) grown in a glasshouse. The obtained samples were laid on the filter paper that was moistened with MES buffer. The upper side of the leaf was in contact with the filter paper. The leaves were covered with a second filter paper also moistened with MES buffer. The air bubbles between the two filter papers were removed and the leaf discs were incubated between the wetted filter papers in a container at <NUM>. After three, five and ten days of incubation of the leaf samples the wound-induced surface discoloration was scored by one person for the presence and intensity of wound-induced surface discoloration represented by the pink color around the edges of the leaf samples. An example of a scale used in this phenotypic identification is from <NUM> to <NUM>, wherein <NUM> means that the edges of the leaf disc have no discoloration, score <NUM> means that the leaf disc has a very slight pink discoloration around the edges, score <NUM> means that the leaf disc has a thin ring of red/pink discoloration around the edges, score <NUM> means that the leaf disc has a darker and thicker ring of red/pink discoloration around the edges as compared to a leaf disc having score <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> and score <NUM> means that the leaf disc has a very dark red and thick ring of discoloration around the edges. While different scales can be used to evaluate the intensity of wound-induced surface discoloration, the scale should range from no wound-induced surface discoloration to the highest intensity of wound-induced surface discoloration. The most interesting candidates were grown for seeds and resown and retested according to the same protocol.

Reduced wound-induced discoloration was confirmed for some candidates. The results of the phenotypic test for wound-induced surface discoloration on the leaf discs at days <NUM>, <NUM> and <NUM> after sampling are shown in <FIG>, <FIG> and <FIG> and the scores are represented in Table <NUM>; At day <NUM> (<FIG>) the WT showed wound-induced surface discoloration (score <NUM>) , the plants comprising mutation <NUM> showed a reduced wound-induced surface discoloration (score <NUM>) as compared to the wound-induced surface discoloration of the wild type plants and plants comprising mutation <NUM> and mutation <NUM>, <NUM>, <NUM>, <NUM> or <NUM> did not show any wound-induced surface discoloration (score <NUM>). At day <NUM> (<FIG>) the WT showed wound-induced surface discoloration (score <NUM>), the plants that were treated one time with EMS and had mutation <NUM> in their F5H1 gene homolog (Table <NUM>) showed a reduced wound-induced surface discoloration (score <NUM>) as compared to the WT (score <NUM>) and plants that have been treated two rounds of EMS and had a mutation <NUM> in their F5H1 gene homolog (Table <NUM>) and mutation <NUM>, <NUM>, <NUM>, <NUM> or <NUM> in their F5H2 gene homolog (Table <NUM>) did not show any wound-induced surface discoloration or a reduced wound-induced surface discoloration as compared to the plants comprising mutation <NUM> (scores <NUM> to <NUM>). At day <NUM> (<FIG>) the plants that were treated one time with EMS and had mutation <NUM> in their F5H1 gene homolog showed a wound-induced surface discoloration (score <NUM>) and plants that have been treated two rounds of EMS and had a mutation <NUM> in their F5H1 gene homolog (Table <NUM>) and mutation <NUM>, <NUM>, <NUM>, <NUM> or <NUM> in their F5H2 gene homolog (Table <NUM>) did not show any wound-induced surface discoloration or a reduced wound-induced surface discoloration as compared to a plant comprising mutation <NUM> (scores <NUM> to <NUM>).

Those plants were selfed in order to produce seeds, which were then deposited with the NCIMB under accession numbers accession numbers NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM>, NCIMB <NUM> and NCIMB <NUM>.

The DNA of the Lactuca sativa plants resulting from the treatment with EMS and showing reduced wound-induced surface discoloration was analyzed to identify mutations in the F5H gene homologs by using standard DNA sequencing techniques. A number of mutations in the gene homologs F5H1 and F5H2 of the Lactuca sativa plant were identified.

The mutation in lettuce F5H gene homolog on chromosome <NUM>, herein referred to as F5H1, resulted in a premature stop codon in the sequence of the corresponding wild type protein represented in SEQ ID No: <NUM>. The different mutations in lettuce F5H gene on chromosome <NUM>, herein referred to as F5H2 resulted in amino acid changes in the sequence of the corresponding wild type proteins represented in SEQ ID No:<NUM>. The presence of the modified F5H1 protein in a Lactuca sativa plant results in reduced wound-induced surface discoloration. The presence of the modified F5H1 and F5H2 proteins in a Lactuca sativa plant enhances this effect. The mutations listed in the following Table <NUM> and Table <NUM> were identified in the F5H gene homologs of the Lactuca sativa plant. The tables show only parts of the F5H1 and F5H2 sequences comprising the mutated nucleotide (SNP) and <NUM> flanking nucleotides on either side.

F5H gene orthologs in others crop species were identified by using a BLASTN and BLASTP program in order to compare the DNA and the protein sequences of F5H1 and F5H2 of the Lactuca sativa plant with the sequences of other crops species. The best hits per species were identified as candidate F5H1 and F5H2 gene orthologs. A non-limitative list of plants that carry one or more F5H gene orthologs are represented in the following table.

The alignments revealed the presence of highly conserved amino acids amongst the F5H gene orthologs of different species. Examples of highly conserved amino acid regions highlighted within the sequences of F5H gene orthologs in the protein sequence alignment <FIG> are listed in Table <NUM>.

A lettuce plant numbered 15E238260 showing reduced wound-induced discoloration after <NUM> days of incubation and found by the phenotypic test described in Example <NUM> was selfed. By use of DNA-marker tests based on SNPs markers in Example <NUM> plant 15E238260 was shown to be homozygous for the mutation in the F5H1 gene homolog and a mutation in the F5H2 gene homolog. Flowers of plant 15E238260 were used as a pollen donor to make a cross with a plant of the lettuce variety Hofnar that showed wound-induced surface discoloration in the phenotypic test described in Example <NUM> just like the wild type plant. The above-mentioned cross result in an F1-seed lot numbered 15E97481.

Four F1-seeds were sown and the resulting plants numbered 15E748101, 15E748102, 15E748103, and 15E748104, were selfed to produce F2-seeds. These F2-seeds are sown and the individual F2-plants were tested for reduced wound-induced surface discoloration by the phenotypic test described in Example <NUM>. Three out of each sixteen F2-plants were expected to show reduced wound-induced surface discoloration after <NUM> to <NUM> days and one out of each sixteen F2-plants was expected to show reduced wound-induced surface discoloration ten days of incubation of the leaf discs at <NUM> as described in Example <NUM>.

The F2 plants that showed a reduced wound-induced surface discoloration after <NUM> days of incubation were therefore expected to be homozygous for the mutation in the F5H1 gene homolog as well as for a mutation in the F5H2 gene homolog. The selected F2-plant was selfed to produce F3-seeds. By growing ten F3-plants out of this seed lot and observing a reduced wound-induced surface discoloration for each of them after ten days of incubation (as in Example <NUM>), the homozygous presence of the two mutated genes in the selected F2-plant was confirmed. The selection of the plant and the confirmation of the selected genotype was done by using a molecular marker recognizing the difference between the wild type and the mutant genes.

To introgress both the mutation of the F5H1 gene homolog and the mutation of the F5H2 gene homolog in the lettuce variety Hofnar a backcross with variety Hofnar as recurrent parent is performed. For this purpose, the selected F2-plant showing a reduced wound-induced surface discoloration after ten days of incubation by using the phenotypic test as described in Example <NUM> was used as a parent in a cross with Hofnar. The resulting BC1-seed was sown and a BC1-plant was used as a parent in a cross with Hofnar to generate BC2-seeds. <NUM> BC2-seeds were sown and each of them was selfed to generate BC2. These <NUM> BC2. S1-seed lots (i.e. BC2. S1families) were sown, <NUM> BC2. S1 plants per seed lot, to select a BC2. S1-family, which was segregating for both the mutation of the F5H1 gene homolog and the mutation of the F5H2 gene homolog. For this purpose, each of the <NUM> plants per BC2. S1-family was tested with the phenotypic test as described in Example <NUM> with an incubation time of ten days. After these ten days a BC2. S1-family is selected which showed segregation for the reduced wound-induced discoloration phenotype. From such a family a BC2. S1-plant with the reduced wound-induced surface discoloration phenotype was selected and used as a parent in a BC3-cross with Hofnar. S1-plant was also selfed to produce BC2. S2- seeds were grown into plants and tested in the phenotypic test as described in Example <NUM>, to confirm the reduced wound-induced discoloration in all of them, after <NUM> days, they indeed show no or reduced wound-induce discoloration in the phenotypic test. This confirmed the homozygous presence of the mutation in F5H1 gene homolog as well as the homozygous presence of the mutation in F5H2 gene homolog. The selection of the plant and the confirmation of the selected genotype could have also been done by using a molecular marker recognizing the difference between the wild type and the mutant genes.

The resulting BC3-seed was sown and a BC3-plant was used as a parent in a cross with Hofnar to generate BC4-seeds. <NUM> BC4-seeds were sown and each of them was selfed to generate BC4. These <NUM> BC4. S1-seed lots (i.e. BC4. S1families) were sown, <NUM> BC4. S1 plants per seed lot, to select a BC4. S1-family, which was segregating for both the mutation of the F5H1 gene homolog and the mutation of the F5H2 gene homolog. For this purpose, each of the <NUM> plants per BC4. S1-family was tested with the wound-induced discoloration test as described in Example <NUM> with an incubation time of ten days. After these ten days a BC4. S1-family was selected which showed segregation for the reduced wound-induced surface discoloration phenotype. From such a family a BC4. S1-plant with the reduced wound-induced surface discoloration phenotype was selected and could be used as a parent in a BC5-cross with Hofnar. S1-plant was also selfed to produce BC4. S2-seeds, of which ten seeds were sown to confirm the reduced wound-induced surface discoloration in all of them. The selection of the plant and the confirmation of the selected genotype could also be done by using a molecular marker recognizing the difference between the wild type and the mutant genes, e.g. markers based on the SNPs as described in Example <NUM>. The genotype of each selected plant could also be confirmed by using molecular markers.

S2-seeds were sown in a trial and the resulting plants are compared with plants of the variety Hofnar grown in the same trial. In this way it is established whether the BC4-generation is sufficiently similar to Hofnar to be used by growers in practice.

A lettuce plant numbered 15E238260 showing reduced wound-induced discoloration and comprising mutation <NUM> (C<NUM> >T<NUM> in the F5H1 gene homolog) and mutation <NUM> (G<NUM> > A<NUM> in the F5H2 gene homolog) in a homozygous state was used for the segregation analysis. Flowers of plant 15E238260 were used as a pollen donor to make a cross with a plant of the lettuce variety Troubadour that showed wound-induced surface discoloration in the phenotypic test described in Example <NUM> just like the wild type plant in order to obtain F1-seeds.

The F1 plants grown from the F1 seeds were sown and selfed to produce F2-seeds. The plants grown from the F2 seeds were used to analyze the segregation of the trait of the invention.

The mutations were homozygously absent, heterozygously present or homozygously present in a plant. The different genotypes and their denomination in this application are represented in Table <NUM>. The reduced wound-induced surface discoloration was evaluated by using the leaf disc test as described in Example <NUM>, but for the segregation analysis, the leaf discs were taken from young plants (approximately <NUM> weeks old). The score for each leaf disc is represented in Table <NUM> and as an example the leafs discs of a lettuce plant comprising mutation <NUM> and mutation <NUM> are represented in <FIG>.

The results in Table <NUM> show that in order to show reduced wound-induced surface discoloration the plant needed to carry mutation <NUM> on the F5H1 gene homolog (C<NUM> >T<NUM> in the F5H1 gene homolog). Preferably the plant carried mutation <NUM> homozygously. A plant that carried mutation <NUM> homozygously (BB) and mutation <NUM> heterozygously (AB) or homozygously (BB) showed a reduced wound-induced surface discoloration as compared to the wild-type (AA/AA) and to plants that carried only mutation <NUM>. The most reduced wound-induced surface discoloration was visible in a plant comprising mutation <NUM> in the F5H1 gene homozygously and mutation <NUM> in F5H2 gene homozygously (BB/BB).

The lettuce plants were harvested at maturity stage (approximately <NUM> months old), preferably the plants in the middle of the plot were chosen for the phenotypic analysis. The harvested plants were stored in a cooling room at <NUM> over night in boxes that were wrapped in plastic to avoid drying out. The plants were then cut under cold temperature (around <NUM>). Old leaves, leaves showing tipburn symptoms and outer leaves were removed from the heads. Two or three plant heads comprising mutation <NUM>, mutation <NUM> and mutation <NUM>, mutation <NUM> and mutation <NUM>, mutation <NUM> and mutation <NUM>, mutation <NUM> and mutation <NUM>, mutation <NUM> and mutation <NUM> and a wild type plant (Troubadour) were vertically cut in four parts to remove the core and the four parts were further cut <NUM> or <NUM> times horizontally. For the phenotypic analysis <NUM> grams of the cut lettuce leaves were washed with a washing machine (Washing step with air bubbles: <NUM>, time of centrifuge: <NUM> with the highest speed) and the water was replaced after each washing step. For washing on a small scale, water should be as cold as possible and the washing should be done in a large sink. The cut and washed leaves were dried in a salad spinner.

The cut leaves were filled in dry plastic bags without ethylene that were folded <NUM> times and stored in boxes and stored at <NUM>-<NUM>. The bags were not stocked on top of each other.

An example of the phenotypic analysis on whole lettuce heads is shown in <FIG> for the wild type lettuce variety Troubadour (wild type) that does not comprise a modified F5H gene homolog, a lettuce plant comprising mutation <NUM> (C<NUM> >T<NUM> in the F5H1 gene homolog) ("Mutation <NUM>") and a lettuce plant comprising mutation <NUM> and mutation <NUM> (G<NUM> > A<NUM> in the F5H2 gene homolog) ("Mutation <NUM> and mutation <NUM>"). The pictures were taken after <NUM> and <NUM> days after after washing. The wild type plant showed wound-induced surface discoloration and the cutting edges after <NUM> days, the plant comprising mutation <NUM> showed a reduced wound-induced surface discoloration at the cutting edges after <NUM> or <NUM> days as compared to the wild type, the plant comprising mutation <NUM> and mutation <NUM> do not show any wound-induced surface discoloration even after <NUM> days and has therefore a reduced wound-induced surface discoloration as compared to plants comprising mutation <NUM>.

Another analysis has been performed with an evaluation of the wound-induced surface discoloration at <NUM>, <NUM> and <NUM> days after washing. The results are represented in Table <NUM>. A plant that comprises mutation <NUM> showed reduced wound-induced surface discoloration as compared to the wild type. Plants that comprise mutation <NUM> and mutation <NUM>, <NUM>, <NUM> or <NUM> showed reduced wound-induced surface discoloration as compared to the wild-type and the plant comprising mutation <NUM>. Plants comprising mutation <NUM> and mutation <NUM> showed the same reduction in wound induced surface discoloration as a plant comprising mutation <NUM> at <NUM> days, but the wound-induced surface discoloration develops slower than in a plant comprising mutation <NUM> as at <NUM> Days a plant comprising mutation <NUM> has a score of <NUM> and a plant comprising mutation <NUM> and mutation <NUM> scores <NUM>.

Claim 1:
A Lactuca sativa plant comprising two modified F5H gene homologs, wherein said gene homolog comprises a modification as compared to its corresponding wild type F5H gene homolog, wherein the modification leads to a reduction or absence of the protein expression and/or activity of the F5H protein homolog as compared to the expression and/or activity of the protein produced by the corresponding wild type F5H gene homolog, wherein the presence of the modified F5H gene homolog in the plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homolog, and wherein the plant comprises a first modified F5H gene homolog called F5H1, the wild type of which has SEQ ID No. <NUM>, and a second modified F5H gene homolog called F5H2, the wild type of which has SEQ ID No. <NUM>.