PLANTS SHOWING A REDUCED WOUND-INDUCED SURFACE DISCOLORATION

Abstract

The present invention relates a plant which may comprise a modified F5H gene homolog, wherein said gene homolog may comprise a modification 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. The invention also relates to a modified F5H gene homolog that leads to the reduced wound-induced surface discoloration. The invention further relates to use of the gene in breeding and producing plants that show reduced wound-induced surface discoloration.

Description

SEQUENCE STATEMENT

The instant application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. Said ASCII copy, is named 00368SL.txt and is 695 kbytes in size.

FIELD OF THE INVENTION

The present invention relates to a plant that shows reduced wound-induced surface discoloration. The invention further relates to a modified gene homolog that leads to the reduced wound-induced surface discoloration in a plant, to its sequences and the use of these sequences for identifying the presence of the modified gene homolog. The invention also relates to the seeds and progeny of such plants and to propagation material for obtaining such plants.

BACKGROUND OF THE INVENTION

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 (Malta 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.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

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 which may comprise such modifications in their corresponding wild type F5H gene homologs. The F5H gene homologs code for Ferulate 5-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 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 plant which may comprise a modified F5H gene homolog, wherein said gene homolog may comprise a modification 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 which may comprise the modified F5H gene homolog.

Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U. S.C. § 112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

In the examples reference is made to the following figures:

FIG. 1: Leaf discs of the phenotypic test described in Example 2 at 3 days of incubation of Lactuca sativa samples of the WT and different mutants. Line 1 and line 8 are leaf disc samples taken from wild type lettuce plants, line 2 and line 9 are leaf disc samples taken from lettuce plants which may comprise mutation 1, line 3 are leaf disc samples taken from lettuce plants which may comprise mutation 1 and mutation 2, line 4 are leaf disc samples taken from lettuce plants which may comprise mutation 1 and mutation 3, line 5 are leaf disc samples taken from lettuce plants which may comprise mutation and mutation 4, line 6 are leaf disc samples taken from lettuce plants which may comprise mutation and mutation 5 and line 7 are leaf disc samples taken from lettuce plants which may comprise mutation and mutation 6. The mutations are represented in Table 3.

FIG. 2: Leaf discs of the phenotypic test described in Example 2 at 5 days of incubation of Lactuca sativa samples of the WT and different mutants. Line 1 and line 8 are leaf disc samples taken from wild type lettuce plants, line 2 and line 9 are leaf disc samples taken from lettuce plants which may comprise mutation 1, line 3 are leaf disc samples taken from lettuce plants which may comprise mutation 1 and mutation 2, line 4 are leaf disc samples taken from lettuce plants which may comprise mutation 1 and mutation 3, line 5 are leaf disc samples taken from lettuce plants which may comprise mutation and mutation 4, line 6 are leaf disc samples taken from lettuce plants which may comprise mutation and mutation 5 and line 7 are leaf disc samples taken from lettuce plants which may comprise mutation and mutation 6. The mutations are represented in Table 3.

FIG. 3: Leaf discs of the phenotypic test described in Example 2 at 10 days of incubation of Lactuca sativa samples of the WT and different mutants. Line 1 and line 8 are leaf disc samples taken from wild type lettuce plants, line 2 and line 9 are leaf disc samples taken from lettuce plants which may comprise mutation 1, line 3 are leaf disc samples taken from lettuce plants which may comprise mutation 1 and mutation 2, line 4 are leaf disc samples taken from lettuce plants which may comprise mutation 1 and mutation 3, line 5 are leaf disc samples taken from lettuce plants which may comprise mutation and mutation 4, line 6 are leaf disc samples taken from lettuce plants which may comprise mutation and mutation 5 and line 7 are leaf disc samples taken from lettuce plants which may comprise mutation and mutation 6. The mutations are represented in Table 3.

FIG. 4: Alignment of the ortholog proteins having the wild type sequences with the SEQ ID numbers listed in Table 2 (CLUSTAL multiple sequence alignment by MUSCLE (3.8)).

The motifs of Table 1 are highlighted in the sequences.

The following symbols are used below the alignment:

* —all residues in that column are identical

: —conserved substitutions have been observed

. —semi-conserved substitutions have been observed

—no match (space)

FIG. 5: Example of a scale used for the evaluation of wound-induced surface discoloration on leaf discs represented with a description of each score and a figure of a leaf disc having the given score.

FIG. 6: Leaf discs of a phenotypic test of the segregation analysis of the F2 lines of lettuce plants which may comprise mutation 1 and mutation 3, as described in Example 7. The pictures represent the leaf discs after 1, 2, 3, 4 and 7 days of incubation after sampling.

FIG. 7-1-7-3: Phenotypic analysis of whole lettuce heads which may comprise mutation 1 and mutation 3, as described in Example 8.

DEPOSITS

Seeds of lettuce plants (Lactuca sativa) of the invention that may 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 Feb. 19, 2016 under deposit accession the NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK on Feb. 19, 2016.

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.

The Deposits with the NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK on Feb. 19, 2016, under deposit accession the NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK on Feb. 19, 2016 were made pursuant to the terms of the Budapest Treaty. Upon issuance of a patent, all restrictions upon the deposit will be removed, and the deposit is intended to meet the requirements of 37 CFR §§ 1.801-1.809. The deposit will be irrevocably and without restriction or condition released to the public upon the issuance of a patent and for the enforceable life of the patent. The deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced if necessary during that period.

DETAILED DESCRIPTION OF THE INVENTION

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 which may comprise 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 which may comprise 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.

The present invention further provides the sequences of the modified F5H gene homologs in order to identify plants which may comprise 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 may comprise two F5H gene homologs. One homolog called herein F5H1, is located on chromosome 4 and has the wild type DNA coding sequence (CDS) represented in SEQ ID No: 115 and encodes the wild type F5H1 protein having SEQ ID No: 1. The other lettuce homolog herein called F5H2, is located on chromosome 3 and has the wild type DNA coding sequence represented in SEQ ID No: 116 and encodes the wild type F5H2 protein having SEQ ID No: 2.

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 may comprise two F5H gene homologs the wild type SEQ ID numbers of which are listed in Table 2. The species chicory (Cichorium intybus), endive (Cichorium endivia), celery and celeriac (Apium graveolens) and apple (Malus domestica) may comprise three F5H gene homologs the wild type SEQ ID numbers of which are listed in Table 2. The genome of banana (Musa acuminata) may comprise four F5H homologs the wild type SEQ ID numbers of which are listed in Table 2. The genomes of soy (Glycine max), pear (Pyrus x bretschneiden), wheat (Triticum aestivum), radish (Raphanus sativus) and cabbage and cauliflower (Brassica oleracea) may comprise five F5H homologs the wild type SEQ ID numbers of which are listed in Table 2, whereas the genomes of potato (Solanum tuberosum) and onion (Allium cepa) may comprise one F5H gene homolog in their genomes, the homolog the wild type SEQ ID numbers of which are listed in Table 2.

In a preferred embodiment, the invention relates to plants belonging to the genus Lactuca. In particular, the invention relates to a Lactuca sativa plant which may comprise two F5H gene homologs that are modified as compared to the nucleotide sequence of the wild type genes (SEQ ID No: 115 and 116), encoding the wild type proteins (SEQ ID No: 1 and 2). Other plants which may comprise 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, are also part of the invention.

In another embodiment, the invention relates to 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 may comprise 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.

In one embodiment, the invention relates to a modified F5H1 gene homolog having the sequence represented by SEQ ID No: 174, that leads to a reduction of wound-induced surface discoloration in comparison to a plant not which may comprise the modified F5H1 gene homolog.

In one embodiment, the invention relates to a modified F5H1 gene homolog having the sequence represented by SEQ ID No:174 and a modified F5H2 gene homolog having the sequence represented by SEQ ID No: 175, 176, 177, 178 or 179, leading to a reduction of wound-induced surface discoloration in comparison to a plant not which may comprise the modified F5H gene homologs.

The invention relates to a plant which may comprise a modified F5H gene homolog, wherein said gene homolog may comprise 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 which may comprise the modified F5H gene homolog.

In one embodiment the invention relates to a plant which may comprise a modified F5H gene homolog, wherein said gene homolog may comprise 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 which may comprise the modified F5H gene homolog and wherein the wild type F5H gene sequence is represented by any one of SEQ ID Nos: 58 to 114.

In a particular embodiment the invention relates to a plant which may comprise a modified F5H gene homolog of the invention, 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 2. It is not intended to claim a Arabidopsis thaliana plant which may comprise a modified F5H gene, in particular the one disclosed in the publication of Meyer et al. in National Academy of Sciences (1996) or in Huang et al. in Planta; an international journal of plant biology (2009), or a plant which may comprise the sequence of the NCBI database with the accession no. XP 011028697 (Predicted: cytochrome P450 84A1-like [Populus euphratica]), in this application.

In another embodiment the invention relates to a plant which may comprise 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 which may comprise the modified F5H gene homologs.

In a particular embodiment, the invention relates to a plant which may comprise 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 2.

In another embodiment the invention relates to a plant which may comprise 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 which may comprise the modified F5H gene homologs.

In a particular embodiment, the invention relates to a plant which may comprise 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 2.

In another embodiment the invention relates to a plant which may comprise 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 which may comprise the modified F5H gene homologs.

In a particular embodiment, the invention relates to a plant which may comprise 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 2.

In another embodiment the invention relates to a plant which may comprise 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 which may comprise the modified F5H gene homologs.

In a particular embodiment, the invention relates to a plant which may comprise 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 2.

The invention relates to edible plants such as vegetables, fruits and cereals which may comprise a modified F5H gene homolog, wherein said gene homolog may comprise 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 which may comprise the modified F5H gene homolog.

A “plant of the invention” as used herein, is a plant that may comprise a modified F5H gene homolog, wherein said gene homolog may comprise 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 which may comprise the modified F5H gene homolog. A plant of the invention is an edible plant such as a vegetable, fruit or cereal. Preferably the plant of the invention 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 2. Even more preferably the plant of the invention is a lettuce (Lactuca sativa) plant.

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 to which the invention relates are listed in Table 2. 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 1 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 of the invention may comprise the motifs represented in Table 1 with an identity percentage of at least 54%, more preferably with an identity percentage of at least 60%, even more preferably with an identity percentage of at least 65%, even more preferably with an identity percentage of at least 70%, even more preferably with an identity percentage of at least 75%, even more preferably with an identity percentage of at least 80%, even more preferably with an identity percentage of at least 85%, even more preferably with an identity percentage of at least 90%, even more preferably with an identity percentage of at least 95% and most preferably with an identity percentage of 100%. The consensus sequences of the five motifs are listed in the Table 1 and are highlighted in the alignment of the F5H protein orthologs in FIG. 4.

TABLE 1
Motifs present in all the F5H protein homolog
sequences
Number Motif
1      IFSNRPATIAISYLTYDRADMAFAHYGPFWRQMRKLCVM
SEQ    KLFSRKRAESW
ID
No:
169
2      DFKGSNFEFIPFGSGRRSCPGMQLGLYALEMAVAHLLHC
SEQ    FTWELPDGMKP
ID
No:
170
3      TRDNIKAIIMDVMFGGTETVASAIEWAMTELMHSPEDLK
SEQ    RVQQELADVVG
ID
No:
171
4      YLKCCIKETLRLHPPIPLLLHETAEDCEVAGYHIPKGSR
SEQ    VMINAWAIGRD
ID
No:
172
5      PYPPGPKGWPIIGNMLMMDQLTHRGLAKLAKQYGGICHL
SEQ    RMGFLHMVAVS
ID
No:
173

“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. The present invention thus provides for 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 to which the invention relates are listed in Table 2.

In this application a “gene” may comprise exonic sequences and regulatory sequences such as a promoter sequence, UTR and polyadenylation signals and if present it also may comprise 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 may comprise 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 of the invention that may comprise 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 which may comprise a modified F5H gene homolog” may comprise 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 F5H homolog that may comprise any modification that leads in a plant to reduced wound-induced surface discoloration. Preferably the modified F5H gene homolog of the invention may comprise a modification represented in Table 3. “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 relates to a plant of the invention, wherein reduction of the endogenous level of the F5H1 protein is due to a premature stop codon in the wild-type F5H1 sequences listed in Table 2.

The invention relates to a method for producing a plant exhibition reduced wound-induced surface discoloration, which may comprise reducing the endogenous level of F5H1 protein in the plant.

The invention further relates to a method for producing a plant exhibition reduced wound-induced surface discoloration, which may comprise 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 may comprise 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 may comprise 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.

In one embodiment the invention relates to a plant which may comprise 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 which may comprise the modified F5H gene homolog.

In a particular embodiment the invention relates to a plant which may comprise a modified F5H gene homolog, wherein said gene homolog may comprise 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 which may comprise the modified F5H gene homolog.

In one embodiment, the invention relates to a plant or plant part which may comprise a modified F5H1 gene homolog of the invention, wherein the modification leads to a reduction or absence of the protein expression of the F5H1 protein homolog as compared to the expression of the protein produced by the corresponding wild type F5H1 gene homolog.

In a particular embodiment, the invention relates to a Lactuca sativa plant or plant part which may comprise a modified F5H1 gene homolog, wherein the modification leads to a reduction or absence of the protein expression of the F5H1 protein homolog as compared to the expression of the protein produced by the corresponding wild type F5H1 gene homolog.

In one embodiment, the invention relates to a plant or plant part which may comprise a modified F5H2 gene homolog of the invention, wherein the modification leads to a reduction or absence or increase of the protein expression of the F5H2 protein homolog as compared to the expression of the protein produced by the corresponding wild type F5H2 gene homolog.

In a particular embodiment, the invention relates to a Lactuca sativa plant or plant part which may comprise a modified F5H2 gene homolog, wherein the modification leads to a reduction or absence or increase of the protein expression of the F5H2 protein homolog as compared to the expression of the protein produced by the corresponding wild type F5H2 gene homolog.

The invention further relates to a method for producing a plant of the invention, which may comprise reducing the endogenous level of F5H1 protein in the plant, 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 also relates to a plant exhibiting reduced wound-induced surface discoloration and showing a reduced F5H1 expression, wherein the reduction or absence is caused by a method described in this application.

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.

In one embodiment, the invention relates to a plant showing reduced wound-induced surface discoloration which may comprise a modified F5H gene homolog, wherein the modification leads to a reduction or absence of the protein activity of the F5H protein homolog as compared to the activity of the protein produced by the corresponding wild type F5H gene homolog.

In one particular embodiment, the invention relates to a plant showing reduced wound-induced surface discoloration which may comprise a modified F5H1 gene homolog, wherein the modification leads to a reduction or absence of the protein activity of the F5H1 protein homolog as compared to the activity of the protein produced by the corresponding wild type F5H1 gene homolog.

In one embodiment, the invention relates to a plant showing reduced wound-induced surface discoloration which may comprise a modified F5H2 gene homolog, wherein the modification leads to a reduction or absence of the protein activity of the F5H2 protein homolog as compared to the activity of the protein produced by the corresponding wild type F5H2 gene homolog.

The plants of the invention were created by using the mutagenic agent EMS, as described in Example 1. Plants grown from seeds treated one time with the mutagenic agent and selected for their ability to show reduced wound-induced surface discoloration, may 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. These types of modifications are also part of the invention.

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 3.

In one embodiment, the invention relates to a plant which may comprise a F5H gene homolog that may comprise a modification as compared to the corresponding wild type sequence, wherein the modification results in a different phenotype displaying reduced wound-induced surface discoloration.

In one embodiment, the invention relates to a plant which may comprise a F5H gene homolog that may comprise 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 which may comprise 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 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 and NCIMB 42551. The mutations are described in Table 3.

Seed of seed lot 16E.607_B01 was deposited with the NCIMB under accession number NCIMB 45546. The deposited seeds comprise mutation 1 in the F5H1 gene homolog.

Seed of seed lot 16E.607_B02 was deposited with the NCIMB under accession number NCIMB 42547. The deposited seeds comprise mutation 1 in the F5H1 gene homolog and mutation 2 in the F5H2 gene homolog.

Seed of seed lot 16E.607_B03 was deposited with the NCIMB under accession number NCIMB 42548. The deposited seeds comprise mutation 1 in the F5H1 gene homolog and mutation 3 in the F5H2 gene homolog.

Seed of seed lot 16E.607_B04 was deposited with the NCIMB under accession number NCIMB 42549. The deposited seeds comprise mutation 1 in the F5H1 gene homolog and mutation 4 in the F5H2 gene homolog.

Seed of seed lot 16E.607_B05 was deposited with the NCIMB under accession number NCIMB 42550. The deposited seeds comprise mutation 1 in the F5H1 gene homolog and mutation 5 in the F5H2 gene homolog.

Seed of seed lot 16E.607_B06 was deposited with the NCIMB under accession number NCIMB 42551. The deposited seeds comprise mutation 1 in the F5H1 gene homolog and mutation 6 in the F5H2 gene homolog.

The lettuce F5H1 gene homolog which may comprise mutation 1 is represented by SEQ ID No: 174.

The lettuce F5H2 gene homolog which may comprise mutation 2 is represented by SEQ ID No: 175.

The lettuce F5H2 gene homolog which may comprise mutation 3 is represented by SEQ ID No: 176.

The lettuce F5H2 gene homolog which may comprise mutation 4 is represented by SEQ ID No: 177.

The lettuce F5H2 gene homolog which may comprise mutation 5 is represented by SEQ ID No: 178.

The lettuce F5H2 gene homolog which may comprise mutation 6 is represented by SEQ ID No: 179.

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 1 and optionally a mutation in the F5H2 gene homolog such as mutation 3 or mutation 6.

The invention relates to a plant that may comprise a modified F5H gene homolog wherein said gene homolog may comprise 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 which may comprise the modified F5H gene homolog. The invention relates to a plant which may comprise a modified F5H gene of the invention, wherein the modification leads to a premature stop codon.

The invention relates to a Lactuca sativa plant which may comprise a first modified F5H gene homolog called F5H1, the wild type of which has SEQ ID No. 115, and optionally a second modified F5H gene homolog called F5H2, the wild type of which has SEQ ID No. 116.

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 may comprise a premature stop codon.

The invention further relates to a Lactuca sativa plant, wherein the modified F5H1 gene may comprise a premature stop codon that is caused by a mutation C>T at position 370 of SEQ ID No: 115.

In one embodiment, the invention relates to another plant of the invention listed in FIG. 4 and the premature stop codon is caused by a mutation at a position that corresponds to position 370 of SEQ ID No:115 in Lactuca sativa.

The invention further relates to a Lactuca sativa plant, which may comprise a first modified F5H gene homolog called F5H1, the wild type of which has SEQ ID No. 115, and optionally a second modified F5H gene homolog called F5H2, the wild type of which has SEQ ID No. 116, wherein the modified F5H2 gene encodes a protein having one or more amino acid substitutions.

The invention further relates to a Lactuca sativa plant, which may comprise a first modified F5H gene homolog called F5H1, the wild type of which has SEQ ID No. 115, and optionally a second modified F5H gene homolog called F5H2, the wild type of which has SEQ ID No. 116, wherein the modified F5H2 gene encodes a protein having an amino acid substitution of Threonine to Isoleucine at position 154 of the encoded F5H2 protein of SEQ ID No: 2, and/or an amino acid substitution of Glycine to Glutamic acid at position 165 of the encoded F5H2 protein of SEQ ID No: 2, and/or an amino acid substitution of Serine to Phenylalanine at position 308 of the encoded F5H2 protein of SEQ ID No: 2, and/or an amino acid substitution of Glycine to Glutamic acid at position 434 of the encoded F5H2 protein of SEQ ID No: 2, and/or an amino acid substitution of Glycine to Glutamic acid at position 436 of the encoded F5H2 protein of SEQ ID No: 2.

The invention further relates to a Lactuca sativa plant, which may comprise a first modified F5H gene homolog called F5H1, the wild type of which has SEQ ID No. 115, and optionally a second modified F5H gene homolog called F5H2, the wild type of which has SEQ ID No. 116, wherein the amino acid substitution of Threonine to Isoleucine at position 154 of the encoded F5H2 protein is the result of a nucleotide change C>T at position 461 of SEQ ID No: 116, the amino acid substitution of Glycine to Glutamic acid at position 165 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 494 of SEQ ID No: 116, the amino acid substitution of Serine to Phenylalanine at position 308 of the encoded F5H2 protein is the result of a nucleotide change C>T at position 923 of SEQ ID No: 116, the amino acid substitution of Glycine to Glutamic acid at position 434 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 1301 of SEQ ID No: 116 and the amino acid substitution of Glycine to Glutamic acid at position 436 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 1307 of SEQ ID No: 116.

In one embodiment the invention relates to another plant of the invention listed in FIG. 4 and the amino acid substitution is at a position that corresponds to the position in Lactuca sativa.

The invention also relates to a Lactuca sativa plant, which may comprise a modified F5H1 gene, which gene may comprise a premature stop codon and a F5H2 gene which may comprise an amino acid substitution.

The invention relates to a Lactuca sativa plant, which may comprise a modified F5H1 gene, which gene may comprise a premature stop codon and a F5H2 gene which may comprise an amino acid substitution, wherein the premature stop codon in the F5H1 gene is caused by a mutation C>T at position 370 of SEQ ID No: 115 and the amino acid substitution in the F5H2 gene selected from Threonine to Isoleucine at position 154 of the encoded F5H2 protein is the result of a nucleotide change C>T at position 461 of SEQ ID No: 116, the amino acid substitution of Glycine to Glutamic acid at position 165 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 494 of SEQ ID No: 116, the amino acid substitution of Serine to Phenylalanine at position 308 of the encoded F5H2 protein is the result of a nucleotide change C>T at position 923 of SEQ ID No: 116, the amino acid substitution of Glycine to Glutamic acid at position 434 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 1301 of SEQ ID No: 116 or the amino acid substitution of Glycine to Glutamic acid at position 436 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 1307 of SEQ ID No: 116.

In one embodiment, the invention relates to a Lactuca sativa plant of the invention, which may comprise a modified F5H1 gene, which gene may comprise a premature stop codon and a F5H2 gene which may comprise an amino acid substitution, wherein the premature stop codon in the F5H1 gene is caused by a mutation C>T at position 370 of SEQ ID No: 115 and the amino acid substitution in the F5H2 gene Glycine to Serine at position 159 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 475 of SEQ ID No: 116.

In particular, the invention relates to a Lactuca sativa plant that may comprise 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 which may comprise these mutations shows an delayed wound-induced surface discoloration. Therefore, a plant which may comprise a modified F5H1 gene homolog and a modified F5H2 gene homolog shows delayed wound-induced surface discoloration, as compared to a plant which may comprise 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 which may comprise 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 may comprise the mutation(s) in a homozygous state, heterozygous state or do not may comprise 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 may comprise the mutation 1 on the F5H1 homolog (C₃₇₀>T₃₇₀ in the F5H1 gene homolog) preferably in a homozygous state. Plants that may comprise the mutation 1 in the F5H1 homolog in a homozygous state and a mutation in the F5H2 gene homolog such as the mutation 3 in a homozygous or heterozygous state show a reduced wound-induced surface discoloration as compared to plants which may comprise only mutation 1 in the F5H1 gene homolog.

In one embodiment the invention relates to a plant which may comprise a modification to the F5H1 gene homolog of a Lactuca sativa plant as indicated in Table 3.

In yet a further embodiment the invention relates to a combination of mutation 1 of the F5H1 gene homolog and one or more of mutation 2, 3, 4, 5 and 6 of the F5H2 gene homolog of a Lactuca sativa plant as indicated in Table 3.

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

The invention further relates to the use of a modified F5H gene homolog 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 and the invention should thus not be limited to the specific modifications described in this application but extends to all other modifications to the gene and/or protein leading to reduced wound-induced surface discoloration. 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 which may comprise 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 to which relates the invention 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 according to the invention, that F5H protein homologs may comprise five conserved motifs as listed in Table 1 and highlighted in the protein alignment (FIG. 4), 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 434 in the lettuce F5H2 protein (SEQ ID No: 2), which is encoded by G₁₃₀₀Gn₁₃₀₁A₁₃₀₂ of the lettuce F5H2 DNA sequence (SEQ ID No: 116), with a Glutamic acid residue, which is encoded by G₁₃₀₀A₁₃₀₁A₁₃₀₂, leads in combination with mutation 1 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 which may comprise only mutation 1.

More in particular, the substitution of a highly conserved Serine residue at position 308 in the lettuce F5H2 protein (SEQ ID No: 2), which is encoded by T₉₂₂C₉₂₃T₉₂₄ of the lettuce F5H2 DNA sequence (SEQ ID No: 116), with a Phenylalanine residue, which is encoded by T₉₂₂T₉₂₃T₉₂₄, leads in combination with mutation 1 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 which may comprise only mutation 1.

More in particular, the substitution of a highly conserved Glycine residue at position in the lettuce F5H2 protein (SEQ ID No: 2), which is encoded by G₁₃₀₆G₁₃₀₇A₁₃₀₈ of the lettuce F5H2 DNA sequence (SEQ ID No: 116), with a Glutamic acid residue, which is encoded by G₁₃₀₆A₁₃₀₇A₁₃₀₈, leads in combination with mutation 1 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 which may comprise only mutation 1.

The present invention 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: 1 and 2) 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 2. Multiple sequence alignments of the protein sequences using CLUSTAL confirmed that the candidate genes were orthologous F5H genes (FIG. 4). 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.

The invention thus also relates to 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, which may comprise 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 may comprise screening a plant or a population of plants for the presence of a modified F5H gene homolog 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 2 and/or the use of molecular markers as characterized in Example 3. Both methods may be used to directly or indirectly detect and select the plants exhibiting a reduction of the wound-induced surface discoloration and which may comprise 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 which may comprise a modified F5H gene homolog.

The trait of the invention can phenotypically be determined in a leaf disc test as described in Example 2 may comprise 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 7.5° C., 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 2 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 9 to 0, wherein 9 means that no discoloration on the edges is visible, score 8 means that the leaf disc has a very slight pink discoloration around the edges, score 5 means that the leaf disc has a thin ring of red/pink discoloration around the edges, score 2 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 9, 8, 7, 6, 5, 4 or 3 and score 0 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. 5. 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 3, 5 and 10 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 9 or score 8. A plant which may comprise a modified F5H1 gene and a modified F5H2 gene has a higher score than a plant which may comprise 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 5-6° C. in a cool cell in plastic bags. After 1, 2, 3, 4, 7 and 10 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 9 to 4, wherein 9 means that no signs of wound-induced surface discoloration are visible, 8 means that first little traces of pink discoloration (nearly not visible, only a glow) are visible, 7 means the pink discoloration is appearing on some edges, 6 means that the pink discoloration is visible on all cutting edges, 5 means that a strong pink discoloration is visible on all cutting edges and 4 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.

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 marker is a mutation in any of the wild type sequences, the SEQ ID numbers of which are shown in Table 2.

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 a nucleotide change of C>T at position 370 of SEQ ID No: 115.

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 154 of the encoded protein as a result of a change C>T at position 461 of SEQ ID No: 116, and/or an amino acid substitution of Glycine to Glutamic acid at position 165 of the encoded protein as a result of a change G>A at position 494 of SEQ ID No: 116 and/or an amino acid substitution of Serine to Phenylalanine at position 308 of the encoded protein as a result of a change C>T at position 923 of SEQ ID No: 116, and/or an amino acid substitution of Glycine to Glutamic acid at position 434 of the encoded protein as a result of a change G>A at position 1301 of SEQ ID No: 116, and/or an amino acid substitution Glycine to Glutamic acid at position 436 of the encoded protein as a result of a change G>A SNP at position 1307 of SEQ ID No: 116.

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, which may comprise:

a) isolating DNA from a plant and from one or both parents of said plant;

b) screening for molecular markers in a region of said DNA at or near sequence corresponding to SEQ ID Nos: 174, 175, 176, 177, 178 or 179; and

c) determining co-inheritance of said markers with the reduced wound-induced phenotype from one or both parents of said plant.

The invention also relates to 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 C₃₇₀>T₃₇₀ SNP in the F5H1 gene of lettuce (Lactuca sativa) represented in Table 5 (Example 3).

In a particular embodiment, one suitable molecular marker is the C₄₆₁>T₄₆₁ SNP in the F5H2 gene of lettuce (Lactuca sativa) represented in Table 6 (Example 3).

In a particular embodiment, one suitable molecular marker is the G₄₉₄>A₄₉₄ SNP in the F5H2 gene of lettuce (Lactuca sativa) represented in Table 6 (Example 3).

In a particular embodiment, one suitable molecular marker is the C₉₂₃>T₉₂₃ SNP in the F5H2 gene of lettuce (Lactuca sativa) represented in Table 6 (Example 3).

In a particular embodiment, one suitable molecular marker is the G₁₃₀₁>A₁₃₀₁ SNP in the F5H2 gene of lettuce (Lactuca sativa) represented in Table 6 (Example 3).

In a particular embodiment, one suitable molecular marker is the G₁₃₀₇>A₁₃₀₇ SNP in the F5H2 gene of lettuce (Lactuca sativa) represented in Table 6 (Example 3).

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 which may comprise 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, which may comprise 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 which may comprise 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 2.

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 may comprise 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.

The invention also relates to a plant grown or regenerated from the said propagation material, which plant may comprise in its genome one or more modified F5H genes as defined herein, providing the plant with reduced wound-induced surface discoloration.

In particular, the plant produced from the propagation material may comprise 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 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 and NCIMB 42551.

The invention also relates to the use of a plant of the invention that may comprise a modified F5H gene, which provides to a plant the reduction of wound-induced surface discoloration, in plant breeding to confer this trait.

The invention relates to a method for producing a plant showing reduced wound-induced surface discoloration which may comprise:

a) crossing a plant which may comprise a modified F5H gene homolog of claim 1, with another plant;

b) optionally performing one or more rounds of selfing and/or crossing; and

c) optionally selecting after each round of selfing or crossing for a plant that may comprise said reduced wound-induced surface discoloration.

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

In one aspect the invention relates to a method for production of a plant that shows reduced wound-induced surface discoloration, which may comprise

a) crossing a plant which may comprise a modified F5H gene homolog of the invention that leads to the trait with another plant;

b) selfing the resulting F1 for obtaining F2 plants;

c) selecting plants that have the trait in the F2;

d) optionally performing one or more additional rounds of selfing or crossing, and subsequently selecting, for a plant which may comprise/showing the trait of the invention.

In one aspect, the invention relates to a method for production of a plant that shows reduced wound-induced surface discoloration, which may comprise

a) crossing a plant which may comprise a modified F5H gene homolog of the invention that leads to the trait with another plant;

b) optionally backcrossing the resulting F1 with the preferred parent;

c) selecting for plants that have the trait in the F2;

d) optionally performing one or more additional rounds of selfing or crossing, and subsequently selecting, for a plant which may comprise the trait.

The invention additionally provides a method of introducing another desired trait into a plant which has the trait of the invention, which may comprise:

a) crossing a plant that may comprise a modified F5H gene homolog of the invention and that shows reduced wound-induced surface discoloration, with a second plant that may comprise a desired trait to produce F1 progeny;

b) selecting an F1 progeny plant that may comprise said trait of reduced wound-induced surface discoloration and the desired trait;

c) crossing the selected F1 progeny with either parent, to produce backcross progeny;

d) selecting backcross progeny which may comprise the desired trait and showing reduced wound-induced surface discoloration; and

e) optionally repeating steps c) and d) one or more times in succession to produce selected fourth or higher backcross progeny that may comprise a modified F5H gene homolog and that shows reduced wound-induced surface discoloration. The invention includes a plant produced by this method.

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 3. In another aspect selection for the trait of the invention 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.

In one embodiment selection for plants that show reduced wound-induced surface discoloration is started in the F3 or a later generation.

In one embodiment the plant which may comprise a F5H gene homolog of the invention is a plant of an inbred line, a hybrid, a doubled haploid, or of a segregating population.

The invention further provides 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 which may comprise the said trait.

The invention further relates to 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 which may comprise 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.

The invention further relates to a method for producing a hybrid plant that that shows reduced wound-induced surface discoloration, which may comprise 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.

The invention also relates to a method of producing a hybrid plant seed which may comprise 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 may comprise a modified F5H gene homolog of the invention.

The invention also relates to a method for the production of a plant that shows reduced wound-induced surface discoloration, which may comprise growing the plant from a seed that may comprise 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 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 and NCIMB 42551.

The invention also relates to a method for seed production which may comprise growing plants from seeds of which a representative sample was deposited with the NCIMB under accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 and NCIMB 42551, allowing the plants to produce seeds, and harvesting those seeds. Production of the seeds is suitably done by crossing or selfing.

The invention relates to a method for the production of a plant that shows reduced wound-induced surface discoloration by tissue culture using a plant of the invention as described herein as the source of the tissue.

The invention furthermore relates to a method for the production of a plant that shows reduced wound-induced surface discoloration by vegetative reproduction of parts of a plant of the invention as described herein.

In one embodiment, the invention relates to 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. Genetic modification may comprise 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.

The invention also relates to a breeding method for the development of plants that show reduced wound-induced surface discoloration wherein germplasm which may comprise said trait is used. Representative seed of said plant which may comprise the modified F5H gene homolog and being representative for the germplasm was deposited with the NCIMB under accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 and NCIMB 42551.

In a further embodiment the invention relates to a method for the production of a plant that shows reduced wound-induced surface discoloration wherein progeny or propagation material of a plant which may comprise 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 which may comprise the modified F5H gene homolog was deposited with the NCIMB under accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 and NCIMB 42551.

The invention provides preferably 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.

The invention further relates to a method for producing an agronomically elite plant that shows the reduced wound-induced surface discoloration of the invention, which may comprise 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. The invention also includes a plant produced by this method.

In yet a further embodiment the agronomically elite plant of the invention 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 F₁ hybrid variety.

In one embodiment the plant of the invention, i.e. a plant which may comprise the modified F5H gene of the invention, is 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 which may comprise 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 may comprise a modified F5H gene homolog in its genome.

The invention further relates to a cell of a plant of the invention, which cell may comprise 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.

The invention also relates to 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 19 Feb. 2016 under NCIMB and having one of the accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550, or NCIMB 42551.

The invention also relates to a cell of a plant, which plant may comprise a modified F5H gene and shows reduced wound-induced surface discoloration.

The invention also relates to a cell of a plant that may comprise a modified F5H gene and that shows reduced wound-induced surface discoloration, which plant is obtainable by crossing a plant which may comprise a modified F5H gene and selecting for a plant that shows a reduced wound-induced surface discoloration.

The invention also relates to 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 19 Feb. 2016 under NCIMB and having one of the accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550, or NCIMB 42551, which lettuce plant is obtainable by crossing a lettuce plant with a lettuce plant grown from seed as deposited with the NCIMB on 19 Feb. 2016 under NCIMB and having one of the accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550, or NCIMB 42551, and selecting for a lettuce plant that shows a reduced wound-induced surface discoloration.

The invention also relates to the use of seeds that were deposited under NCIMB under accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 and NCIMB 42551, for developing plants showing reduced wound-induced surface discoloration into another lettuce plant (Lactuca sativa).

The invention also relates to the use of the seeds of which a representative sample was deposited under NCIMB under accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 and NCIMB 42551 for transferring the reduced wound-induced surface discoloration trait into another agronomically valuable lettuce plant.

The invention also relates to the use of a plant that may comprise a modified F5H gene homolog of the invention and shows reduced wound-induced surface discoloration, as a crop.

In particular the invention relates to 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 19 Feb. 2016 and having one of the accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 or NCIMB 42551, as a crop.

The invention also relates to the use of a plant that may comprise a modified F5H gene homolog of the invention and shows reduced wound-induced surface discoloration, as a source of seed.

In particular the invention relates to 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 19 Feb. 2016 and having one of the accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 or NCIMB 42551, as a source of seed.

The invention also relates to the use of a plant that may comprise a modified F5H gene homolog of the invention and shows reduced wound-induced surface discoloration, as a source of propagation.

In particular the invention relates to 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 19 Feb. 2016 and having one of the accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 or NCIMB 42551, as a source of propagating material.

The invention also relates to the use of a plant that may comprise a modified F5H gene homolog of the invention and shows reduced wound-induced surface discoloration, for consumption.

In particular the invention relates to 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 19 Feb. 2016 and having one of the accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, 42550 or NCIMB 42551, for consumption. The invention also relates to the use of a modified F5H gene homolog of the invention, for conferring reduced wound-induced surface discoloration to a plant.

In particular, the invention relates to the use of a F5H gene homolog as found in seeds that were deposited with the NCIMB on 19 Feb. 2016 and having one of the accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 or NCIMB 42551, for conferring reduced wound-induced surface discoloration to a lettuce plant (Lactuca sativa).

The invention relates to the use of a plant as a recipient of modified F5H gene homologs of the invention.

In particular, the invention relates to 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 19 Feb. 2016 and having one of the accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 or NCIMB 42551.

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

In particular, the invention relates to the use of a modified F5H gene homolog as found in seeds that were deposited with the NCIMB on 19 Feb. 2016 and having one of the accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550, or NCIMB 42551, conferring reduced wound-induced surface discoloration to a lettuce plant (Lactuca sativa).

The invention also relates to the use of seeds that were deposited with the NCIMB on 19 Feb. 2016 and having one of the accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550, or NCIMB 42551, for transferring reduced wound-induced surface discoloration into another lettuce plant (Lactuca sativa).

TABLE 2
Overview of the F5H orthologs. The overview indicates
which SEQ ID Nos are linked to which plant species.
		Common		SEQ
Name	Species	Name	Detail	ID No
>Lettuce_LsF5H_1_1	Lactuca sativa	Lettuce	Protein	1
>Lettuce_LsF5H_2_1	Lactuca sativa	Lettuce	Protein	2
>Endive_ce_gene1	Cichorium	Endive	Protein	3
	endivia
>Endive_ce_gene2	Cichorium	Endive	Protein	4
	endivia
>Endive_ce_kethel_v0.1_EVM71979	Cichorium	Endive	Protein	5
	endivia
>Chicory_ci_vitessa_fr_v1_EVM170737	Cichorium	Chicory	Protein	6
	intybus
>Chicory_ci_vitessa_fr_v1_EVM170780	Cichorium	Chicory	Protein	7
	intybus
>Chicory_ci_vitessa_fr_v1_EVM158591	Cichorium	Chicory	Protein	8
	intybus
>Celery_RZ_draft_99.605_EVM363634	Apium	Celery/Celariac	Protein	11
	graveolens
>Celery_RZ_draft_99.605_EVM348724	Apium	Celery/Celariac	Protein	12
	graveolens
>Celery_c17480_g1_i1|m.26831	Apium	Celery/Celariac	Protein	13
	graveolens
>Brassica_Bolg005770	Brassica	Cabbage/	Protein	14
	oleracea	Cauliflower
>Brassica_Bo7g117840	Brassica	Cabbage/	Protein	15
	oleracea	Cauliflower
>Brassica_Bo7g119430	Brassica	Cabbage/	Protein	16
	oleracea	Cauliflower
>Brassica_Bo3g093960	Brassica	Cabbage/	Protein	17
	oleracea	Cauliflower
>Brassica_XP_013607401.1	Brassica	Cabbage/	Protein	18
	oleracea	Cauliflower
>Eggplant_c18725_g1_i1|m.19489	Solanum	Eggplant	Protein	19
	melongena
>Eggplant_sm_67_3_v1_EVM112823_1—	Solanum	Eggplant	Protein	20
	melongena
>Potato_XP_006340697.1	Solanum	Potato	Protein	21
	tuberosum
>Apple_XP_008372753.1	Malus	Apple	Protein	22
	domestica
>Apple_XP_008337913.1	Malus	Apple	Protein	23
	domestica
>Apple_XP_008391587.1	Malus	Apple	Protein	24
	domestica
>Pear_XP_009378215.1	Pyrus ×	Pear	Protein	25
	bretschneideri
>Pear_XP_009338655.1	Pyrus ×	Pear	Protein	26
	bretschneideri
>Pear_AGR44939.1	Pyrus ×	Pear	Protein	27
	bretschneideri
>Pear_XP_009346304.1	Pyrus ×	Pear	Protein	28
	bretschneideri
>Pear_XP_009378429.1	Pyrus ×	Pear	Protein	29
	bretschneideri
>Peach_XP_007199246.1	Prunus persica	Peach	Protein	30
>Peach_XP_007203643.1	Prumis persica	Peach	Protein	31
>Banana_XP_009384541.1	Musa	Banana	Protein	32
	acuminata
>Banana_XP_009384087.1	Musa	Banana	Protein	33
	acuminata
>Banana_XP_009411495.1	Musa	Banana	Protein	34
	acuminata
>Banana_XP_009403617.1	Musa	Banana	Protein	35
	acuminata
>Wheat_A0A077RPS5	Triticum	Wheat	Protein	36
	aestivum
>Wheat_W5A2I1	Triticum	Wheat	Protein	37
	aestivum
>Wheat_W5B6W3	Triticum	Wheat	Protein	38
	aestivum
>Wheat_A0A077RQ37	Triticum	Wheat	Protein	39
	aestivum
>Wheat_W5AC21	Triticum	Wheat	Protein	40
	aestivum
>Rice_Os03g0112900	Oryza sativa	Rice	Protein	41
>Rice_Os10g0512400	Oryza sativa	Rice	Protein	42
>Maize_A0A0B4J2X1	Zea mays	Corn	Protein	43
>Maize_B4FWF9	Zea mays	Corn	Protein	44
>Soybean_G3E7M3	Glycine max	Soy	Protein	45
>Soybean_I1J8P0	Glycine max	Soy	Protein	46
>Soybean_Q2LAL3	Glycine max	Soy	Protein	47
>Soybean_K7MH28	Glycine max	Soy	Protein	48
>Soybean_I1LHY5	Glycine max	Soy	Protein	49
>Artichoke_KVI02897.1	Cynara	Artichoke	Protein	50
	cardunculus
	var. scolymus
>Artichoke_KVH92322.1	Cynara	Artichoke	Protein	51
	cardunculus
	var. scolymus
>Radish_GSRAST00026355001	Raphanus	Radish	Protein	52
	sativus
>Radish_GSRAST00007419001	Raphanus	Radish	Protein	53
	sativus
>Radish_GSRAST00001088001	Raphanus	Radish	Protein	54
	sativus
>Radish_GSRAST00042054001	Raphanus	Radish	Protein	55
	sativus
>Radish_rs_Aokubi_v1_EVM13601	Raphanus	Radish	Protein	56
	sativus
>Onion_AC.SP3B.Locus_5396.1.10	Allium cepa	Onion	Protein	57
>Lettuce_LsF5H_1_1	Lactuca sativa	Lettuce	Genomic	58
			DNA
>Lettuce_LsF5H_2_1	Lactuca sativa	Lettuce	Genomic	59
			DNA
>Endive_ce_gene1	Cichorium	Endive	Genomic	60
	endivia		DNA
>Endive_ce_gene2	Cichorium	Endive	CDS	61
	endivia
>Endive_ce_kethel_v0.1_EVM71979	Cichorium	Endive	Genomic	62
	endivia		DNA
>Chicory_ci_vitessa_fr_v1_EVM170737	Cichorium	Chicory	Genomic	63
	intybus		DNA
>Chicory_ci_vitessa_fr_v1_EVM170780	Cichorium	Chicory	Genomic	64
	intybus		DNA
>Chicory_ci_vitessa_fr_v1_EVM158591	Cichorium	Chicory	Genomic	65
	intybus		DNA
>Celery_RZ_draft_99.605_EVM363634	Apium	Celery/Celariac	Genomic	68
	graveolens		DNA
>Celery_RZ_draft_99.605_EVM348724	Apium	Celery/Celariac	Genomic	69
	graveolens		DNA
>Celery_c17480_g1_i1|m.26831	Apium	Celery/Celariac	CDS	70
	graveolens
>Brassica_Bo1g005770	Brassica	Cabbage/	Genomic	71
	oleracea	Cauliflower	DNA
>Brassica_Bo7g117840	Brassica	Cabbage/	Genomic	72
	oleracea	Cauliflower	DNA
>Brassica_Bo7g119430	Brassica	Cabbage/	Genomic	73
	oleracea	Cauliflower	DNA
>Brassica_Bo3g093960	Brassica	Cabbage/	Genomic	74
	oleracea	Cauliflower	DNA
>Brassica_XP_013607401.1	Brassica	Cabbage/	Genomic	75
	oleracea	Cauliflower	DNA
>Eggplant_c18725_g1_i1|m. 19489	Solanum	Eggplant	CDS	76
	melongena
>Eggplant_sm_67_3_v1_EVM112823_1—	Solanum	Eggplant	Genomic	77
	melongena		DNA
>Potato_XP_006340697.1	Solanum	Potato	Genomic	78
	tuberosum		DNA
>Apple_XP_008372753.1	Malus	Apple	Genomic	79
	domestica		DNA
>Apple_XP_008337913.1	Malus	Apple	Genomic	80
	domestica		DNA
>Apple_XP_008391587.1	Malus	Apple	Genomic	81
	domestica		DNA
>Pear_XP_009378215.1	Pyrus ×	Pear	Genomic	82
	bretschneideri		DNA
>Pear_XP_009338655.1	Pyrus ×	Pear	Genomic	83
	bretschneideri		DNA
>Pear_AGR44939.1	Pyrus ×	Pear	CDS	84
	bretschneideri
>Pear_XP_009346304.1	Pyrus ×	Pear	Genomic	85
	bretschneideri		DNA
>Pear_XP_009378429.1	Pyrus ×	Pear	Genomic	86
	bretschneideri		DNA
>Peach_XP_007199246.1	Prunus persica	Peach	Genomic	87
			DNA
>Peach_XP_007203643.1	Prunus persica	Peach	Genomic	88
			DNA
>Banana_XP_009384541.1	Musa	Banana	Genomic	89
	acuminata		DNA
>Banana_XP_009384087.1	Musa	Banana	Genomic	90
	acuminata		DNA
>Banana_XP_009411495.1	Musa	Banana	Genomic	91
	acuminata		DNA
>Banana_XP_009403617.1	Musa	Banana	Genomic	92
	acuminata		DNA
>Wheat_A0A077RPS5	Triticum	Wheat	Genomic	93
	aestivum		DNA
>Wheat_W5A2I1	Triticum	Wheat	Genomic	94
	aestivum		DNA
>Wheat_W5B6W3	Triticum	Wheat	Genomic	95
	aestivum		DNA
>Wheat_A0A077RQ37	Triticum	Wheat	Genomic	96
	aestivum		DNA
>Wheat_W5AC21	Triticum	Wheat	Genomic	97
	aestivum		DNA
>Rice_Os03g0112900	Oryza sativa	Rice	Genomic	98
	Japonica		DNA
>Rice_Os10g0512400	Oryza sativa	Rice	Genomic	99
	Japonica		DNA
>Maize_A0A0B4J2X1	Zea mays	Corn	Genomic	100
			DNA
>Maize_B4FWF9	Zea mays	Corn	Genomic	101
			DNA
>Soybean_G3E7M3	Glycine max	Soy	Genomic	102
			DNA
>Soybean_I1J8P0	Glycine max	Soy	Genomic	103
			DNA
>Soybean_Q2LAL3	Glycine max	Soy	Genomic	104
			DNA
>Soybean_K7MH28	Glycine max	Soy	Protein	105
>Soybean_I1LHY5	Glycine max	Soy	Genomic	106
			DNA
>Artichoke_KVI02897.1	Cynara	Artichoke	Genomic	107
	cardunculus		DNA
	var. scolymus
>Artichoke_KVH92322.1	Cynara	Artichoke	Genomic	108
	cardunculus		DNA
	var. scolymus
>Radish_GSRAST00026355001	Raphanus	Radish	Genomic	109
	sativus		DNA
>Radish_GSRAST00007419001	Raphanus	Radish	Genomic	110
	sativus		DNA
>Radish_GSRAST00001088001	Raphanus	Radish	Genomic	111
	sativus		DNA
>Radish_GSRAST00042054001	Raphanus	Radish	Genomic	112
	sativus		DNA
>Radish_rs_Aokubi_v1_EVM13601	Raphanus	Radish	Genomic	113
	sativus		DNA
>Onion_AC.SP3B.Locus_5396.1.10	Allium cepa	Onion	Genomic	114
			DNA

TABLE 3
Modifications of the F5H1 and F5H2 gene homologs of Lactuca sativa
Mutation			Pos. CDS
number	F5H gene	Nt. change	Nt. change	Codon change	AA change	AA change Pos.
1	F5H1	C > T	370	C370A371A372 >	Gln > stop	124
				T370A371A372
2	F5H2	C > T	461	A460C461C462 >	Thr > Ile	154
				A460T461C462
3	F5H2	G > A	494	G493G494A495 >	Gly > Glu	165
				G493A494A495
4	F5H2	C > T	923	T922C923T924 >	Ser > Phe	308
				T922T923T924
5	F5H2	G > A	1301	G1300G1301A1302 >	Gly > Glu	434
				G1300A1301A1302
6	F5H2	G > A	1307	G1306G1307A1308 >	Gly > Glu	436
				G1306A1307A1308
(AA = Amino acid, Nt = Nucleotide, CDS = DNA coding sequence, Pos. = Position)

Table 3 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: 115 (F5H1 CDS sequence wild type), SEQ ID No: 116 (F5H2 CDS sequence wild type), SEQ ID No: 1 (F5H1 protein sequence wild type) and SEQ ID No: 2 (F5H2 protein sequence wild type).

Lengthy table referenced here
US20200165624A1-20200528-T00001
Please refer to the end of the specification for access instructions.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

EXAMPLES

Example 1: Creation of a Lactuca sativa Plant Mutant by Treatment with EMS

In order to create Lactuca sativa plant mutants, approximately 2000 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 0.05% (w/v) or 0.07% (w/v) EMS for 24 hours at room temperature. Following EMS treatment, the M1 seeds were rinsed with water, germinated and grown in a greenhouse at 20° C. at 16 hours light, 8 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 2.

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 3 to 5 days incubation of a leaf disc sample in the phenotypic test described in Example 2 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 5000 seeds of the line 11K200310, in the same manner as previously described in the first EMS treatment, but using a concentration of 0.14% 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 2.

Example 2: Phenotypic Identification of a Plant that Show Reduced Wound-Induced Surface Discoloration

Plants from Example 1 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 6 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 2 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 7.5° C. 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 9 to 0, wherein 9 means that the edges of the leaf disc have no discoloration, score 8 means that the leaf disc has a very slight pink discoloration around the edges, score 5 means that the leaf disc has a thin ring of red/pink discoloration around the edges, score 2 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 9, 8, 7, 6, 5, 4 or 3 and score 0 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 3, 7 and 10 after sampling are shown in FIGS. 1, 2 and 3 and the scores are represented in Table 4; At day 3 (FIG. 1) the WT showed wound-induced surface discoloration (score 0), the plants comprising mutation 1 showed a reduced wound-induced surface discoloration (score 7) as compared to the wound-induced surface discoloration of the wild type plants and plants comprising mutation 1 and mutation 2, 3, 4, 5 or 6 did not show any wound-induced surface discoloration (score 9). At day 5 (FIG. 2) the WT showed wound-induced surface discoloration (score 0), the plants that were treated one time with EMS and had mutation 1 in their F5H1 gene homolog (Table 3) showed a reduced wound-induced surface discoloration (score 2) as compared to the WT (score 0) and plants that have been treated two rounds of EMS and had a mutation 1 in their F5H1 gene homolog (Table 3) and mutation 2, 3, 4, 5 or 6 in their F5H2 gene homolog (Table 3) did not show any wound-induced surface discoloration or a reduced wound-induced surface discoloration as compared to the plants comprising mutation 1 (scores 7 to 9). At day 10 (FIG. 3) the plants that were treated one time with EMS and had mutation 1 in their F5H1 gene homolog showed a wound-induced surface discoloration (score 0) and plants that have been treated two rounds of EMS and had a mutation 1 in their F5H1 gene homolog (Table 3) and mutation 2, 3, 4, 5 or 6 in their F5H2 gene homolog (Table 3) did not show any wound-induced surface discoloration or a reduced wound-induced surface discoloration as compared to a plant comprising mutation 1 (scores 5 to 9).

Those plants were selfed in order to produce seeds, which were then deposited with the NCIMB under accession numbers accession numbers NCIMB 42546, NCIMB 42547, NCIMB 42548, NCIMB 42549, NCIMB 42550 and NCIMB 42551.

TABLE 4
Scores of the leaf discs for phenotypic identification of a
plant that show reduced wound-induced surface discoloration
by at 3, 4, 5, 6, 7 and 10 days after sampling the leaf discs
		3	4	5	6	7	10
Line	Plant	days	days	days	days	days	days
1	Wild type	0	0	0	0	0	0
2	Mutation 1	7	5	2	0	0	0
3	Mutation 1 +	9	9	8	7	7	5
	Mutation 2
4	Mutation 1 +	9	9	9	9	9	9
	Mutation 3
5	Mutation 1 +	9	9	7	7	5	5
	Mutation 4
6	Mutation 1 +	9	9	9	9	9	9
	Mutation 5
7	Mutation 1 +	9	9	9	9	9	9
	Mutation 6
8	Wild type	0	0	0	0	0	0
9	Mutation 1	7	5	2	0	0	0

Example 3: Modifications of the Lactuca sativa F5H Homologs

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 4, 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: 1. The different mutations in lettuce F5H gene on chromosome 3, herein referred to as F5H2 resulted in amino acid changes in the sequence of the corresponding wild type proteins represented in SEQ ID No:2. 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 5 and Table 6 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 50 flanking nucleotides on either side.

TABLE 5
Sequence data of SNP mutations in the F5H1 gene
homolog of Lactuca sativa.
Desig-
nation	WT Sequence
WT	CTTACAACGGCGTAGATTTGGCTTTTGCTAATT
SEQ ID	ATGGACCTTTCTGGCGACAAATGCGAAAGCTT
No: 143	TGTGTCATGAAGCTGTTCAGCCGGAAACGAGC
	GGAG
Desig-		SNP
nation	SNP Sequence	Position
1	CTTACAACGGCGTAGATTTGGCTTTTGCTAATT	F5H1
SEQ ID	ATGGACCTTTCTGGCGATAAATGCGAAAGCTT	Chr.4
No: 117	TGTGTCATGAAGCTGTTCAGCCGGAAACGAGC	370 bp
	GGAG

TABLE 6
Sequence data of SNP mutations in the F5H2 gene
homolog of Lactuca sativa
Desig-
nation	WT Sequence
WT	TGAGTCTTGGGACTCCGTCAGAGACGAAGTTG
SEQ ID	TCTCCATGGTCAAAATCACCGCTGCAAGCTCC
No: 150	GGCACCGCTGTTAACCTTGGAGAGCTTGTTTT
	CGGGT
Desig-		SNP
nation	SNP Sequence	Position
8	TGAGTCTTGGGACTCCGTCAGAGACGAAGTTG	F5H2
SEQ ID	TCTCCATGGTCAAAATCATCGCTGCAAGCTCC	Chr. 3
No: 124	GGCACCGCTGTTAACCTTGGAGAGCTTGTTTT	461 bp
	CGGGT
Desig-
nation	WT Sequence
WT	CTCCATGGTCAAAATCACCGCTGCAAGCTCCG
SEQ ID	GCACCGCTGTTAACCTTGGAGAGCTTGTTTTC
No: 151	GGGTTAACCCATGATATCATTTACCGAGCAGC
	TTTCG
Desig-		SNP
nation	SNP Sequence	Position
9	CTCCATGGTCAAAATCACCGCTGCAAGCTCCG	F5H2
SEQ ID	GCACCGCTGTTAACCTTGAAGAGCTTGTTTTC	Chr. 3
No: 125	GGGTTAACCCATGATATCATTTACCGAGCAGC	494 bp
	TTTCG
Desig-
nation	WT Sequence
WT	CAAAGCCATTATTATGGATGTAATGTTTGGTG
SEQ ID	GGACTGAAACTGTTGCTTCTGCTATCGAATGG
No: 152	GCTTTAACTGAGCTAATGCACACCCCAGAATC
	CTTAA
Desig-		SNP
nation	SNP Sequence	Position
10	CAAAGCCATTATTATGGATGTAATGTTTGGTG	F5H2
SEQ ID	GGACTGAAACTGTTGCTTTTGCTATCGAATGG	Chr. 3
No: 126	GCTTTAACTGAGCTAATGCACACCCCAGAATC	923 bp
	CTTAA
Desig-
nation	WT Sequence
WT	AGATGGGGCACCCGACTTTAAAGGAAGCAATT
SEQ ID	ATGAGTTTCTTCCATTTGGATCTGGACGTAGA
No: 153	TCATGTCCTGGAATGCAACTTGGATTGTACGC
	AATGG
Desig-		SNP
nation	SNP Sequence	Position
11	AGATGGGGCACCCGACTTTAAAGGAAGCAATT	F5H2
SEQ ID	ATGAGTTTCTTCCATTTGAATCTGGACGTAGAT	Chr. 3
No: 127	CATGTCCTGGAATGCAACTTGGATTGTACGCA	1301 bp
	ATGG
Desig-
nation	WT Sequence
WT	GGCACCCGACTTTAAAGGAAGCAATTATGAGT
SEQ ID	TTCTTCCATTTGGATCTGGACGTAGATCATGTC
No: 154	CTGGAATGCAACTTGGATTGTACGCAATGGAG
	ATGG
Desig-		SNP
nation	SNP Sequence	Position
12	GGCACCCGACTTTAAAGGAAGCAATTATGAGT	F5H2
SEQ ID	TTCTTCCATTTGGATCTGAACGTAGATCATGTC	Chr. 3
No: 128	CTGGAATGCAACTTGGATTGTACGCAATGGAG	1307 bp
	ATGG

Example 4: Identification of F5H Gene Orthologs and Conserved Regions

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.

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.

TABLE 7
List of plant species that carry a F5H gene ortholog and number of
gene orthologs present in the genome of these plants. The SEQ ID
numbers of the F5H gene ortholog sequences are listed in Table 2.
                                 Number of F5H gene
Plant crops                      orthologs
Potato (Solanum tuberosum)       1
Onion (Allium cepa)              1
Artichocke (Cynara cardunculus var. Scolymus) 2
Rice (Oryza sativa)              2
Corn (Zea mays)                  2
Peach (Prunus persica)           2
Eggplant (Solanum melongena)     2
Chicory (Cichorium intybus)      3
Endive (Cichorium endivia)       3
Celery and celeriac (Apium graveolens) 3
Apple (Malus domestica)          3
Banana (Musa acuminata)          4
Soy (Glycine max)                5
Pear (Pyrus × bretschneideri)    5
Wheat (Triticum aestivum)        5
Radish (Raphanus sativus)        5
Cabbage and cauliflower (Brassica oleracea) 5

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. 4 are listed in Table 1.

Example 5: Development of New Plants Having the Trait of the Invention

A lettuce plant numbered 15E238260 showing reduced wound-induced discoloration after 10 days of incubation and found by the phenotypic test described in Example 2 was selfed. By use of DNA-marker tests based on SNPs markers in Example 3 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 2 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 2. Three out of each sixteen F2-plants were expected to show reduced wound-induced surface discoloration after 3 to 5 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 7.5° C. as described in Example 2.

The F2 plants that showed a reduced wound-induced surface discoloration after 10 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 2), 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.

Example 6: Introgression of the Trait of the Invention by Backcrossing

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 2 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. 20 BC2-seeds were sown and each of them was selfed to generate BC2.S1-seeds. These 20 BC2.S1-seed lots (i.e. BC2. S1families) were sown, 20 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 20 plants per BC2.S1-family was tested with the phenotypic test as described in Example 2 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. This BC2.S1-plant was also selfed to produce BC2. S2-seeds. Ten BC2. S2-seeds were grown into plants and tested in the phenotypic test as described in Example 2, to confirm the reduced wound-induced discoloration in all of them, after 10 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. 20 BC4-seeds were sown and each of them was selfed to generate BC4.S1-seeds. These 20 BC4.S1-seed lots (i.e. BC4.S1 families) were sown, 20 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 20 plants per BC4.S1-family was tested with the wound-induced discoloration test as described in Example 2 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. This BC4.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 3. The genotype of each selected plant could also be confirmed by using molecular markers.

The BC4.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.

Example 7: Segregation Analysis of the Trait of the Invention

A lettuce plant numbered 15E238260 showing reduced wound-induced discoloration and comprising mutation 1 (C₃₇₀>T₃₇₀ in the F5H1 gene homolog) and mutation 3 (G₄₉₄>A₄₉₄ 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 2 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 8. The reduced wound-induced surface discoloration was evaluated by using the leaf disc test as described in Example 2, but for the segregation analysis, the leaf discs were taken from young plants (approximately 2 weeks old). The score for each leaf disc is represented in Table 9 and as an example the leafs discs of a lettuce plant comprising mutation 1 and mutation 3 are represented in FIG. 6.

TABLE 8
Representation of the genotype of the plants used
for the segregation analysis represented in FIG. 6
Mutation 1	Mutation 3	Genotype
Homozygously absent	Homozygously absent	AA/AA
Heterozygously present	Homozygously absent	AB/AA
Homozygously absent	Heterozygously present	AA/AB
Heterozygously present	Heterozygously present	AB/AB
Homozygously present	Heterozygously present	BB/AB
Heterozygously present	Homozygously present	AB/BB
Homozygously absent	Homozygously present	AA/BB
Homozygously present	Homozygously absent	BB/AA
Homozygously present	Homozygously present	BB/BB

TABLE 9
Phenotypic analysis of the F2 population of plants
comprising mutation 1 and mutation 3 The genotypes
of the plants are represented in Table 8:
Genotype
Mutation	Mutation	Phenotype (Scores)
1	3	1 Day	2 Days	3 Days	4 Days
AA	AA	9	2	0	0
AB	AA	9	2	1	0
AA	AB	9	2	1	0.5
AB	AB	9	2	1	0
AB	BB	9	2	0	0
AA	BB	9	1	1	0
BB	AA	9	8	8	7
BB	BB	9	9	9	9
BB	AB	9	9	9	8

The results in Table 9 show that in order to show reduced wound-induced surface discoloration the plant needed to carry mutation 1 on the F5H1 gene homolog (C₃₇₀>T₃₇₀ in the F5H1 gene homolog). Preferably the plant carried mutation 1 homozygously. A plant that carried mutation 1 homozygously (BB) and mutation 3 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 1. The most reduced wound-induced surface discoloration was visible in a plant comprising mutation 1 in the F5H1 gene homozygously and mutation 3 in F5H2 gene homozygously (BB/BB).

Example 8: Phenotypic Analysis of Whole Lettuce Heads

The lettuce plants were harvested at maturity stage (approximately 3 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 5° C. over night in boxes that were wrapped in plastic to avoid drying out. The plants were then cut under cold temperature (around 15° C.). Old leaves, leaves showing tipburn symptoms and outer leaves were removed from the heads. Two or three plant heads comprising mutation 1, mutation 1 and mutation 2, mutation 1 and mutation 3, mutation 1 and mutation 4, mutation 1 and mutation 5, mutation 1 and mutation 6 and a wild type plant (Troubadour) were vertically cut in four parts to remove the core and the four parts were further cut 2 or 3 times horizontally. For the phenotypic analysis 100 grams of the cut lettuce leaves were washed with a washing machine (Washing step with air bubbles: 3 min, time of centrifuge: 2.5 min 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 2 times and stored in boxes and stored at 5-6° C. The bags were not stocked on top of each other.

An example of the phenotypic analysis on whole lettuce heads is shown in FIG. 7 for the wild type lettuce variety Troubadour (wild type) that does not comprise a modified F5H gene homolog, a lettuce plant comprising mutation 1 (C₃₇₀>T₃₇₀ in the F5H1 gene homolog) (“Mutation 1”) and a lettuce plant comprising mutation 1 and mutation 3 (G₄₉₄>A₄₉₄ in the F5H2 gene homolog) (“Mutation 1 and mutation 3”). The pictures were taken after 7 and 10 days after after washing. The wild type plant showed wound-induced surface discoloration and the cutting edges after 7 days, the plant comprising mutation 1 showed a reduced wound-induced surface discoloration at the cutting edges after 7 or 10 days as compared to the wild type, the plant comprising mutation 1 and mutation 3 do not show any wound-induced surface discoloration even after 10 days and has therefore a reduced wound-induced surface discoloration as compared to plants comprising mutation 1.

Another analysis has been performed with an evaluation of the wound-induced surface discoloration at 3, 7 and 14 days after washing. The results are represented in Table 10. A plant that may comprise mutation 1 showed reduced wound-induced surface discoloration as compared to the wild type. Plants that comprise mutation 1 and mutation 3, 4, 5 or 6 showed reduced wound-induced surface discoloration as compared to the wild-type and the plant comprising mutation 1. Plants comprising mutation 1 and mutation 2 showed the same reduction in wound induced surface discoloration as a plant comprising mutation 1 at 14 days, but the wound-induced surface discoloration develops slower than in a plant comprising mutation 1 as at 7 Days a plant comprising mutation 1 has a score of 7 and a plant comprising mutation 1 and mutation 2 scores 8.

TABLE 10
Phenotypic analysis of whole lettuce heads after
cutting the heads, washing the leaves and storing
the leaves for 3, 7 and 9 or 14 days at 5-6° C.:
	Days of storage
	Plant type	3 Days	7 Days	14 Days
	Wild type	9	5	4
	Mutation 1	9	7	5
	Mutation 1 + mutation 2	9	8	5
	Mutation 1 + mutation 3	9	9	7.5
	Mutation 1 + mutation 4	9	8	6.5
	Mutation 1 + mutation 5	9	8.5	6
	Mutation 1 + mutation 6	9	8	7

The invention is further described by the following numbered paragraphs:

1. 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 the plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homolog.

2. Plant of paragraph 1, wherein the wild type F5H gene sequence is represented by any one of SEQ ID Nos: 58 to 114.

3. Plant of any of the paragraphs 1-2, 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 2.

4. Plant of any one of the paragraphs 1-2, wherein the plant comprises two modified F5H gene homologs.

5. Plant of paragraph 4, 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 2.

6. Plant of any one of the paragraphs 1-2, wherein the plant comprises three modified F5H gene homologs.

7. Plant of paragraph 6, 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 2.

8. Plant of any one of the paragraphs 1-2, wherein the plant comprises four modified F5H gene homologs.

9. Plant of paragraph 8, wherein the plant is selected from the group consisting ofMusa 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 2.

10. Plant of any one of the paragraphs 1-2, wherein the plant comprises five or more modified F5H gene homologs.

11. Plant of paragraph 10, 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 2.

12. Plant of any one of the paragraphs 1-11, wherein the modification leads to a reduction or absence of the protein expression of the F5H1 protein homolog as compared to the expression of the protein produced by the corresponding wild type F5H1 gene homolog.

13. Plant of any one of the paragraphs 1-12, wherein the modification leads to a reduction or absence of the protein activity of the F5H1 protein homolog as compared to the activity of the protein produced by the corresponding wild type F5H1 gene homolog.

14. Plant of any one of the paragraphs 1-13, wherein the modification leads to a premature stop codon.

15. Plant of any one of the paragraphs 1-5 and 12-14, wherein the plant is a Lactuca sativa plant and comprises a first modified F5H gene homolog called F5H1, the wild type of which has SEQ ID No. 115, and optionally a second modified F5H gene homolog called F5H2, the wild type of which has SEQ ID No. 116.

16. Plant of paragraph 15, wherein the modified F5H1 gene homolog is homozygouly present and the modified F5H2 gene homolog is either heterozygously or homozygously present.

17. Plant as paragraphed any one of the paragraphs 15-16, wherein the modified F5H1 gene comprises a premature stop codon.

18. Plant of paragraph 17, wherein the plant is a Lactuca sativa plant and the premature stop codon is caused by a mutation C>T at position 370 of SEQ ID No: 115.

19. Plant according to paragraph 17, wherein the plant is another plant listed in FIG. 4 and the premature stop codon is caused by a mutation at a position that corresponds to position 370 of SEQ ID No:115 in Lactuca sativa.

20. Plant of any one of the paragraphs 15-19, wherein the modified F5H2 gene encodes a protein having one or more amino acid substitutions.

21. Plant of any one of the paragraphs 15-20, wherein the plant is a Lactuca sativa plant and the modified F5H2 gene comprises a mutation resulting in an amino acid substitution of Threonine to Isoleucine at position 154 of the encoded F5H2 protein of SEQ ID No: 2, and/or an amino acid substitution of Glycine to Glutamic acid at position 165 of the encoded F5H2 protein of SEQ ID No: 2, and/or an amino acid substitution of Serine to Phenylalanine at position 308 of the encoded F5H2 protein of SEQ ID No: 2, and/or an amino acid substitution of Glycine to Glutamic acid at position 434 of the encoded F5H2 protein of SEQ ID No: 2, and/or an amino acid substitution of Glycine to Glutamic acid at position 436 of the encoded F5H2 protein of SEQ ID No: 2.

22. Plant of paragraph 21, wherein the amino acid substitution of Threonine to Isoleucine at position 154 of the encoded F5H2 protein is the result of a nucleotide change C>T at position 461 of SEQ ID No: 116, the amino acid substitution of Glycine to Glutamic acid at position 165 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 494 of SEQ ID No: 116, the amino acid substitution of Serine to Phenylalanine at position 308 of the encoded F5H2 protein is the result of a nucleotide change C>T at position 923 of SEQ ID No: 116, the amino acid substitution of Glycine to Glutamic acid at position 434 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 1301 of SEQ ID No: 116 and the amino acid substitution of Glycine to Glutamic acid at position 436 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 1307 of SEQ ID No: 116.

23. Plants according to paragraph 20 or 22, wherein the plant is another plant listed in FIG. 4 and the amino acid substitution is at a position that corresponds to the position in Lactuca sativa.

24. Part of a plant of any one of the paragraphs 1-23, wherein the part is a leaf, a whole head of a plant, a fruit, an inflorescence, a seed, a curd, a stem, a tuber, a bulb or a root, optionally in processed form.

25. A food product comprising a part of a plant of paragraph 24.

26. A seed capable of developing into a plant of any one of the paragraphs 1-23.

27. Seed of a plant of any one of the paragraphs 1-23, wherein the seed comprises a modified F5H gene homolog in its genome.

28. Propagation material capable of developing into and/or derived from a plant of any one of the paragraphs 1-23, wherein the propagation material is selected from the group consisting of 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.

29. A modified F5H gene homolog as defined in any one of the paragraphs 1-23 that confers reduced wound-induced surface discoloration to the plant.

30. Use of a modified F5H gene homolog as defined in any one of the paragraphs 1-23 for the development of a plant exhibiting reduced wound-induced surface discoloration.

31. A plant of any of the paragraphs 1-23, wherein reduction of the endogenous level of the F5H1 protein is due to a premature stop codon in the wild-type F5H sequences listed in Table 2.

32. Method for producing a plant exhibiting reduced wound-induced surface discoloration, comprising reducing the endogenous level of F5H1 protein in the plant.

33. Method of paragraph 32, wherein the endogenous level of F5H1 protein in the plant is reduced by mutating a wild type F5H1 gene homolog.

34. The method of paragraphs 32 or 33, wherein the mutation is effected by CRISPR, by a chemical agent, radiation, or a combination thereof.

35. The method of paragraph 32 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.

36. The method of any one of the paragraphs 32-35, wherein the wild type F5H1 gene homolog or homologs have the nucleotide sequence and corresponding amino acid sequence of which the SEQ ID numbers are listed in Table 2.

37. A plant comprising a reduced F5H1 expression, wherein the reduction is caused by one of the methods of paragraphs 32-36.

38. 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 as described in any one of the paragraphs 1-23, optionally applying a phenotypic test to identify plants showing reduced wound-induced surface discoloration, and selecting a plant showing reduced wound-induced surface discoloration.

39. A molecular marker for detecting in the genome of a plant a mutation causative of reduced wound-induced surface discoloration in said plant or a part thereof, wherein the marker is a mutation in any of the F5H wild type sequences, the SEQ ID numbers of which wild type sequences are shown in Table 2.

40. Molecular marker of paragraph 39, wherein the mutation is a nucleotide change of C>T at position 370 of SEQ ID No: 115.

41. Molecular marker of paragraph 39, wherein the mutation is an amino acid substitution of Threonine to Isoleucine at position 154 of the encoded protein as a result of a change C>T at position 461 of SEQ ID No: 116, and/or an amino acid substitution of Glycine to Glutamic acid at position 165 of the encoded protein as a result of a change G>A at position 494 of SEQ ID No: 116 and/or an amino acid substitution of Serine to Phenylalanine at position 308 of the encoded protein as a result of a change C>T at position 923 of SEQ ID No: 116, and/or an amino acid substitution of Glycine to Glutamic acid at position 434 of the encoded protein as a result of a change G>A at position 1301 of SEQ ID No: 116, and/or an amino acid substitution of Glycine to Glutamic acid at position 436 of the encoded protein as a result of a change G>A at position 1307 of SEQ ID No: 116.

42. Use of a molecular marker of any of the paragraphs 39-41, to identify or develop a plant showing reduced wound-induced surface discoloration, or develop other markers linked to a modified F5H gene homolog as defined in any of the paragraphs 1-23.

43. A method for identifying molecular markers linked to reduced wound-induced surface discoloration of a plant, comprising:

a) isolating DNA from a plant and from one or both parents of said plant;

b) screening for molecular markers in a region of said DNA at or near a sequence corresponding to SEQ ID Nos: 175, 176, 177, 178 or 179.

c) determining co-inheritance of said markers with the reduced wound-induced surface discoloration phenotype from one or both parents of said plant.

44. A method for producing a plant showing reduced wound-induced surface discoloration comprising:

(a) crossing a plant comprising a modified F5H gene homolog of paragraph 1, with another plant;

(b) optionally performing one or more rounds of selfing and/or crossing; and

(c) optionally selecting after each round of selfing or crossing for a plant that comprises said reduced wound-induced surface discoloration.

45. Method of paragraph 44, wherein the plant is phenotypically selected and/or selected by use of molecular markers.

46. 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 as defined in paragraph 1.

47. A method of determining the presence of a modified F5H gene homolog in a plant according to paragraph 1, 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 F5H gene homolog.

48. Method of paragraph 47, wherein the wild type F5H gene homolog is any one of the sequences of which the SEQ ID numbers are listed in Table 2.

Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

LENGTHY TABLES
The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

Claims

1. 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 the plant leads to a reduction of wound-induced surface discoloration in comparison to a plant not comprising the modified F5H gene homolog.

2. The plant as claimed in claim 1, wherein the wild type F5H gene sequence is represented by any one of SEQ ID Nos: 58 to 114.

3. The plant as claimed in claim 1, 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 2.

4. The plant as claimed in claim 1, wherein the plant comprises two modified F5H gene homologs.

5. The plant as claimed in claim 4, 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 2.

6. The plant as claimed in claim 1, wherein the plant comprises three modified F5H gene homologs.

7. The plant as claimed in claim 6, 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 2.

8. The plant as claimed in claim 1, wherein the plant comprises four modified F5H gene homologs.

9. The plant as claimed in claim 8, 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 2.

10. The plant as claimed in claim 1, wherein the plant comprises five or more modified F5H gene homologs.

11. The plant as claimed in claim 10, 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 2.

12. The plant as claimed in claim 1, wherein the modification leads to a reduction or absence of the protein expression of the F5H1 protein homolog as compared to the expression of the protein produced by the corresponding wild type F5H1 gene homolog.

13. The plant as claimed in claim 1, wherein the modification leads to a reduction or absence of the protein activity of the F5H1 protein homolog as compared to the activity of the protein produced by the corresponding wild type F5H1 gene homolog.

14. The plant as claimed in claim 1, wherein the modification leads to a premature stop codon.

15. The plant as claimed in claim 1, wherein the plant is a Lactuca sativa plant and comprises a first modified F5H gene homolog called F5H1, the wild type of which has SEQ ID No. 115, and optionally a second modified F5H gene homolog called F5H2, the wild type of which has SEQ ID No. 116.

16. The plant as claimed in claim 15, wherein the modified F5H1 gene homolog is homozygouly present and the modified F5H2 gene homolog is either heterozygously or homozygously present.

17. The plant as claimed in claim 15, wherein the modified F5H1 gene comprises a premature stop codon.

18. The plant as claimed in claim 17, wherein the plant is a Lactuca sativa plant and the premature stop codon is caused by a mutation C>Tat position 370 of SEQ ID No: 115.

19. The plant according to claim 17, wherein the plant is another plant listed in FIG. 4 and the premature stop codon is caused by a mutation at a position that corresponds to position 370 of SEQ ID No:115 in Lactuca sativa.

20. The plant as claimed in claim 15, wherein the modified F5H2 gene encodes a protein having one or more amino acid substitutions.

21. The plant as claimed in claim 15, wherein the plant is a Lactuca sativa plant and the modified F5H2 gene comprises a mutation resulting in an amino acid substitution of Threonine to Isoleucine at position 154 of the encoded F5H2 protein of SEQ ID No: 2, and/or an amino acid substitution of Glycine to Glutamic acid at position 165 of the encoded F5H2 protein of SEQ ID No: 2, and/or an amino acid substitution of Serine to Phenylalanine at position 308 of the encoded F5H2 protein of SEQ ID No: 2, and/or an amino acid substitution of Glycine to Glutamic acid at position 434 of the encoded F5H2 protein of SEQ ID No: 2, and/or an amino acid substitution of Glycine to Glutamic acid at position 436 of the encoded F5H2 protein of SEQ ID No: 2.

22. The plant as claimed in claim 21, wherein the amino acid substitution of Threonine to Isoleucine at position 154 of the encoded F5H2 protein is the result of a nucleotide change C>Tat position 461 of SEQ ID No: 116, the amino acid substitution of Glycine to Glutamic acid at position 165 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 494 of SEQ ID No: 116, the amino acid substitution of Serine to Phenylalanine at position 308 of the encoded F5H2 protein is the result of a nucleotide change C>Tat position 923 of SEQ ID No: 116, the amino acid substitution of Glycine to Glutamic acid at position 434 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 1301 of SEQ ID No: 116 and the amino acid substitution of Glycine to Glutamic acid at position 436 of the encoded F5H2 protein is the result of a nucleotide change G>A at position 1307 of SEQ ID No: 116.

23. The plants according to claim 20, wherein the plant is another plant listed in FIG. 4 and the amino acid substitution is at a position that corresponds to the position in Lactuca sativa.

24. A part of a plant as claimed in claim 1, wherein the part is a leaf, a whole head of a plant, a fruit, an inflorescence, a seed, a curd, a stem, a tuber, a bulb or a root, optionally in processed form.

25. A food product comprising a part of a plant as claimed in claim 24.

26. A seed capable of developing into a plant as claimed in claim 1.

27. A seed of a plant as claimed in claim 1, wherein the seed comprises a modified F5H gene homolog in its genome.

28. A propagation material capable of developing into and/or derived from a plant as claimed in claim 1, wherein the propagation material is selected from the group consisting of 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

29. A modified F5H gene homolog as defined in claim 1 that confers reduced wound-induced surface discoloration to the plant.

30. A plant as claimed in claim 1, wherein reduction of the endogenous level of the F5H1 protein is due to a premature stop codon in the wild-type F5H sequences listed in Table 2.

31. A method for producing a plant exhibiting reduced wound-induced surface discoloration, comprising reducing the endogenous level of F5H1 protein in the plant.

32. The method of claim 31, wherein the endogenous level of F5H1 protein in the plant is reduced by mutating a wild type F5H1 gene homolog.

33. The method of claim 31, wherein the mutation is effected by CRISPR, by a chemical agent, radiation, or a combination thereof.

34. The method of claim 31 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.

35. The method as claimed in claim 31, wherein the wild type F5H1 gene homolog or homologs have the nucleotide sequence and corresponding amino acid sequence of which the SEQ ID numbers are listed in Table 2.

36. A plant comprising a reduced F5H1 expression, wherein the reduction is caused the method of claim 31.

37. 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 as described in claim 1, optionally applying a phenotypic test to identify plants showing reduced wound-induced surface discoloration, and selecting a plant showing reduced wound-induced surface discoloration.

38. A molecular marker for detecting in the genome of a plant a mutation causative of reduced wound-induced surface discoloration in said plant or a part thereof, wherein the marker is a mutation in any of the F5H wild type sequences, the SEQ ID numbers of which wild type sequences are shown in Table 2.

39. The molecular marker as claimed in claim 38, wherein the mutation is a nucleotide change of C>Tat position 370 of SEQ ID No: 115.

40. The molecular marker as claimed in claim 38, wherein the mutation is an amino acid substitution of Threonine to Isoleucine at position 154 of the encoded protein as a result of a change C>T at position 461 of SEQ ID No: 116, and/or an amino acid substitution of Glycine to Glutamic acid at position 165 of the encoded protein as a result of a change G>A at position 494 of SEQ ID No: 116 and/or an amino acid substitution of Serine to Phenylalanine at position 308 of the encoded protein as a result of a change C>Tat position 923 of SEQ ID No: 116, and/or an amino acid substitution of Glycine to Glutamic acid at position 434 of the encoded protein as a result of a change G>A at position 1301 of SEQ ID No: 116, and/or an amino acid substitution of Glycine to Glutamic acid at position 436 of the encoded protein as a result of a change G>A at position 1307 of SEQ ID No: 116.

41. A method for identifying molecular markers linked to reduced wound-induced surface discoloration of a plant, comprising: a) isolating DNA from a plant and from one or both parents of said plant;b) screening for molecular markers in a region of said DNA at or near a sequence corresponding to SEQ ID Nos: 175, 176, 177, 178 or 179.c) determining co-inheritance of said markers with the reduced wound-induced surface discoloration phenotype from one or both parents of said plant.

42. A method for producing a plant showing reduced wound-induced surface discoloration comprising: (a) crossing a plant comprising a modified F5H gene homolog of claim 1, with another plant;(b) optionally performing one or more rounds of selfing and/or crossing; and(c) optionally selecting after each round of selfing or crossing for a plant that comprises said reduced wound-induced surface discoloration.

43. The method of claim 42, wherein the plant is phenotypically selected and/or selected by use of molecular markers.

44. 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 as defined in claim 1.

45. A method of determining the presence of a modified F5H gene homolog in a plant according to claim 1, 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 F5H gene homolog.

46. The method of claim 45, wherein the wild type F5H gene homolog is any one of the sequences of which the SEQ ID numbers are listed in Table 2.