Reduced browning in banana fruit

Abstract

The present invention relates to compositions and methods for the reduced browning in banana fruit by genetically editing a Polyphenol Oxidase (PPO).

Description

[0001] REDUCED BROWNING IN BANANA FRUIT

[0002] FIELD OF THE INVENTION

[0003] The present invention, according to some embodiments, relates to gene-edited banana plants characterised by reduced browning in banana fruit flesh, and the associated method of making such banana plants. The reduced browning in banana fruit flesh is achieved by generating edits in the gene encoding Polyphenol Oxidase 1 (PPO1), with those edits resulting in premature stop codons leading to the expression of truncated proteins.

[0004] BACKGROUND OF THE INVENTION

[0005] Cultivated bananas are giant herbaceous plants within the genus Musa. They are both sterile and parthenocarpic, so the fruit develops without seed.

[0006] During harvesting and packaging, bruising of bananas can occur which can give rise to notable wastage since bruised bananas are frequently discarded rather than being exported. Further, when banana fruit becomes over-ripe, brown, or spoiled it is often discarded by consumers, thus increasing waste and lowering its market value. There is therefore a need to control fruit browning in banana. In particular, the ability to reduce fruit browning would facilitate the harvesting / transport of banana fruit and would improve banana fruit shelf life. Hence it may subsequently, for example, be more practical to incorporate banana in pre-made fruit salad.

[0007] Polyphenol oxidases (PPOs), as described in detail herein, are enzymes found throughout the plant and animal kingdoms, including in most fruits and vegetables, such as banana. PPOs are important in the food industry because they catalyse enzymatic browning when tissues are damaged from bruising or compression, making produce less marketable and causing economic loss. Enzymatic browning due to PPO action can also lead to loss of nutritional content of produce, further lowering its value. Exposure to oxygen when fruit is sliced or pureed also leads to enzymatic browning by PPOs. Accordingly, it would be desirable to develop banana plants with a reduced level or activity of targeted PPOs active in fruit, in an effort to reduce fruit browning.

[0008] As has been described in Tropic Biosciences’ earlier patent application, WO 2023 / 275255 (incorporated herein by reference), reducing the expression of PPO genes to reduce PPO level or activity by introducing targeted genetic changes is an efficient way of inhibiting banana browning.The present invention is based, in part, on the inventors’ work to generate a banana plant gene edited in each copy of the PPO1 gene, with the inventors identifying that plants with such edits have truncated PPO1 proteins that result in a particularly useful reduced browning phenotype in banana fruit flesh. According to the present invention, there is provided banana plant cells, and the plant and banana fruit itself, in which each copy of the PPO1 gene is edited to generate stop codons at particular locations in said genes, which results in banana fruits with reduced browning in the banana fruit flesh.

[0009] SUMMARY OF THE INVENTION

[0010] The banana plant cells of the invention are gene-edited banana plant cells, with the associated banana plants and banana fruits of the invention also being gene-edited.

[0011] In one aspect, the invention provides a banana plant cell, wherein the banana plant cell comprises three copies of a PPO1 gene, and wherein:

[0012] • a first copy of the PPO1 gene comprises a premature stop-codon TGA (SEQ ID NO:

[0013] 29) in a nucleotide sequence of the PPO1 gene comprising TGACCT (SEQ ID NO: 31);

[0014] • a second copy of the PPO1 gene comprises a premature stop-codon TAG (SEQ ID NO: 30) in a nucleotide sequence of the PPO1 gene comprising TAGGCA (SEQ ID NO: 32); and

[0015] • a third copy of the PPO1 gene comprises a premature stop-codon TGA (SEQ ID NO:

[0016] 29) in a nucleotide sequence of the PPO1 gene comprising TGAGGG (SEQ ID NO: 33).

[0017] In some embodiments, each copy of the PPO1 gene comprises one or more indel, wherein the indel causes a frameshift in the wildtype PPO1 gene sequence leading to each premature stop-codon. In a particular embodiment, the indel is a deletion of 1, 2, 4, 5 or 7 nucleotides or an addition of 1, 2, 4, 5 or 7 nucleotides. In a further embodiment, the one or more indel is at a polynucleotide sequence of GAGACCA (SEQ ID NO: 34) of the PPO1 gene and / or is at a polynucleotide sequence of ACCCGTC (SEQ ID NO: 35) of the PPO1 gene. In a specific embodiment, the one or more indel is selected from the group consisting of:

[0018] • an insertion of any one nucleotide in GAGACCA (SEQ ID NO: 34) of the PPO1 gene, preferably the insertion of any one nucleotide after position 3 or 4 in GAGACCA (SEQ ID NO: 34), most preferably the insertion of an adenine;• a deletion of GAGACCA (SEQ ID NO: 34) of the PP01 gene; or

[0019] • a deletion of any one nucleotide from ACCCGTC (SEQ ID NO: 35) of the PPO1 gene, preferably the deletion of a cytosine, most preferably the cytosine at position 4 from ACCCGTC (SEQ ID NO: 35).

[0020] In some embodiments:

[0021] • the first PPO1 gene comprises an insertion of any one nucleotide in GAGACCA (SEQ ID NO: 34), preferably the insertion of any one nucleotide after position 3 or 4 in GAGACCA (SEQ ID NO: 34), most preferably the insertion of an adenine; and / or

[0022] • the second PPO1 gene comprises a deletion of GAGACCA (SEQ ID NO: 34); and / or • the third PPO1 gene comprises a deletion of any one nucleotide from ACCCGTC (SEQ ID NO: 35), preferably the deletion of a cytosine, most preferably the cytosine at position 4 of ACCCGTC (SEQ ID NO: 35).

[0023] In a further aspect, the invention provides a banana plant cell, wherein the banana plant cell comprises three copies of a PPO1 gene, and wherein:

[0024] • a first PPO1 gene comprises: (1 ) an insertion of any one nucleotide in a nucleotide sequence GAGACCA (SEQ ID NO: 34) of the PPO1 gene, preferably the insertion of any one nucleotide after position 3 or 4 in GAGACCA (SEQ ID NO: 34), most preferably the insertion of an adenine; and (2) a premature stop-codon TGA (SEQ ID NO: 29) in a nucleotide sequence of the PPO1 gene comprising TGACCT (SEQ ID NO: 31); and / or

[0025] • a second PPO1 gene comprises: (1 ) a deletion of a nucleotide sequence GAGACCA (SEQ ID NO: 34) of the PPO1 gene; and (2) a premature stop-codon TAG (SEQ ID NO: 30) in a nucleotide sequence of the PPO1 gene comprising TAGGCA (SEQ ID NO: 32); and / or

[0026] • a third PPO1 gene comprises: (1) a deletion of any one nucleotide from in a nucleotide sequence ACCCGTC (SEQ ID NO: 35) of the PPO1 gene, preferably the deletion of a cytosine, most preferably the cytosine at position 4 from ACCCGTC (SEQ ID NO: 35); and (2) a premature stop-codon TGA (SEQ ID NO: 29) in a nucleotide sequence of the PPO1 gene comprising TGAGGG (SEQ ID NO: 33), wherein said insertions and / or deletions cause a frameshift in the wildtype PPO1 gene sequence leading to each premature stop-codon.

[0027] In some embodiments,• GAGACCA (SEQ ID NO: 34) is in a nucleotide sequence of the PPO1 gene comprising GAGACCACAACCAACCCCTCGT (SEQ ID NO: 36) or CCAGAGACCACAA (SEQ ID NO: 37); and / or

[0028] • ACCCGTC (SEQ ID NO: 35) is in a nucleotide sequence of the PPO1 gene comprising ACCCGTCCTCGCCCTTGAGG (SEQ ID NO: 38) or CCGACCCGTCCTC (SEQ ID NO: 39). In a specific embodiment, the wildtype PPO1 gene sequence comprises a polynucleotide sequence selected from the list consisting of:

[0029] • SEQ ID NO: 16, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 16 wherein said polynucleotide sequence with at least 90% identity comprises TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33);

[0030] • a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 18, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity to SEQ ID NO: 18 wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity comprises TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33); or

[0031] • a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 19, or a polynucleotide sequence that encodes a polypeptide comprising at least 90% identity to SEQ ID NO: 19 wherein said polynucleotide sequence that encodes a polypeptide comprising at least 90% identity comprises TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33).

[0032] In some embodiments:

[0033] • the first PPO1 gene encodes a polypeptide of about 95 to about 100 amino acids in length, preferably about 97 amino acids in length; and / or

[0034] • the second PPO1 gene encodes a polypeptide of about 65 to about 70 amino acids in length, preferably about 67 amino acids in length; and / or

[0035] • the third PPO1 gene encodes a polypeptide of about 130 to about 135 amino acids in length, preferably about 132 amino acids in length.

[0036] In a further embodiment:

[0037] • the first copy of the PPO1 gene comprises AGAGAACCAC (SEQ ID NO: 40); and / or • the second copy of the PPO1 gene comprises CCACAA (SEQ ID NO: 41); and / or • the third copy of the PPO1 gene comprises ACCGTC (SEQ ID NO: 42).

[0038] In a specific embodiment:• the first copy of the PP01 gene comprises a polynucleotide sequence of SEQ ID NO: 83, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 83 wherein said polynucleotide sequence with at least 90% identity comprises AGAGAACCAC (SEQ ID NO: 40);

[0039] • the second copy of the PPO1 gene comprises a polynucleotide sequence of SEQ ID NO: 84, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 84 wherein said polynucleotide sequence with at least 90% identity comprises CCACAA (SEQ ID NO: 41); and

[0040] • the third copy of the PPO1 gene comprises a polynucleotide sequence of SEQ ID NO: 85, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 85 wherein said polynucleotide sequence that encodes a polypeptide comprising at least 90% identity comprises ACCGTC (SEQ ID NO: 42).

[0041] In a specific embodiment:

[0042] • the first copy of the PPO1 gene comprises a polynucleotide sequence selected from the list consisting of:

[0043] a. SEQ ID NO: 20, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 20 wherein said polynucleotide sequence with at least 90% identity comprises TGACCT (SEQ ID NO: 31) and AGAGAACCAC (SEQ ID NO: 40); b. a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 23, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity to SEQ ID NO: 23 wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity comprises TGACCT (SEQ ID NO: 31) and AGAGAACCAC (SEQ ID NO: 40); or

[0044] c. a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO:

[0045] 26, or a polynucleotide sequence that encodes a polypeptide comprising at least 90% identity to SEQ ID NO: 26 wherein said polynucleotide sequence that encodes a polypeptide comprising at least 90% identity comprises TGACCT (SEQ ID NO: 31) and AGAGAACCAC (SEQ ID NO: 40), and / or

[0046] • the second copy of the PPO1 gene comprises a polynucleotide sequence selected from the list consisting of:

[0047] a. SEQ ID NO: 21, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 21 wherein said polynucleotide sequence with at least 90% identity comprises TAGGCA (SEQ ID NO: 32) and CCACAA (SEQ ID NO: 41);b. a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 24, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity to SEQ ID NO: 24 wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity comprises TAGGCA (SEQ ID NO: 32) and CCACAA (SEQ ID NO: 41); or

[0048] c. a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO:

[0049] 27, or a polynucleotide sequence that encodes a polypeptide comprising at least 90% identity to SEQ ID NO: 27 wherein said polynucleotide sequence that encodes a polypeptide comprising at least 90% identity comprises TAGGCA (SEQ ID NO: 32) and CCACAA (SEQ ID NO: 41), and / or

[0050] • the third copy of the PPO1 gene comprises a polynucleotide sequence selected from the list consisting of:

[0051] a. SEQ ID NO: 22, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 22 wherein said polynucleotide sequence with at least 90% identity comprises TGAGGG (SEQ ID NO: 33) and ACCGTC (SEQ ID NO: 42);

[0052] b. a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 25, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity to SEQ ID NO: 25 wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity comprises TGAGGG (SEQ ID NO: 33) and ACCGTC (SEQ ID NO: 42); or

[0053] c. a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO:

[0054] 28, or a polynucleotide sequence that encodes a polypeptide comprising at least 90% identity to SEQ ID NO: 28 wherein said polynucleotide sequence that encodes a polypeptide comprising at least 90% identity comprises TGAGGG (SEQ ID NO: 33) and ACCGTC (SEQ ID NO: 42).

[0055] In a further embodiment, the second PPO1 gene comprises a deletion of any one nucleotide from ACCCGTC (SEQ ID NO: 35), preferably the deletion of a cytosine, most preferably the cytosine at position 4 from ACCCGTC (SEQ ID NO: 35).

[0056] In a further aspect, the invention provides a banana plant or a banana fruit comprising a banana plant cell of the invention.

[0057] In a preferred embodiment, the banana plant cell or the banana plant or banana fruit is an autotriploid, preferably an autotriploid Musa acuminata.In a further aspect, the invention provides a processed banana product comprising a banana fruit of the invention.

[0058] BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The invention can be more fully understood from the following description, sequence listing and the accompanying drawings, of which:

[0060] Figure 1 - Clustal analysis aligning the polynucleotide sequences of the wildtype PPO1 gene from the DH Pahang V2 genome (SEQ ID NO: 1 - identifiable as Ma06_g31080); the DH Pahang V4 genome (SEQ ID NO: 2 - identifiable as Macma4_06_g32590); and the three chromosomes sequenced by Tropic Biosciences (SEQ ID NOs: 3-5, respectively).

[0061] Figure 2 - Clustal analysis aligning the polynucleotide sequences of the protein coding sequences (CDS) of the wildtype PPO1 gene from the DH Pahang V2 genome (SEQ ID NO: 6 - identifiable as Ma06_t31080.1); the DH Pahang V4 genome (SEQ ID NO: 7 -identifiable as Macma4_06_g32590.1 ); and the three chromosomes sequenced by Tropic Biosciences (SEQ ID NOs: 8-10, respectively).

[0062] Figure 3 - Clustal analysis aligning the polypeptide sequences encoded by the wildtype PPO1 gene from the DH Pahang V2 genome (SEQ ID NO: 11 - identifiable as Ma06_t31080.1); the DH Pahang V4 genome (SEQ ID NO: 12 - identifiable as Macma4_06_g32590.1 ); and the three chromosomes sequenced by Tropic Biosciences (SEQ ID NOs: 13-15, respectively).

[0063] Figure 4 - Map of the pMOL_0019 (A) plasmid and (B) T-DNA used in the genetic manipulation of the gene-edited line. Protein coding sequences are highlighted in dark grey (e.g. “8. Cas9 Coding Sequence + SV40 NLS”) with the sgRNA coding sequences highlighted in light grey (e.g. “11. sgRNA Coding Sequence”). The T-DNA region is enclosed within the Left and Right Borders and encodes the Nptll antibiotic resistance marker, Cas9 and the sgRNAs. Outside of the T-DNA, the pVS1 Short Fragment and pMB1 rep. regions contain the elements required for plasmid replication in Agrobacterium tumefaciens and Escherichia coli, respectively. The plasmid backbone also encodes Nptlll, the antibiotic resistance marker for selection in bacteria.

[0064] Figure 5 - Edits at the sgRNA target sites identified in the initial plant of the gene-edited banana line. PCR products were cloned into the pJET vector, purified and Sanger sequenced to confirm the edits in each PP01 allele. (A) Sanger sequencing traces for each allele and sgRNA target site alongside a visual representation of the three edited allele sequences. (B) Table outlining the nature of the edits in PP01 at the sg857 and sg858 target sites. The edits are described by their nucleotide position in the wild-type consensus PPO1 sequence with nucleotide position 1 being the first nucleotide of the first exon. Note, as the expected cut site for sg857 is between nucleotides 151 and 152, the A insertion edit in Allele 1 is called as being at this position. This edit is indistinguishable from an A insertion between positions 152 and 153, which is how the edit is presented in (A) by the alignment software.

[0065] Figure 6 - The region of the PPO1 genomic sequence highlighting the sgRNA target sequences, PAM sites, intended cut sites (black arrows) and genotyping primers (light grey, and not marked as a sgRNA target sequence or PAM site). SEQ ID NO: 80 is the top-strand genomic region, and SEQ ID NO: 81 is the lower-strand genomic region.

[0066] Figure 7 - Sequence alignment of the gene-edited line alleles with the consensus wild-type sequence for the targeted region of PPO1. Sequences are shown in the 5’ - 3’ direction for the forward strand only. The sgRNA target sites and protospacer adjacent motif (PAM) sites are annotated with the expected double-stranded break sites indicated by black arrows. The sequences shown are numbered relative to the first nucleotide (nucleotide position 1) of the first exon. Allele 1 is SEQ ID NO: 86; Allele 2 is SEQ ID NO: 87; and Allele 3 is SEQ ID NO: 88. The wildtype consensus sequence is SEQ ID NO: 89.

[0067] Figure 8 - Alignment of predicted protein sequences from wild-type PPO1 consensus and edited PPO1 alleles, with premature stop codons (*) indicated. Allele 1 is SEQ ID NO: 26; Allele 2 is SEQ ID NO: 27; and Allele 3 is SEQ ID NO: 28. The wildtype consensus sequence is SEQ ID NO: 82.

[0068] Figure 9 - Visualization of coverage for the whole genome sequencing reads aligned to the pMOL_0019 plasmid map for the gene-edited line, a wild-type banana and a transgenic control banana. Coverage of reads aligning to the plasmid map was visualized in IGV (Robinson et al., 2011, Nature Biotechnology 29, 24-26). The pMOL_0019 plasmid map is shown along the horizontal axis at the top whilst sequence coverage (x) is shown on the y-axis with the coverage range shown adjusted for each sample and stated.

[0069] Figure 10 - Exemplary photographs from the 60-minute homogenisation assay, comparing the gene-edited banana line with the wildtype control (Grande Naine).

[0070] Figure 11 - Graph showing the statistically significant reduction in browning in the gene-edited line when compared to the wildtype control (Grande Naine), at the time points of 30 and 60 minutes. The error bars represent standard error.

[0071] Figure 12 - Exemplary photographs from the 24-hour homogenisation assay, comparing the gene-edited banana line with the wildtype control (Grande Naine).Figure 13 - Graph showing the statistically significant reduction in browning in the gene-edited line when compared to the wildtype control (Grande Naine), at the time points of 60-minutes, 4 hours, and 24 hours. The error bars represent standard error.

[0072] Figure 14 - A comparison of the browning of the gene-edited line and the wildtype control (Grande Naine) when peeled and cut, and left for 12 hours at room temperature.

[0073] Figure 15 - A comparison of the browning of the gene-edited line and the wildtype control (Grande Naine) when peeled and sliced, and stored for up to 12 days under refrigeration.

[0074] Figure 16 - A comparison of the browning of the gene-edited line and the wildtype control (Grande Naine) when peeled and sliced, and included in fruit salad stored for up to 12 days under refrigeration.

[0075] Figure 17 - A comparison of the browning of the gene-edited line and the wildtype control (Grande Naine) when made into a banana juice, and stored for up to 48 hours under refrigeration.

[0076] Figure 18 - A comparison of the browning of the gene-edited line and the wildtype control (Grande Naine) when made into a banana -strawberry smoothie, and stored for up to 48 hours under refrigeration.

[0077] Figure 19 - A comparison of the browning of the gene-edited line and the wildtype control (Grande Naine) when peeled and sliced then treated with calcium ascorbate, and stored for up to 12 days under refrigeration.

[0078] Figure 20 - A comparison of the browning of the gene-edited line and the wildtype control (Grande Naine) when peeled and sliced then treated with calcium ascorbate, and included in fruit salad stored for up to 12 days under refrigeration.

[0079] LIST OF SEQUENCES

[0080] In respect of the SEQ ID NOs in Figures 1-3 (i.e. SEQ ID NOs: 1-15), that sequence information shows Clustal alignment analysis, in which at some positions a nucleotide or amino acid in a given sequence is absent when compared to the other sequences, with the absence being denoted by

[0081]

[0082] As would be known to the skilled person, that notation should simply be ignored when interpreting a full complete sequence in isolation, so the exemplary sequence of CCT---TCC would be read as CCTTCC.

[0083] SEQ ID NO: 1 is the polynucleotide sequence of the wildtype PPO1 gene from the DH Pahang V2 genome (identifiable as Ma06_g31080) - shown in Figure 1.SEQ ID NO: 2 is the polynucleotide sequence of the wildtype PPO1 gene from the DH Pahang V4 genome (identifiable as Macma4_06_g32590) - shown in Figure 1.

[0084] SEQ ID NO: 3 is the polynucleotide sequence of the wildtype PPO1 gene from the first of three chromosomes sequenced by Tropic Biosciences (identifiable as contig_834) - shown in Figure 1.

[0085] SEQ ID NO: 4 is the polynucleotide sequence of the wildtype PPO1 gene from the second of three chromosomes sequenced by Tropic Biosciences (identifiable as scaffold_141) - shown in Figure 1.

[0086] SEQ ID NO: 5 is the polynucleotide sequence of the wildtype PPO1 gene from the third of three chromosomes sequenced by Tropic Biosciences (identifiable as contig_835) - shown in Figure 1.

[0087] SEQ ID NO: 6 is the polynucleotide sequence of the protein coding sequence (CDS) of the wildtype PPO1 gene from the from the DH Pahang V2 genome (identifiable as Ma06_t31080.1 ) - shown in Figure 2.

[0088] SEQ ID NO: 7 is the polynucleotide sequence of the CDS of the wildtype PPO1 gene from the DH Pahang V4 genome (identifiable as Macma4_06_g32590.1) - shown in Figure 2.

[0089] SEQ ID NO: 8 is the polynucleotide sequence of the CDS of the wildtype PPO1 gene from the first of three chromosomes sequenced by Tropic Biosciences (identifiable as contig_834) - shown in Figure 2.

[0090] SEQ ID NO: 9 is the polynucleotide sequence of the CDS of the wildtype PPO1 gene from the second of three chromosomes sequenced by Tropic Biosciences (identifiable as scaffold_141) - shown in Figure 2.

[0091] SEQ ID NO: 10 is the polynucleotide sequence of the CDS of the wildtype PPO1 gene from the third of three chromosomes sequenced by Tropic Biosciences (identifiable as contig_835) - shown in Figure 2.

[0092] SEQ ID NO: 11 is the polypeptide sequence encoded by the wildtype PPO1 gene from the DH Pahang V2 genome (identifiable as Ma06_t31080.1) - shown in Figure 3.

[0093] SEQ ID NO: 12 is the polypeptide sequence encoded by the wildtype PPO1 gene from the DH Pahang V4 genome (identifiable as Macma4_06_g32590.1) - shown in Figure 3.

[0094] SEQ ID NO: 13 is the polypeptide sequence encoded by the wildtype PPO1 gene from the first of three chromosomes sequenced by Tropic Biosciences (identifiable as contig_834) - shown in Figure 3.SEQ ID NO: 14 is the polypeptide sequence encoded by the wildtype PPO1 gene from the second of three chromosomes sequenced by Tropic Biosciences (identifiable as scaffold_141) - shown in Figure 3.

[0095] SEQ ID NO: 15 is the polypeptide sequence encoded by the wildtype PPO1 gene from the third of three chromosomes sequenced by Tropic Biosciences (identifiable as contig_835) - shown in Figure 3.

[0096] SEQ ID NO: 16 is a consensus polynucleotide sequence of the wildtype PPO1 gene sequences from the three chromosomes sequenced by Tropic Biosciences (i.e. SEQ ID NOs: 3-5):

[0097] ATGGCTTCTATCTC^CAGCTAATCACTACAAGCATCC^TTCATGCCCCTTCTCCCCTCCGAGAACCA CGGTATCGATCTCCGGCT^CAAACACCACCACC^CGTTTCCCCCATCTCATGCTCC^CCAGAGACC ACAACCAACCCCTCGT^GATCCCACAGTCGA^CGCCGCCACGTCCTCGTCGGACTAGGCAGCCTCTA CGGCGCCTCCGCTGC^CTAACGTCCCTCC^CGAGGCCAG^GCGGCACCGATCGCGGCGCCCGACCT GTCCGCATGCGGGCCGGCTGACCTCCCTCCGGATGCCACTCCGACGAACTGCTGCC^GCCGTCCGCG GGCGACGCGACCGAGTTCGTCATTCCCGACCCGTCCTCGCCCTTGAGGGTGCGCCCGGCGGCCCACTC GGTCGACAA^GATTACATAGCTAAGTTCGCGAAGGGCGTCGCTCTCATGAAGGCGCTTCCGGCCGAC GACCCCCGGAACTTCACTCAGCATGCCAACGTGCACTGCGCCTACTGCGACGGGGCGTACAGCCAAGT CGGCTTCCCGGACCTTGAGCTCCAGGTGCACAACTCATGGCTCTTCCTGCCATGGCACCGCTGCTACC TCTACTTCTTCGAGAGAATACTCGGGAAGTTGATCGGCGA^GACAGCTTCGCGATTCCGTTCTGGAA CTGGGACGCCCCTGACGGGATGCGATTGCCAGCGATGTACGTGGATCCCACGTCGCCGCTTTACGATC CCCTAAGGGATGCACAGCATCAGCCGCCGACGTT^GTGGATTTGGACTTCGGAGGGAT^GATCCTTC TTTCAGTGATAAGCAGCAGATTGATCACAACCTCAAGGTTATGTACAGGCAGGTAATTAATGGACTCC AA^ACC ACT GAAT C AGT CAT GC AT TAT GT TAT T^T^T GT AC GAGCAT C AT AAAAT^AT GAT T GAT AGGC GAC AT C AGC C AT AAT T AT CCT GC AT T AAT T GAC GAAT CT T GT AAAC AAT G^C AT CT T GT T GGG CTTTCCATGGCTCATGGAGTTGATTGATGTGATTCTACGTCAATGATCGCTTGGAGAACGTAACTTAA GCTTGATGGGTCAAACTTCTTGTCTCGATGACGACAGAATACTGTCTTTTGATGCTTAGTTTTGTGT^ -ATATTAACTGCAAGATTTTTCTGCAGATCGTCTCGAATGCACCGACACCGAGGCTCTTCTTCGGAAA CCC^TACCGAGCCGGCGACAATCCGAACCCCGGTGGCGGCTCGCTTGAGAACGTCCCCCACGGACCG GTCCACGTCTGGACCGGCGACCGCAGCCAGTCGGAACTGGAGGACATGGGCAACCTGTACTCCGCCGC TCGCGACCCCGTCTTCTT^GCCCACCACTCCAACATCGACCGCATCTGGAACGTGTGGAAGGG^CTC GGT^GCCGGCGCAAGGACCTGGCCGACCCCGACTGGCTCGACGCCTCCTTCGTCTTCTACGACGAGA ACGCCAACCTCGTCAAGATCCGA^TTCGCGACTGCATCGACTCAGACAAGCTACGCTACGAGTACCA GGACGTCGGTAACC^ATGGCTCAACACACGCCCGACGGTGACGTCCGGAGTGAGGCCGAGAGTGGCC GGAGTGGCGCATGCAAACGT^GTGGAGCCGAAGTTTCCGATAAAGTTGGACTCGGTGGTGACTGCCA AGGTGAAGAGGCCAAAGGCGGCGAGGACCAAGGAGGAGAAGGAGGAGAAGGAGGAGGTGCTGGTGGTT GAAGGGAT C GAGC T GGAT C GA GAC GT GC AC GT C AAAT T C GAC GT GT T C GT GAAC GT GAC C GA^C AC G GGAAGGTCGGGCCGGGGGGCCGGGAGCTCGCCGGGAGCTTCGTGAACGTGCCTCACAGGCACAAGCAT GACAAGATGAGCAAGC^GCTGAAGACCAGGCTGCAGCTGGGCTTGACTGAGCTGTTGGAGGATCTCA AGG^TGAAGGAGATGGGAGCATCATGGTGACTTTGGTGCCGAGGCAGGGGAAGGGGAAGGTGAAGGT TA4Ga3AGT CT C AAGAT C GAGT T AGT T GAT T GA

[0098] whereina1is T or G; whereina2is CCACCAC (SEQ ID NO: 75) or CCACCACCTTTCCTCTCTCCTC (SEQ ID NO: 76) or CCACCACCTTTTCTCTCTCCTA (SEQ ID NO: 77); whereina3is C or T; whereina4is G or A; whereina5is A or T; whereina6is A or C; whereina7is C or G; whereina8is T or A or G.

[0099] SEQ ID NO: 17 is a consensus polynucleotide sequence of exon 1 of the wildtype PPO1 gene sequences from the three chromosomes sequenced by Tropic Biosciences:ATGGCTTCTATCTC^CAGCTAATCACTACAAGCATCC^TTCATGCCCCTTCTCCCCTCCGAGAACCA CGGTATCGATCTCCGGCT^CAAACACCACCACC^CGTTTCCCCCATCTCATGCTCC^CCAGAGACC ACAACCAACCCCTCGT^GATCCCACAGTCGA^CGCCGCCACGTCCTCGTCGGACTAGGCAGCCTCTA CGGCGCCTCCGCTGC^CTAACGTCCCTCC^CGAGGCCAG^GCGGCACCGATCGCGGCGCCCGACCT GTCCGCATGCGGGCCGGCTGACCTCCCTCCGGATGCCACTCCGACGAACTGCTGCC^GCCGTCCGCG GGCGACGCGACCGAGTTCGTCATTCCCGACCCGTCCTCGCCCTTGAGGGTGCGCCCGGCGGCCCACTC GGTCGACAA^GATTACATAGCTAAGTTCGCGAAGGGCGTCGCTCTCATGAAGGCGCTTCCGGCCGAC GACCCCCGGAACTTCACTCAGCATGCCAACGTGCACTGCGCCTACTGCGACGGGGCGTACAGCCAAGT CGGCTTCCCGGACCTTGAGCTCCAGGTGCACAACTCATGGCTCTTCCTGCCATGGCACCGCTGCTACC TCTACTTCTTCGAGAGAATACTCGGGAAGTTGATCGGCGA^GACAGCTTCGCGATTCCGTTCTGGAA CTGGGACGCCCCTGACGGGATGCGATTGCCAGCGATGTACGTGGATCCCACGTCGCCGCTTTACGATC CCCTAAGGGATGCACAGCATCAGCCGCCGACGTT^GTGGATTTGGACTTCGGAGGGAT^GATCCTTC T TTCAGT GAT AAGCAGCAGAT T GAT CACAACCT CAAGGTT AT GT ACAGGCAG

[0100] whereina1is T or G; whereina2is CCACCAC (SEQ ID NO: 75) or CCACCACCTTTCCTCTCTCCTC (SEQ ID NO: 76) or CCACCACCTTTTCTCTCTCCTA (SEQ ID NO: 77); whereina3is C or T; whereina4is G or A; whereina5is A or T; whereina6is A or C; whereina7is C or G.

[0101] It will be appreciated that from SEQ ID NO: 17 the skilled person would be able to directly and unambiguously understand the polynucleotides sequences of exon 1 from the corresponding polynucleotide sequences of the gene-edited PPO1 gene from the three chromosomes sequenced by Tropic Biosciences (i.e. SEQ ID NOs: 20-22).

[0102] SEQ ID NO: 18 is a consensus polynucleotide sequence of the CDS of the wildtype PPO1 gene sequences from the three chromosomes sequenced by Tropic Biosciences (i.e. SEQ ID NOs: 8-10). ATGGCTTCTATCTC^CAGCTAATCACTACAAGCATCC^CACCACTTCATGCCCCTTCTCCCCTCCGA GAACCACGGTATCGATCTCCGGCT^CAAACACCACCACC^CGTTTCCCCCATCTCATGCTCC^CCA GAGACCACAACCAACCCCTCGT^GATCCCACAGTCGA^CGCCGCCACGTCCTCGTCGGACTAGGCAG CCTCTACGGCGCCTCCGCTGC^CTAACGTCCCTCC^CGAGGCCAG^GCGGCACCGATCGCGGCGCC

[0103]

[0104] CGACCTGTCCGCATGCGGGCCGGCTGACCTCCCTCCGGATGCCACTCCGACGAACTGCTGCC^GCCG TCCGCGGGCGACGCGACCGAGTTCGTCATTCCCGACCCGTCCTCGCCCTTGAGGGTGCGCCCGGCGGC CCACTCGGTCGACAA^GATTACATAGCTAAGTTCGCGAAGGGCGTCGCTCTCATGAAGGCGCTTCCG GCCGACGACCCCCGGAACTTCACTCAGCATGCCAACGTGCACTGCGCCTACTGCGACGGGGCGTACAG CCAAGTCGGCTTCCCGGACCTTGAGCTCCAGGTGCACAACTCATGGCTCTTCCTGCCATGGCACCGCT GCTACCTCTACTTCTTCGAGAGAATACTCGGGAAGTTGATCGGCGA^GACAGCTTCGCGATTCCGTT CTGGAACTGGGACGCCCCTGACGGGATGCGATTGCCAGCGATGTACGTGGATCCCACGTCGCCGCTTT ACGATCCCCTAAGGGATGCACAGCATCAGCCGCCGACGTT^GTGGATTTGGACTTCGGAGGGAT^GA TCCTTCTTTCAGTGATAAGCAGCAGATTGATCACAACCTCAAGGTTATGTACAGGCAGATCGTCTCGA ATGCACCGACACCGAGGCTCTTCTTCGGAAACCC^TACCGAGCCGGCGACAATCCGAACCCCGGTGG CGGCTCGCTTGAGAACGTCCCCCACGGACCGGTCCACGTCTGGACCGGCGACCGCAGCCAGTCGGAAC TGGAGGACATGGGCAACCTGTACTCCGCCGCTCGCGACCCCGTCTTCTT^GCCCACCACTCCAACAT CGACCGCATCTGGAACGTGTGGAAGGG^CTCGGT^GCCGGCGCAAGGACCTGGCCGACCCCGACTGG CTCGACGCCTCCTTCGTCTTCTACGACGAGAACGCCAACCTCGTCAAGATCCGA^TTCGCGACTGCA TCGACTCAGACAAGCTACGCTACGAGTACCAGGACGTCGGTAACC^ATGGCTCAACACACGCCCGAC GGTGACGTCCGGAGTGAGGCCGAGAGTGGCCGGAGTGGCGCATGCAAACGT^GTGGAGCCGAAGTTT CCGATAAAGTTGGACTCGGTGGTGACTGCCAAGGTGAAGAGGCCAAAGGCGGCGAGGACCAAGGAGGA GAAGGAGGAGAAGGAGGAGGTGCTGGTGGTTGAAGGGATCGAGCTGGATCGAGACGTGCACGTCAAAT TCGACGTGTTCGTGAACGTGACCGA^CACGGGAAGGTCGGGCCGGGGGGCCGGGAGCTCGCCGGGAG CTTCGTGAACGTGCCTCACAGGCACAAGCATGACAAGATGAGCAAGCA8GCTGAAGACCAGGCTGCAGCTGGGCTTGACTGAGCTGTTGGAGGATCTCAAGG^TGAAGGAGATGGGAGCATCATGGTGACTTTGG T GCC GAGGC AGGGGAAGGGGAAGGT GAAGGT T^G^AGT C T C AAGAT C GAGT T AGT T GAT T GA whereina1is T or G; whereina2is CCACCAC (SEQ ID NO: 75) or CCACCACCTTTCCTCTCTCCTC (SEQ ID NO: 76) or CCACCACCTTTTCTCTCTCCTA (SEQ ID NO: 77); whereina3is C or T; whereina4is G or A; whereina5is A or T; whereina6is A or C; whereina7is C or G; whereina8is T or A or G.

[0105] SEQ ID NO: 19 is a consensus polypeptide sequence encoded by the wildtype PPO1 gene from the three chromosomes sequenced by Tropic Biosciences (i.e. SEQ ID NOs: 13-15): MASISQLITTSIPTT^SCPFSPPRTTVSISG^KHHH^VSPISCS^RDHNQPLVDPTVDRRHVLVG LGSLYGASAALTSIrJ^EASAAPIAAPDLSACGPADLPPDATPTNCC^PSAGDATEFVIPDPSSPLRVR

[0106]

[0107] PAAHSVD^DYIAKFAKGVALMKALPADDPRNFTQHANVHCAYCDGAYSQVGFPDLELQVHNSWLFLP WHRCYLYFFERILGKLIGDDSFAIPFWNWDAPDGMRLPAMYVDPTSPLYDPLRDAQHQPPTLVDLDFG

[0108] G^DPSFSDKQQIDHNLKVMYRQIVSNAPTPRLFFGNPYRAGDNPNPGGGSLENVPHGPVHVWTGDRS QSELEDMGNLYSAARDPVFFAHHSNIDRIWNVWKGLG^RRKDLADPDWLDASFVFYDENANLVKIR_^ -RDCIDSDKLRYEYQDVGN J^WLNTRPTVTSGVRPRVAGVAHANVVEPKFPIKLDSVVTAKVKRPKAA RTKEEKEEKEEVLWEGIELDRDVHVKFDVFVNVTDHGKVGPGGRELAGSFVNVPHRHKHDKMSK^L KTRLQLGLTELLEDLK*13EGDGSIMVTLVPRQGKGKVKV*14SLKIELVD

[0109] wherein *1is FPLSS (SEQ ID NO: 78); FSLSY (SEQ ID NO: 79) or no amino acids; wherein *2is F or S; wherein *3is H or R; wherein *4is S or T; wherein *5is H or R; wherein *6is L or P; wherein *7is N or K; wherein *8is I or M; wherein *9is S or G; wherein *10is V or I; wherein *11is L or P; wherein *12is L or Q or R; wherein *13is A or V; wherein *14is G or S.

[0110] SEQ ID NO: 20 is the polynucleotide sequence of the gene-edited PPO1 gene from the first of three chromosomes sequenced by Tropic Biosciences - the inserted A nucleotide is underlined and emboldened:

[0111] ATGGCTTCTATCTCTCAGCTAATCACTACAAGCATCCCCACCACTTCATGCCCCTTCTCCCCTCCGAG AACCACGGTATCGATCTCCGGCTCCAAACACCACCACCGCGTTTCCCCCATCTCATGCTCCACCAGAG AACCACAACCAACCCCTCGTCGATCCCACAGTCGACCGCCGCCACGTCCTCGTCGGACTAGGCAGCCT CTACGGCGCCTCCGCTGCACTAACGTCCCTCCGCGAGGCCAGTGCGGCACCGATCGCGGCGCCCGACC TGTCCGCATGCGGGCCGGCTGACCTCCCTCCGGATGCCACTCCGACGAACTGCTGCCCGCCGTCCGCG GGCGACGCGACCGAGTTCGTCATTCCCGACCCGTCCTCGCCCTTGAGGGTGCGCCCGGCGGCCCACTC GGT C GAG AAAGAT T AC AT AGC T AAGT T C GC GAAGGGC GT C GCT C T CAT GAAGGC GC T T C C GGC C GAC G ACCCCCGGAACTTCACTCAGCATGCCAACGTGCACTGCGCCTACTGCGACGGGGCGTACAGCCAAGTC GGCTTCCCGGACCTTGAGCTCCAGGTGCACAACTCATGGCTCTTCCTGCCATGGCACCGCTGCTACCT CTACTTCTTCGAGAGAATACTCGGGAAGTTGATCGGCGACGACAGCTTCGCGATTCCGTTCTGGAACT GGGACGCCCCTGACGGGATGCGATTGCCAGCGATGTACGTGGATCCCACGTCGCCGCTTTACGATCCC CTAAGGGATGCACAGCATCAGCCGCCGACGTTAGTGGATTTGGACTTCGGAGGGATCGATCCTTCTTT CAGTGATAAGCAGCAGATTGATCACAACCTCAAGGTTATGTACAGGCAGGTAATTAATGGACTCCAAT ACC AC T GAAT C AGT CAT GC AT TAT GT TAT T GT C T GT AC GAGCAT C AT AAAAT AAT GAT T GAT AGGC GA CATCAGCCATAATTATCCTGCATTAATTGACGAATCTTGTAAACAATGTCATCTTGTTGGGCTTTCCA TGGCTCATGGAGTT GAT T GAT GTGATTCTACGT C AAT GAT C GC T T GGAGAAC GTAACTTAAGCTTGAT GGGTCAAACTTCTTGTCTCGATGACGACAGAATACTGTCTTTTGATGCTTAGTTTTGTGTCATATTAA CTGCAAGATTTTTCTGCAGATCGTCTCGAATGCACCGACACCGAGGCTCTTCTTCGGAAACCCCTACC GAGCCGGCGACAATCCGAACCCCGGTGGCGGCTCGCTTGAGAACGTCCCCCACGGACCGGTCCACGTC TGGACCGGCGACCGCAGCCAGTCGGAACTGGAGGACATGGGCAACCTGTACTCCGCCGCTCGCGACCC CGTCTTCTTTGCCCACCACTCCAACATCGACCGCATCTGGAACGTGTGGAAGGGCCTCGGTGGCCGGCGCAAGGACCTGGCCGACCCCGACTGGCTCGACGCCTCCTTCGTCTTCTACGACGAGAACGCCAACCTC GTCAAGATCCGAGTTCGCGACTGCATCGACTCAGACAAGCTACGCTACGAGTACCAGGACGTCGGTAA CCCATGGCTCAACACACGCCCGACGGTGACGTCCGGAGTGAGGCCGAGAGTGGCCGGAGTGGCGCATG CAAACGTCGTGGAGCCGAAGTTTCCGATAAAGTTGGACTCGGTGGTGACTGCCAAGGTGAAGAGGCCA AAGGCGGCGAGGACCAAGGAGGAGAAGGAGGAGAAGGAGGAGGTGCTGGTGGTTGAAGGGATCGAGCT GGATCGAGACGTGCACGTCAAATTCGACGTGTTCGTGAACGTGACCGACCACGGGAAGGTCGGGCCGG GGGGCCGGGAGCTCGCCGGGAGCTTCGTGAACGTGCCTCACAGGCACAAGCATGACAAGATGAGCAAG CTGCTGAAGACCAGGCTGCAGCTGGGCTTGACTGAGCTGTTGGAGGATCTCAAGGCTGAAGGAGATGG GAGCATCATGGTGACTTTGGTGCCGAGGCAGGGGAAGGGGAAGGTGAAGGTTGGTAGTCTCAAGATCG AGTTAGTTGATTGA SEQ ID NO: 21 is the polynucleotide sequence of the gene-edited PPO1 gene from the second of three chromosomes sequenced by Tropic Biosciences:

[0112] ATGGCTTCTATCTCGCAGCTAATCACTACAAGCATCCCCACCACCTTTCCTCTCTCCTCTTCATGCCC CTTCTCCCCTCCGAGAACCACGGTATCGATCTCCGGCTTCAAACACCACCACCACGTTTCCCCCATCT CATGCTCCTCCACAACCAACCCCTCGTTGATCCCACAGTCGATCGCCGCCACGTCCTCGTCGGACTAG GCAGCCTCTACGGCGCCTCCGCTGCTCTAACGTCCCTCCACGAGGCCAGCGCGGCACCGATCGCGGCG CCCGACCTGTCCGCATGCGGGCCGGCTGACCTCCCTCCGGATGCCACTCCGACGAACTGCTGCCTGCC GTCCGCGGGCGACGCGACCGAGTTCGTCATTCCCGACCGTCCTCGCCCTTGAGGGTGCGCCCGGCGGC CCACTCGGTCGACAACGATTACATAGCTAAGTTCGCGAAGGGCGTCGCTCTCATGAAGGCGCTTCCGG CCGACGACCCCCGGAACTTCACTCAGCATGCCAACGTGCACTGCGCCTACTGCGACGGGGCGTACAGC CAAGTCGGCTTCCCGGACCTTGAGCTCCAGGTGCACAACTCATGGCTCTTCCTGCCATGGCACCGCTG CTACCTCTACTTCTTCGAGAGAATACTCGGGAAGTTGATCGGCGACGACAGCTTCGCGATTCCGTTCT GGAACTGGGACGCCCCTGACGGGATGCGATTGCCAGCGATGTACGTGGATCCCACGTCGCCGCTTTAC GATCCCCTAAGGGATGCACAGCATCAGCCGCCGACGTTGGTGGATTTGGACTTCGGAGGGATCGATCC TTCTTTCAGTGATAAGCAGCAGATTGATCACAACCTCAAGGTTATGTACAGGCAGGTAATTAATGGAC T CC AAT ACC ACT GAAT C AGT C AT GC AT T AT GT TAT TAT T T GT AC GAGCAT C AT AAAAT GAT GAT T GAT AGGC GAC AT C AGC C AT AAT T AT CCT GC AT T AAT T GAC GAAT CT T GT AAAC AAT GT C AT C T T GT T GGGC TTTCCATGGCTCATGGAGTTGATTGATGTGATTCTACGTCAATGATCGCTTGGAGAACGTAACTTAAG CTTGATGGGTCAAACTTCTTGTCTCGATGACGACAGAATACTGTCTTTTGATGCTTAGTTTTGTGTAA TATTAACTGCAAGATTTTTCTGCAGATCGTCTCGAATGCACCGACACCGAGGCTCTTCTTCGGAAACC CGTACCGAGCCGGCGACAATCCGAACCCCGGTGGCGGCTCGCTTGAGAACGTCCCCCACGGACCGGTC CACGTCTGGACCGGCGACCGCAGCCAGTCGGAACTGGAGGACATGGGCAACCTGTACTCCGCCGCTCG CGACCCCGTCTTCTTCGCCCACCACTCCAACATCGACCGCATCTGGAACGTGTGGAAGGGTCTCGGTA GCCGGCGCAAGGACCTGGCCGACCCCGACTGGCTCGACGCCTCCTTCGTCTTCTACGACGAGAACGCC AACCTCGTCAAGATCCGAGTTCGCGACTGCATCGACTCAGACAAGCTACGCTACGAGTACCAGGACGT CGGTAACCTATGGCTCAACACACGCCCGACGGTGACGTCCGGAGTGAGGCCGAGAGTGGCCGGAGTGG CGCATGCAAACGTGGTGGAGCCGAAGTTTCCGATAAAGTTGGACTCGGTGGTGACTGCCAAGGTGAAG AGGCCAAAGGCGGCGAGGACCAAGGAGGAGAAGGAGGAGAAGGAGGAGGTGCTGGTGGTTGAAGGGAT CGAGCTGGATCGAGACGTGCACGTCAAATTCGACGTGTTCGTGAACGTGACCGATCACGGGAAGGTCG GGCCGGGGGGCCGGGAGCTCGCCGGGAGCTTCGTGAACGTGCCTCACAGGCACAAGCATGACAAGATG AGCAAGCAGCTGAAGACCAGGCTGCAGCTGGGCTTGACTGAGCTGTTGGAGGATCTCAAGGTTGAAGG AGATGGGAGCATCATGGTGACTTTGGTGCCGAGGCAGGGGAAGGGGAAGGTGAAGGTTAGCAGTCTCA AGATCGAGTTAGTTGATTGA SEQ ID NO: 22 is the polynucleotide sequence of the gene-edited PPO1 gene from the third of three chromosomes sequenced by Tropic Biosciences:

[0113] ATGGCTTCTATCTCGCAGCTAATCACTACAAGCATCCCCACCACCTTTTCTCTCTCCTATTCATGCCC CTTCTCCCCTCCGAGAACCACGGTATCGATCTCCGGCTCCAAACACCACCACCACGTTTCCCCCATCT CATGCTCCACCAGAGACCACAACCAACCCCTCGTCGATCCCACAGTCGACCGCCGCCACGTCCTCGTC GGACTAGGCAGCCTCTACGGCGCCTCCGCTGCACTAACGTCCCTCCGCGAGGCCAGTGCGGCACCGAT CGCGGCGCCCGACCTGTCCGCATGCGGGCCGGCTGACCTCCCTCCGGATGCCACTCCGACGAACTGCT GCCCGCCGTCCGCGGGCGACGCGACCGAGTTCGTCATTCCCGACCGTCCTCGCCCTTGAGGGTGCGCC CGGCGGCCCACTCGGTCGACAAAGATTACATAGCTAAGTTCGCGAAGGGCGTCGCTCTCATGAAGGCG CTTCCGGCCGACGACCCCCGGAACTTCACTCAGCATGCCAACGTGCACTGCGCCTACTGCGACGGGGC GTACAGCCAAGTCGGCTTCCCGGACCTTGAGCTCCAGGTGCACAACTCATGGCTCTTCCTGCCATGGCACCGCTGCTACCTCTACTTCTTCGAGAGAATACTCGGGAAGTTGATCGGCGATGACAGCTTCGCGATT CCGTTCTGGAACTGGGACGCCCCTGACGGGATGCGATTGCCAGCGATGTACGTGGATCCCACGTCGCC GCTTTACGATCCCCTAAGGGATGCACAGCATCAGCCGCCGACGTTAGTGGATTTGGACTTCGGAGGGA TGGATCCTTCTTTCAGTGATAAGCAGCAGATTGATCACAACCTCAAGGTTATGTACAGGCAGGTAATT AAT GGACT C C AAAACC AC T GAAT C AGT CAT GC AT T AT GT TAT TAT T T GT AC GAGCAT C AT AAAAT AAT GATTGATAGGCGACATCAGCCATAATTATCCTGCATTAATTGACGAATCTTGTAAACAATGACATCTT GTTGGGCTTTCCATGGCTCATGGAGTTGATTGATGTGATTCTACGTCAATGATCGCTTGGAGAACGTA ACTTAAGCTTGATGGGTCAAACTTCTTGTCTCGATGACGACAGAATACTGTCTTTTGATGCTTAGTTT TGTGTCATATTAACTGCAAGATTTTTCTGCAGATCGTCTCGAATGCACCGACACCGAGGCTCTTCTTC GGAAACCCCTACCGAGCCGGCGACAATCCGAACCCCGGTGGCGGCTCGCTTGAGAACGTCCCCCACGG ACCGGTCCACGTCTGGACCGGCGACCGCAGCCAGTCGGAACTGGAGGACATGGGCAACCTGTACTCCG CCGCTCGCGACCCCGTCTTCTTTGCCCACCACTCCAACATCGACCGCATCTGGAACGTGTGGAAGGGT CTCGGTAGCCGGCGCAAGGACCTGGCCGACCCCGACTGGCTCGACGCCTCCTTCGTCTTCTACGACGA GAACGCCAACCTCGTCAAGATCCGAATTCGCGACTGCATCGACTCAGACAAGCTACGCTACGAGTACC AGGACGTCGGTAACCCATGGCTCAACACACGCCCGACGGTGACGTCCGGAGTGAGGCCGAGAGTGGCC GGAGTGGCGCATGCAAACGTCGTGGAGCCGAAGTTTCCGATAAAGTTGGACTCGGTGGTGACTGCCAA GGTGAAGAGGCCAAAGGCGGCGAGGACCAAGGAGGAGAAGGAGGAGAAGGAGGAGGTGCTGGTGGTTG AAGGGAT C GAGCT GGAT C GAGAC GT GC AC GT C AAAT T C GAC GT GT T C GT GAAC GT GACC GACC AC GGG AAGGTCGGGCCGGGGGGCCGGGAGCTCGCCGGGAGCTTCGTGAACGTGCCTCACAGGCACAAGCATGA CAAGATGAGCAAGCGGCTGAAGACCAGGCTGCAGCTGGGCTTGACTGAGCTGTTGGAGGATCTCAAGG CTGAAGGAGATGGGAGCATCATGGTGACTTTGGTGCCGAGGCAGGGGAAGGGGAAGGTGAAGGTTGGC AGT CT C AAGAT C GAGT T AGT T GAT T GA SEQ ID NO: 23 is the polynucleotide sequence of the CDS of the gene-edited PPO1 gene from the first of three chromosomes sequenced by Tropic Biosciences - the inserted A nucleotide is underlined and emboldened:

[0114] ATGGCTTCTATCTCTCAGCTAATCACTACAAGCATCCCCACCACTTCATGCCCCTTCTCCCCTCCGAG AACCACGGTATCGATCTCCGGCTCCAAACACCACCACCGCGTTTCCCCCATCTCATGCTCCACCAGAG AACCACAACCAACCCCTCGTCGATCCCACAGTCGACCGCCGCCACGTCCTCGTCGGACTAGGCAGCCT CTACGGCGCCTCCGCTGCACTAACGTCCCTCCGCGAGGCCAGTGCGGCACCGATCGCGGCGCCCGACC TGTCCGCATGCGGGCCGGCTGACCTCCCTCCGGATGCCACTCCGACGAACTGCTGCCCGCCGTCCGCG GGCGACGCGACCGAGTTCGTCATTCCCGACCCGTCCTCGCCCTTGAGGGTGCGCCCGGCGGCCCACTC GGT C GAC AAAGAT T AC AT AGC T AAGT T C GC GAAGGGC GT C GCT C T CAT GAAGGC GC T T C C GGC C GAC G ACCCCCGGAACTTCACTCAGCATGCCAACGTGCACTGCGCCTACTGCGACGGGGCGTACAGCCAAGTC GGCTTCCCGGACCTTGAGCTCCAGGTGCACAACTCATGGCTCTTCCTGCCATGGCACCGCTGCTACCT CTACTTCTTCGAGAGAATACTCGGGAAGTTGATCGGCGACGACAGCTTCGCGATTCCGTTCTGGAACT GGGACGCCCCTGACGGGATGCGATTGCCAGCGATGTACGTGGATCCCACGTCGCCGCTTTACGATCCC CTAAGGGATGCACAGCATCAGCCGCCGACGTTAGTGGATTTGGACTTCGGAGGGATCGATCCTTCTTT C AGT GAT AAGC AGC AGAT T GAT C AC AACC T C AAGGT T AT GT AC AGGC AGAT C GT CT C GAAT GC ACC GA CACCGAGGCTCTTCTTCGGAAACCCCTACCGAGCCGGCGACAATCCGAACCCCGGTGGCGGCTCGCTT GAGAACGTCCCCCACGGACCGGTCCACGTCTGGACCGGCGACCGCAGCCAGTCGGAACTGGAGGACAT GGGCAACCTGTACTCCGCCGCTCGCGACCCCGTCTTCTTTGCCCACCACTCCAACATCGACCGCATCT GGAACGTGTGGAAGGGCCTCGGTGGCCGGCGCAAGGACCTGGCCGACCCCGACTGGCTCGACGCCTCC TTCGTCTTCTAC GAC GAG AAC GC C AAC CTCGTCAAGATCCGAGTTCGC GAC T GC AT C GAC T C AGAC AA GCTACGCTACGAGTACCAGGACGTCGGTAACCCATGGCTCAACACACGCCCGACGGTGACGTCCGGAG TGAGGCCGAGAGTGGCCGGAGTGGCGCATGCAAACGTCGTGGAGCCGAAGTTTCCGATAAAGTTGGAC TCGGTGGTGACTGCCAAGGTGAAGAGGCCAAAGGCGGCGAGGACCAAGGAGGAGAAGGAGGAGAAGGA GGAGGT GCT GGT GGT T GAAGGGAT C GAGC T GGAT C GAGAC GT GC AC GT C AAAT T C GAC GT GT T C GT GA ACGTGACCGACCACGGGAAGGTCGGGCCGGGGGGCCGGGAGCTCGCCGGGAGCTTCGTGAACGTGCCT CACAGGCACAAGCATGACAAGATGAGCAAGCTGCTGAAGACCAGGCTGCAGCTGGGCTTGACTGAGCT GTTGGAGGATCTCAAGGCTGAAGGAGATGGGAGCATCATGGTGACTTTGGTGCCGAGGCAGGGGAAGG GGAAGGT GAAGGT T GGT AGT C T C AAGAT C GAGT T AGT T GAT T GA SEQ ID NO: 24 is the polynucleotide sequence of the CDS of the gene-edited PPO1 gene from the second of three chromosomes sequenced by Tropic Biosciences:ATGGCTTCTATCTCGCAGCTAATCACTACAAGCATCCCCACCACCTTTCCTCTCTCCTCTTCATGCCC CTTCTCCCCTCCGAGAACCACGGTATCGATCTCCGGCTTCAAACACCACCACCACGTTTCCCCCATCT CATGCTCCTCCACAACCAACCCCTCGTTGATCCCACAGTCGATCGCCGCCACGTCCTCGTCGGACTAG GCAGCCTCTACGGCGCCTCCGCTGCTCTAACGTCCCTCCACGAGGCCAGCGCGGCACCGATCGCGGCG CCCGACCTGTCCGCATGCGGGCCGGCTGACCTCCCTCCGGATGCCACTCCGACGAACTGCTGCCTGCC GTCCGCGGGCGACGCGACCGAGTTCGTCATTCCCGACCGTCCTCGCCCTTGAGGGTGCGCCCGGCGGC CCACTCGGTCGACAACGATTACATAGCTAAGTTCGCGAAGGGCGTCGCTCTCATGAAGGCGCTTCCGG CCGACGACCCCCGGAACTTCACTCAGCATGCCAACGTGCACTGCGCCTACTGCGACGGGGCGTACAGC CAAGTCGGCTTCCCGGACCTTGAGCTCCAGGTGCACAACTCATGGCTCTTCCTGCCATGGCACCGCTG CTACCTCTACTTCTTCGAGAGAATACTCGGGAAGTTGATCGGCGACGACAGCTTCGCGATTCCGTTCT GGAACTGGGACGCCCCTGACGGGATGCGATTGCCAGCGATGTACGTGGATCCCACGTCGCCGCTTTAC GATCCCCTAAGGGATGCACAGCATCAGCCGCCGACGTTGGTGGATTTGGACTTCGGAGGGATCGATCC TTCTTTCAGTGATAAGCAGCAGATTGATCACAACCTCAAGGTTATGTACAGGCAGATCGTCTCGAATG CACCGACACCGAGGCTCTTCTTCGGAAACCCGTACCGAGCCGGCGACAATCCGAACCCCGGTGGCGGC TCGCTTGAGAACGTCCCCCACGGACCGGTCCACGTCTGGACCGGCGACCGCAGCCAGTCGGAACTGGA GGACATGGGCAACCTGTACTCCGCCGCTCGCGACCCCGTCTTCTTCGCCCACCACTCCAACATCGACC GCATCTGGAACGTGTGGAAGGGTCTCGGTAGCCGGCGCAAGGACCTGGCCGACCCCGACTGGCTCGAC GCCTCCTTCGTCTTCTACGACGAGAACGCCAACCTCGTCAAGATCCGAGTTCGCGACTGCATCGACTC AGACAAGCTACGCTACGAGTACCAGGACGTCGGTAACCTATGGCTCAACACACGCCCGACGGTGACGT CCGGAGTGAGGCCGAGAGTGGCCGGAGTGGCGCATGCAAACGTGGTGGAGCCGAAGTTTCCGATAAAG TTGGACTCGGTGGTGACTGCCAAGGTGAAGAGGCCAAAGGCGGCGAGGACCAAGGAGGAGAAGGAGGA GAAGGAGGAGGTGCTGGTGGTTGAAGGGATCGAGCTGGATCGAGACGTGCACGTCAAATTCGACGTGT TCGTGAACGTGACCGATCACGGGAAGGTCGGGCCGGGGGGCCGGGAGCTCGCCGGGAGCTTCGTGAAC GT GCC T C AC AGGC AC AAGC AT GAC AAGAT GAGC AAGC AGC T GAAGACC AGGC T GC AGC T GGGCT T GAC TGAGCTGTTGGAGGATCTCAAGGTTGAAGGAGATGGGAGCATCATGGTGACTTTGGTGCCGAGGCAGG GGAAGGGGAAGGTGAAGGTTAGCAGTCTCAAGATCGAGTTAGTTGATTGA SEQ ID NO: 25 is the polynucleotide sequence of the CDS of the gene-edited PPO1 gene from the third of three chromosomes sequenced by Tropic Biosciences: ATGGCTTCTATCTCGCAGCTAATCACTACAAGCATCCCCACCACCTTTTCTCTCTCCTATTCATGCCC CTTCTCCCCTCCGAGAACCACGGTATCGATCTCCGGCTCCAAACACCACCACCACGTTTCCCCCATCT CATGCTCCACCAGAGACCACAACCAACCCCTCGTCGATCCCACAGTCGACCGCCGCCACGTCCTCGTC GGACTAGGCAGCCTCTACGGCGCCTCCGCTGCACTAACGTCCCTCCGCGAGGCCAGTGCGGCACCGAT CGCGGCGCCCGACCTGTCCGCATGCGGGCCGGCTGACCTCCCTCCGGATGCCACTCCGACGAACTGCT GCCCGCCGTCCGCGGGCGACGCGACCGAGTTCGTCATTCCCGACCGTCCTCGCCCTTGAGGGTGCGCC CGGCGGCCCACTCGGTCGACAAAGATTACATAGCTAAGTTCGCGAAGGGCGTCGCTCTCATGAAGGCG CTTCCGGCCGACGACCCCCGGAACTTCACTCAGCATGCCAACGTGCACTGCGCCTACTGCGACGGGGC GTACAGCCAAGTCGGCTTCCCGGACCTTGAGCTCCAGGTGCACAACTCATGGCTCTTCCTGCCATGGC ACCGCTGCTACCTCTACTTCTTCGAGAGAATACTCGGGAAGTTGATCGGCGATGACAGCTTCGCGATT CCGTTCTGGAACTGGGACGCCCCTGACGGGATGCGATTGCCAGCGATGTACGTGGATCCCACGTCGCC GCTTTACGATCCCCTAAGGGATGCACAGCATCAGCCGCCGACGTTAGTGGATTTGGACTTCGGAGGGA TGGATCCTTCTTTCAGTGATAAGCAGCAGATTGATCACAACCTCAAGGTTATGTACAGGCAGATCGTC TCGAATGCACCGACACCGAGGCTCTTCTTCGGAAACCCCTACCGAGCCGGCGACAATCCGAACCCCGG TGGCGGCTCGCTTGAGAACGTCCCCCACGGACCGGTCCACGTCTGGACCGGCGACCGCAGCCAGTCGG AACTGGAGGACATGGGCAACCTGTACTCCGCCGCTCGCGACCCCGTCTTCTTTGCCCACCACTCCAAC ATCGACCGCATCTGGAACGTGTGGAAGGGTCTCGGTAGCCGGCGCAAGGACCTGGCCGACCCCGACTG GCTCGACGCCTCCTTCGTCTTCTACGACGAGAACGCCAACCTCGTCAAGATCCGAATTCGCGACTGCA TCGACTCAGACAAGCTACGCTACGAGTACCAGGACGTCGGTAACCCATGGCTCAACACACGCCCGACG GTGACGTCCGGAGTGAGGCCGAGAGTGGCCGGAGTGGCGCATGCAAACGTCGTGGAGCCGAAGTTTCC GATAAAGTTGGACTCGGTGGTGACTGCCAAGGTGAAGAGGCCAAAGGCGGCGAGGACCAAGGAGGAGA AGGAGGAGAAGGAGGAGGTGCTGGTGGTTGAAGGGATCGAGCTGGATCGAGACGTGCACGTCAAATTC GACGTGTTCGTGAACGTGACCGACCACGGGAAGGTCGGGCCGGGGGGCCGGGAGCTCGCCGGGAGCTT C GT GAACGT GCCT CACAGGCACAAGCAT GACAAGAT GAGC AAGC GGCT GAAGACCAGGCT GCAGCT GG GCTTGACTGAGCTGTTGGAGGATCTCAAGGCTGAAGGAGATGGGAGCATCATGGTGACTTTGGTGCCG AGGCAGGGGAAGGGGAAGGTGAAGGTTGGCAGTCTCAAGATCGAGTTAGTTGATTGASEQ ID NO: 26 is the polypeptide sequence encoded by the gene-edited PPO1 gene from the first of three chromosomes sequenced by Tropic Biosciences:

[0115] MAS I SQLITTS I PTTSCPFSPPRTTVS I SGSKHHHRVSPI SCSTREPQPTPRRSHSRPPPRPRRTRQP L RRL RCT NVP P RGQC GT D RGARP VRMRAG SEQ ID NO: J is the polypeptide sequence encoded by the gene-edited PPO1 gene from the second of three chromosomes sequenced by Tropic Biosciences:

[0116] MAS I SQLITTS I PTTFPLSSSCPFS PPRTTVS I SGFKHHHHVSP I SCS STTNPSLI PQS IAAT SSSD SEQ ID NO: 28 is the polypeptide sequence encoded by the gene-edited PPO1 gene from the third of three chromosomes sequenced by Tropic Biosciences:

[0117] MASISQLITTSIPTTFSLSYSCPFSPPRTTVSISGSKHHHHVSPISCSTRDHNQPLVDPTVDRRHVLV GLGSLYGASAALTSLREASAAPIAAPDLSACGPADLPPDATPTNCCPPSAGDATEFVIPDRPRP SEQ ID NO: 29 and 30 are TGA and TAG, respectively, which are stop-codons. SEQ ID NOs: 31-33 are TGACCT, TAGGCA and TGAGGG, respectively, which are the PPO1 genomic locations of stop-codons.

[0118] SEQ ID NO: 34 and 35 are GAGACCA and ACCCGTC, respectively, which are the PPO1 genomic locations of indels in the gene-edited banana plant cells.

[0119] SEQ ID NO: 36 and 37 are GAGACCACAACCAACCCCTCGT and CCAGAGACCACAA, respectively, which are extended PPO1 genomic locations containing GAGACCA (SEQ ID NO: 34).

[0120] SEQ ID NO: 38 and 39 are ACCCGTCCTCGCCCTTGAGG and CCGACCCGTCCTC, respectively, which are extended PPO1 genomic locations containing ACCCGTC (SEQ ID NO: 35).

[0121] SEQ ID NOs: 40-42 are AGAGAACCAC, CCACAA, and ACCGTC, respectively, which are edits at the exemplified indels of PPO1.

[0122] SEQ ID NO: 43, 90 and 91 is GAGAACCA, TCCACAAC, and GACCGTCC, respectively, which are alternative sequences of the edits of SEQ ID NOs: 40-42, of different lengths.

[0123] SEQ ID NO: 44 and 45 are the variable regions GAGGGGTTGGTTGTGGTCTC and CCTCAAGGGCGAGGACGGGT of the crRNA for the exemplary sg857 and sg858 guides, respectively.

[0124] SEQ ID NOs: 46-49 are the crRNA constant region G I I I I AGAGCTA, the Linker Loop GAAA, the tracer RNA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC and the Poly T terminator I I I I I I I for the exemplary sg857 and sg858 guides.

[0125] SEQ ID NO: 50 is the crRNA for sg857.

[0126] SEQ ID NO: 51 is the crRNA for sg858.

[0127] SEQ ID NO: 52 is sg857.SEQID NO: 53 is sg858.

[0128] SEQ ID NO: 54 and 55 are the sequences in Table 1.

[0129] SEQ ID NOs: 56-74 and 92 are the sequences in Table 2 and 3.

[0130] SEQID NOs: 75-77 are sequences CCACCAC, CCACCACCTTTCCTCTCTCCTC, and CCACCACCl I I ICTCTCTCCTA, respectively, which are relevant to the consensus polynucleotide sequences.

[0131] SEQID NO: 78 and 79 are sequences FPLSS and FSLSY, respectively, which are relevant to the consensus polypeptide sequences.

[0132] SEQ ID NO: 80 is the top-strand genomic region shown in Figure 6.

[0133] SEQID NO: 81 is the lower-strand genomic region shown in Figure 6.

[0134] SEQ ID NO: 82 is the wildtype consensus sequence in Figure 8.

[0135] SEQID NO: 83 is the polynucleotide sequence of the gene-edited PPO1 gene up to first pre-mature stop codon from the first of three chromosomes sequenced by Tropic Biosciences - the inserted A nucleotide is underlined and emboldened:

[0136] ATGGCTTCTATCTCTCAGCTAATCACTACAAGCATCCCCACCACTTCATGCCCCTTCTCCCCTCCGAG AACCACGGTATCGATCTCCGGCTCCAAACACCACCACCGCGTTTCCCCCATCTCATGCTCCACCAGAG AACCACAACCAACCCCTCGTCGATCCCACAGTCGACCGCCGCCACGTCCTCGTCGGACTAGGCAGCCT CTACGGCGCCTCCGCTGCACTAACGTCCCTCCGCGAGGCCAGTGCGGCACCGATCGCGGCGCCCGACC TGTCCGCATGCGGGCCGGCTGA SEQID NO: 84 is the polynucleotide sequence of the gene-edited PPO1 gene up to first pre-mature stop codon from the second of three chromosomes sequenced by Tropic Biosciences:

[0137] ATGGCTTCTATCTCGCAGCTAATCACTACAAGCATCCCCACCACCTTTCCTCTCTCCTCTTCATGCCC CTTCTCCCCTCCGAGAACCACGGTATCGATCTCCGGCTTCAAACACCACCACCACGTTTCCCCCATCT CATGCTCCTCCACAACCAACCCCTCGTTGATCCCACAGTCGATCGCCGCCACGTCCTCGTCGGACTAG SEQID NO: 85 is the polynucleotide sequence of the gene-edited PPO1 gene up to first pre-mature stop codon from the third of three chromosomes sequenced by Tropic Biosciences:

[0138] ATGGCTTCTATCTCGCAGCTAATCACTACAAGCATCCCCACCACCTTTTCTCTCTCCTATTCATGCCC CTTCTCCCCTCCGAGAACCACGGTATCGATCTCCGGCTCCAAACACCACCACCACGTTTCCCCCATCT CATGCTCCACCAGAGACCACAACCAACCCCTCGTCGATCCCACAGTCGACCGCCGCCACGTCCTCGTC GGACTAGGCAGCCTCTACGGCGCCTCCGCTGCACTAACGTCCCTCCGCGAGGCCAGTGCGGCACCGAT CGCGGCGCCCGACCTGTCCGCATGCGGGCCGGCTGACCTCCCTCCGGATGCCACTCCGACGAACTGCT GCCCGCCGTCCGCGGGCGACGCGACCGAGTTCGTCATTCCCGACCGTCCTCGCCCTTGA SEQID NOs: 86-88 are Alleles 1-3 in Figure 7, with SEQID NO: 89 being the wildtype sequence.It will be appreciated that any of the partial defined sequences herein (for example, the premature stop codon sequences, the premature stop codon location sequences (e.g. TGACCT (SEQ ID NO: 31)), the sequences of the genomic locations of indels (e.g. GAGACCA (SEQ ID NO: 34)), and / or the sequences of the edits (e.g. AGAGAACCAC (SEQ ID NO: 40))) can be represented by extended sequences including upstream and downstream nucleotides of the sequences from which they are taken (for example, the wildtype first, second and / or third PPO1 gene sequences, such as SEQ ID NOs: 20-22), as would be directly and unambiguously derivable from herein. In a particular embodiment, those partial defined sequences are extended by one nucleotide upstream and / or one nucleotide downstream, preferably both upstream and downstream. In a further embodiment, those partial defined sequences are extended by two nucleotides upstream and / or two nucleotides downstream, preferably both upstream and downstream. In an additional embodiment, those partial defined sequences are extended by three nucleotides upstream and / or three nucleotides downstream, preferably both upstream and downstream.

[0139] It will also be appreciated by one skilled in the art that any of the sequences herein could be combined with any compatible aspects of the invention; for example, any of the nucleic acid and protein sequences used to describe the reduced browning banana plants of the invention (whether those sequences appear in the description or the Examples) could be combined with any of the aspects herein, such as those describing the banana cells, plants, or methods of the invention.

[0140] DETAILED DESCRIPTION OF THE INVENTION

[0141] The present invention is based on the inventors’ work in developing a banana plant with a particularly desirable phenotype of reduced browning banana fruit flesh, based on targeting an edit to the PPO1 gene. In carefully analysing the genotype of banana cells of said gene-edited banana, the inventors have identified that the phenotype is associated with each of the three copies of the PPO1 gene (i.e. all of the PPO1 genes) comprising a premature stop-codon, which in turn leads to truncated PP01 proteins. Without being bound by theory, it is thought that the absence of the full-length PP01 proteins and their replacement with truncated versions has led to the desirable phenotype.

[0142] Accordingly, in one aspect the invention provides a banana plant cell,wherein the banana plant cell comprises three copies of a PPO1 gene, and wherein:

[0143] • a first copy of the PPO1 gene comprises a premature stop-codon TGA (SEQ ID NO:

[0144] 29) in a nucleotide sequence of the PPO1 gene comprising TGACCT (SEQ ID NO: 31);

[0145] • a second copy of the PPO1 gene comprises a premature stop-codon TAG (SEQ ID NO: 30) in a nucleotide sequence of the PPO1 gene comprising TAGGCA (SEQ ID NO: 32); and

[0146] • a third copy of the PPO1 gene comprises a premature stop-codon TGA (SEQ ID NO:

[0147] 29) in a nucleotide sequence of the PPO1 gene comprising TGAGGG (SEQ ID NO: 33).

[0148] The premature stop-codons described in the above aspect were identified by the inventors as leading to the edited PPO1 gene encoding the truncated proteins that resulted in the desirable reduced browning banana fruit flesh phenotype. The polynucleotide sequences TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33) used to define the locations of the premature stop-codons have been identified as being unique and conserved within the PPO1 gene, so would be understood by the person skilled in genetics to accurately and clearly define the key characteristic that makes the banana cell of the invention advantageous.

[0149] As used herein, the term “polyphenol oxidase” or “PPO” refers to an enzyme belonging to a group of enzymes found throughout the plant and animal kingdoms, including in most fruits and vegetables, such as banana. Correspondingly, the term “polyphenol oxidase gene” or “PPO gene” as used herein refers to gene encoding a polyphenol oxidase or PPO. PPOs, including PP01 as relevant to the present invention, catalyse enzymatic browning, including when tissues are damaged from bruising or compression. Exposure to oxygen when fruit is sliced or pureed also leads to enzymatic browning by PPOs. PPOs are known to accept monophenols and / or o-diphenols as substrates, and work by catalysing the o-hydroxylation of monophenol molecules in which the benzene ring contains a single hydroxyl substituent to o-diphenols (phenol molecules containing two hydroxyl substituents at the 1, 2 positions, with no carbon between). The enzymes can also further catalyse the oxidation of o-diphenols to produce o-quinones. PPOs catalyse the rapid polymerisation of o-quinones to produce black, brown or red pigments (polyphenols) that cause fruit browning.As defined further herein, the banana plant cell of the invention can ultimately be regenerated into a banana plant from which grows banana fruit characterised as having fruit flesh exhibiting reduced browning.

[0150] As used herein, the terms “copies” or “copy” in the context of defining the number of PPO1 genes in the banana cell of the invention relates to the number of said specific PPO1 genes in the genome of said banana cell. Generally, there will be a single copy of a given PPO1 gene on each chromosome in the banana cell’s genome - therefore, if the banana cell has three chromosomes it will comprise three copies of the PPO1 gene. As will be appreciated, genetic variation can occur between the same gene on different chromosomes, so the polynucleotide sequence of the PPO1 gene might be identical or comprise (often limited) variation between different chromosomes. In some scientific literature the copies of a given gene on different chromosomes can be referred to as different alleles, and so it is included that the term “copies” and “alleles” can be used herein interchangeably.

[0151] The term “premature stop-codon” will be well understood by the person skilled in genetics. Herein, by “premature stop-codon” we include a stop-codon (i.e. TAA, TAG, TGA) that is present in the PPO1 gene of the invention prior to (i.e. more 5’ of) the position of the stop-codon in the wildtype PPO1 gene. This might also be referred to as being ‘upstream’. The presence of a premature stop-codon in a gene will result in the translation of a truncated protein, sometimes with a different amino acid sequence, when compared to the wildtype protein. This truncated protein will often not be functional, have an aberrant function or have reduced functionality, when compared to the wildtype protein. Without wanting to be bound by theory, it is thought that this absence or difference in PPO1 protein function leads to the observed difference in banana fruit flesh browning phenotype when banana fruit of the invention is compared to wildtype banana fruit. To explain this in a different manner, without wanting to be bound by theory, it is believed that the truncated PPO1 proteins expressed by the banana plant cell of the invention have a reduced level and / or activity of the endogenous PPO1 gene. By “reduced the level or activity of” endogenous PPO1 it is included that the function of PPO1 is reduced as compared to in “wild-type” banana plants or plant cells. Such reduction in function may be by at least 50%, 60%, 70%, 80%, 90% or 100%. According to some embodiments, reduction is complete loss of function.

[0152] Although premature stop-codons can be introduced into a gene sequence using different gene-editing tools (such as CRISPR / Cas9) to cause a variety of possible edits, the inventors identified that gene-editing that leads to the introduction of an ‘indel’was particularly effective. That is because an indel will often cause a frameshift mutation which alters the reading frame of a gene. That change in reading frame can lead to a triplet of nucleotides that did not encode a stop-codon in the wildtype gene now encoding a so-called “premature stop-codon”. Accordingly, in some embodiments each copy of the PPO1 gene comprises one or more indel, wherein the indel causes a frameshift in the wildtype PPO1 gene sequence leading to each premature stop-codon. In a preferred embodiment, each copy of the PPO1 gene comprises one or two indels, preferably one indel.

[0153] In an alternative embodiment, it might not be necessary for the stop-codons, as defined herein, in the first, second and third copies of the PPO1 gene to be caused by indels, as they could be introduced by other genetic techniques such as base editing, as described herein. Accordingly, in some embodiments only one copy or two copies of the PPO1 gene comprises one or more indel.

[0154] In the context of the present invention, “wildtype” means banana cells (or plants or fruit thereof) with a genetic background the same (or similar to) the banana cells of the invention prior to gene-editing; for example, banana cells from the same original cultivar. The relevant wildtype polynucleotide and polypeptide sequences of PPO1 are described herein.

[0155] In a particular embodiment, the indel is a deletion of 1, 2, 4, 5 or 7 nucleotides or an addition of 1, 2, 4, 5 or 7 nucleotides, preferably a deletion of 1 nucleotide and / or the deletion of 7 nucleotides and / or an insertion of 1 nucleotide. As will be appreciated, the indel in each copy of the PPO1 gene might be of the same size or a different size.

[0156] In a preferred embodiment,

[0157] • the first copy of the PPO1 gene comprises an insertion of one nucleotide; and / or • the second copy of the PPO1 gene comprises a deletion of seven nucleotides;

[0158] and / or

[0159] • the third copy of the PPO1 gene comprises a deletion of one nucleotide.

[0160] The inventors have identified that to generate the premature stop-codons described herein, it can be particularly advantageous to cause indels in unique and conserved upstream PPO1 gene sequences (i.e. to the 5’ of the intended premature stopcodon location), such as GAGACCA (SEQ ID NO: 34) and ACCCGTC (SEQ ID NO: 35). As shown in the Examples, the PPO1 gene sequences that are targeted by the guide RNAs used to generate the exemplified banana plant cells comprise GAGACCA (SEQ ID NO: 34) and ACCCGTC (SEQ ID NO: 35). Accordingly, in a further embodiment the one or more indel is at a polynucleotide sequence of GAGACCA (SEQ ID NO: 34) of the PPO1 geneand / or is at a polynucleotide sequence of ACCCGTC (SEQ ID NO: 35) of the PPO1 gene. In a specific embodiment, the one or more indel is selected from the group consisting of:

[0161] • an insertion of any one nucleotide in GAGACCA (SEQ ID NO: 34), preferably the insertion of any one nucleotide after position 3 or 4 in GAGACCA (SEQ ID NO: 34) of the PPO1 gene, most preferably the insertion of an adenine;

[0162] • a deletion of GAGACCA (SEQ ID NO: 34) of the PPO1 gene; or

[0163] • a deletion of any one nucleotide from ACCCGTC (SEQ ID NO: 35) of the PPO1 gene, preferably the deletion of a cytosine, most preferably the cytosine at position 4 from ACCCGTC (SEQ ID NO: 35).

[0164] In some embodiments:

[0165] • the first PPO1 gene comprises an insertion of any one nucleotide in GAGACCA (SEQ ID NO: 34), preferably the insertion of any one nucleotide after position 3 or 4 in GAGACCA (SEQ ID NO: 34), most preferably the insertion of an adenine; and / or • the second PPO1 gene comprises a deletion of GAGACCA (SEQ ID NO: 34); and / or • the third PPO1 gene comprises a deletion of any one nucleotide from ACCCGTC (SEQ ID NO: 35), preferably the deletion of a cytosine, most preferably the cytosine at position 4 of ACCCGTC (SEQ ID NO: 35).

[0166] In a further aspect, the invention provides a banana plant cell,

[0167] wherein the banana plant cell comprises three copies of a PPO1 gene, and wherein:

[0168] • a first PPO1 gene comprises: (1 ) an insertion of any one nucleotide in a nucleotide sequence GAGACCA (SEQ ID NO: 34) of the PPO1 gene, preferably the insertion of any one nucleotide after position 3 or 4 in GAGACCA (SEQ ID NO: 34), most preferably the insertion of an adenine; and (2) a premature stop-codon TGA (SEQ ID NO: 29) in a nucleotide sequence of the PPO1 gene comprising TGACCT (SEQ ID NO: 31); and / or

[0169] • a second PPO1 gene comprises: (1 ) a deletion of a nucleotide sequence GAGACCA (SEQ ID NO: 34) of the PPO1 gene; and (2) a premature stop-codon TAG (SEQ ID NO: 30) in a nucleotide sequence of the PPO1 gene comprising TAGGCA (SEQ ID NO: 32); and / or

[0170] • a third PPO1 gene comprises: (1) a deletion of any one nucleotide from in a nucleotide sequence ACCCGTC (SEQ ID NO: 35) of the PPO1 gene, preferably the deletion of a cytosine, most preferably the cytosine at position 4 from ACCCGTC (SEQ ID NO: 35); and (2) a premature stop-codon TGA (SEQ ID NO: 29) in a nucleotide sequence of the PPO1 gene comprising TGAGGG (SEQ ID NO: 33),wherein said insertions and / or deletions cause a frameshift in the wildtype PPO1 gene sequence leading to each premature stop-codon.

[0171] In some embodiments,

[0172] • GAGACCA (SEQ ID NO: 34) is in a nucleotide sequence of the PPO1 gene comprising GAGACCACAACCAACCCCTCGT (SEQ ID NO: 36) or CCAGAGACCACAA (SEQ ID NO: 37); and / or

[0173] • ACCCGTC (SEQ ID NO: 35) is in a nucleotide sequence of the PPO1 gene comprising ACCCGTCCTCGCCCTTGAGG (SEQ ID NO: 38) or CCGACCCGTCCTC (SEQ ID NO: 39). These are longer conserved and unique sequences representing the polynucleotide sequences in the PPO1 gene that are targeted by the exemplified guide RNAs. It should be noted that in this embodiment, and as described elsewhere herein, where a first sequence is described as being “in” a second sequence then it would be understood that the first sequence is part of the second sequence; for example GAGACCA (SEQ ID NO: 34) is “in” CCAGAGACCACAA (SEQ ID NO: 37), as shown by the emboldened sequence of SEQ ID NO: 37. Accordingly, it could be considered that the first sequence is “found in” the second sequence; for example, GAGACCA (SEQ ID NO: 34) is found in a nucleotide sequence of the PPO1 gene comprising GAGACCACAACCAACCCCTCGT (SEQ ID NO: 36).

[0174] During the inventors’ analysis of the exemplified banana plant cells, it was also noted that the second PPO1 gene included a further targeted indel at the site of ACCCGTC (SEQ ID NO: 35). However, as this indel was downstream (i.e. to the 3’) of the pre-mature codon it did not contribute to the part of the gene that encoded the truncated protein. Accordingly, in a specific embodiment the first copy of the PPO1 gene comprises one indel, the second copy of the PPO1 gene comprises two indels, and the third copy of the PPO1 gene comprises one indel. In a very specific embodiment, the second PPO1 gene comprises a deletion of any one nucleotide from ACCCGTC (SEQ ID NO: 35), preferably the deletion of a cytosine, most preferably the cytosine at position 4 from ACCCGTC (SEQ ID NO: 35).

[0175] In an alternative aspect, the invention provides a banana plant cell, wherein the banana plant cell comprises three copies of a PPO1 gene, and wherein each copy of the PPO1 gene encodes a truncated polypeptide when compared to the wildtype PPO1 polypeptide.

[0176] In a further alternative aspect, the invention provides a banana plant cell, wherein the banana plant cell comprises three copies of a PPO1 gene, and wherein each copy of the PPO1 gene comprises one or more indel at a polynucleotide sequence ofGAGACCA (SEQ ID NO: 34) of the PPO1 gene and / or is at a polynucleotide sequence of ACCCGTC (SEQ ID NO: 35) of the PPO1 gene, and wherein the indel causes a frameshift in the wildtype PPO1 gene sequence leading to a premature stop-codon.

[0177] In this alternative, and further alternative, aspect, it is included that the three copies of the PPO1 gene might each be independently defined as: the first PPO1 gene; the second PPO1 gene; and / or the third PPO1 gene, as described herein. Accordingly, by way of an example, one, two, or three copies of the PPO1 gene in the alternative aspect may be the first PPO1 gene as described herein; or, byway of another example, two copies of the PPO1 gene in the alternative aspect may be the first PPO1 gene as described herein and the third copy of the PPO1 gene in the alternative aspect may be the second PPO1 gene as described herein.

[0178] PPO1 sequences of the invention

[0179] Information regarding the PPO1 sequences of the invention (both polynucleotide and polypeptide sequences) can be derived from publicly-available reference banana genomes, as well as being provided herein by sequencing undertaken by Tropic Biosciences. The reference banana genomes used are those of Musa acuminata (DH Pahang), of which there are two - Version 2 (V2) (Martin G et al., (2016), Improvement of the banana “Musa acuminata” reference sequence using NGS data and semiautomated bioinformatic methods. BMC Genomics) and Version 4 (V4) (Belser C et al., (2021) Telomere-to-telomere gapless chromosomes of banana using nanopore sequencing, BioRxiv.04.16.440017). Those reference genomes are accessible via the “Banana Genome Hub” (https: / / banana-genome-hub.southgreen.fr).

[0180] The Tropic Biosciences sequencing was undertaken on DNA from nuclei isolated from Musa acuminata ‘Cavendish’ leaf tissue powdered following treatment with liquid nitrogen. The DNA sequencing was undertaken using Oxford Nanopore Technologies’ MinlON and PromethlON machines (based on a library prepared using Oxford Nanopore Technologies’ 1D Ligation), with R9.4.1 flow cells, as well as Illumina’s NovaSeq6000 machine (based on a library prepared using Illumina Nextera (RTM) Flex).

[0181] In some embodiments herein, the PPO1 gene is described with reference to an accession number. An accession number (sometimes referred to as a GenelD, depending on the genome resource being used) is a unique identifier assigned to a particular gene (e.g. PPO1) within a specific sequenced genome (e.g. DH Pahang V2 or DH Pahang V4). Therefore, whilst the accession number used to refer to a particular gene (e.g. PPO1) will differ between specific sequenced genomes the identity of the gene itself will not(e.g. the gene accession number of PPO1 in DH Pahang V2 is Ma06_g31080 and in DH Pahang V4 is Macma4_06_g32590, but the gene itself is the same). Accordingly, when the invention is defined in terms of an accession number it is to be understood as referring to a PP01 gene identifiable by that accession number, and not as a synonym for the specific polynucleotide or polypeptide sequences associated with that accession number in the relevant reference genome.

[0182] A PPO1 gene in any of the embodiments herein can be described as having “sequence identity” or homology to a polynucleotide sequence. The amount of homology or sequence identity can vary, and includes total lengths and / or regions having unit integral values in the ranges of about 1 -20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp or up to and including the total length of the PPO1 gene sequence or polynucleotide sequence. These ranges include every integer within the range, for example, the range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bp. The amount of homology or sequence identity can also be described by percent sequence identity over the full aligned length of the two genes or two polynucleotides, which includes percent sequence identity of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Sufficient homology includes any combination of polynucleotide length, global percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity. For example, sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity to a region of the PPO1 gene sequence or polynucleotide sequence. Sufficient homology can also be described by the predicted ability of two genes or polynucleotides to specifically hybridize under high stringency conditions. In this regard, see, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al., Eds (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and, Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[0183] When reference is made herein to particular sequences, these may also encompass sequences that substantially correspond to their complementary sequences, e.g. including minor sequence variations resulting from, for example, sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or baseaddition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides.

[0184] Any polynucleotide Sequence Identification Number (SEQ ID NO) disclosed herein can refer to either a DNA sequence or an RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or an RNA sequence format. For example, a given SEQ ID NO: is expressed in a DNA sequence format (e.g. reciting T for thymine), but it can refer to either a DNA sequence that corresponds to a given nucleic acid sequence, or the RNA sequence of an RNA molecule nucleic acid sequence. Similarly, though some sequences are expressed in an RNA sequence format (e.g. reciting U for uracil), depending on the actual type of molecule being described, it can refer to either the sequence of an RNA molecule comprising a double-stranded RNA (dsRNA), or the sequence of a DNA molecule that corresponds to the RNA sequence shown. In any event, both DNA and RNA molecules having the sequences disclosed with any substitutes are envisaged. The names of the nucleotides and amino acids used herein with reference to their one-letter code would be well known to one skilled in genetics. For example, for nucleotides: A is adenine; G is guanine; C is cytosine; and T is thymine. For example, for amino acids: Alanine is A; Arginine is R; Asparagine is N; Aspartic acid is D; Cysteine is C; Glutamine is Q; Glutamic acid is E; Glycine is G; Histidine is H; Isoleucine is I; Leucine is L; Lysine is K; Methionine is M; Phenylalanine is F; Proline is P; Serine is S; Threonine is T; Tryptophan is W; Tyrosine is Y; and Valine is V.

[0185] When percentage sequence identity is used in reference to polypeptides, residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given ascore of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g. according to the algorithm of Henikoff and Henikoff (1992), Proc. Natl. Acad. Sci. U. S. A., 89(22): 10915-9. Identity can be determined using any homology comparison software, including for example, the “BlastN” software of the National Center of Biotechnology Information (NCBI) such as by using default parameters. In some embodiments, the identity is a global identity, i.e. an identity over an entire nucleic acid sequence and not over portions thereof.

[0186] The polynucleotide sequences of the wildtype PPO1 gene described herein are SEQ ID NOs: 1-5, with a consensus polynucleotide sequence being SEQ ID NO: 16. The polynucleotide sequences of the CDS of the wildtype PPO1 gene described herein are SEQ ID NOs: 6-10, with a consensus polynucleotide sequence being SEQ ID NO: 18. The polypeptide sequences encoded by the wildtype PPO1 gene described herein are SEQ ID NOs: 11-15, with a consensus polypeptide sequence being SEQ ID NO: 19. Accordingly, in one embodiment the wildtype PPO1 gene sequence comprises a polynucleotide sequence selected from the list consisting of:

[0187] • SEQ ID NO: 16, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 16, preferably wherein said polynucleotide sequence with at least 70% identity comprises TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33), more preferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34) and ACCCGTC (SEQ ID NO: 35);

[0188] • a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 18, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity to SEQ ID NO: 18, preferably wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity comprises TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33), more preferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34) and ACCCGTC (SEQ ID NO: 35); or

[0189] • a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 19, or a polynucleotide sequence that encodes a polypeptide comprising at least 70% identity to SEQ ID NO: 19, preferably wherein said polynucleotide sequence that encodes a polypeptide comprising at least 70% identity comprises TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33), morepreferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34) and ACCCGTC (SEQ ID NO: 35), optionally wherein the at least 70% identity is: at least 75%; at least 80%; at least 85%; at least 90%; at least 95%; or at least 99% identity, preferably at least 90% identity, and more preferably at least 95% identity.

[0190] In one embodiment the wildtype PPO1 gene sequence comprises a polynucleotide sequence of SEQ ID NO: 17, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 17, preferably wherein said polynucleotide sequence with at least 70% identity comprises TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33), more preferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34) and ACCCGTC (SEQ ID NO: 35), optionally wherein the at least 70% identity is: at least 75%; at least 80%; at least 85%; at least 90%; at least 95%; or at least 99% identity, preferably at least 90% identity, and more preferably at least 95% identity.

[0191] In a more specific embodiment, the wildtype PPO1 gene sequence comprises a polynucleotide sequence selected from the list consisting of:

[0192] • any one or more of SEQ ID NOs: 1-5, or a polynucleotide sequence with at least 70% identity to any one or more of SEQ ID NOs: 1-5, preferably wherein said polynucleotide sequence with at least 70% identity comprises TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33); more preferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34) and ACCCGTC (SEQ ID NO: 35);

[0193] • a polynucleotide sequence that encodes a CDS polynucleotide sequence of any one or more of SEQ ID NOs: 6-10, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity to any one or more of SEQ ID NOs: 6-10, preferably wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity comprises TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33), more preferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34) and ACCCGTC (SEQ ID NO: 35); or

[0194] • a polynucleotide sequence that encodes a polypeptide comprising of any one or more of SEQ ID NOs: 11-15, or a polynucleotide sequence that encodes a polypeptide comprising at least 70% identity to of any one or more of SEQ ID NOs: 11-15, preferably wherein said polynucleotide sequence that encodes a polypeptide comprising at least 70% identity comprises TGACCT (SEQ ID NO: 31),TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33), more preferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34) and ACCCGTC (SEQ ID NO: 35), optionally

[0195] wherein the at least 70% identity is: at least 75%; at least 80%; at least 85%; at least 90%; at least 95%; or at least 99% identity, preferably at least 90% identity, and more preferably at least 95% identity.

[0196] In a particularly specific embodiment:

[0197] • the wildtype PPO1 gene sequence of the first copy of the PPO1 gene comprises a polynucleotide sequence selected from the list consisting of:

[0198] a. SEQ ID NO: 3, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 3, preferably wherein said polynucleotide sequence with at least 70% identity comprises TGACCT (SEQ ID NO: 31), more preferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34);

[0199] b. a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 8, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity to SEQ ID NO: 8, preferably wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity comprises TGACCT (SEQ ID NO: 31), more preferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34); or

[0200] c. a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO:

[0201] 13, or a polynucleotide sequence that encodes a polypeptide comprising at least 70% identity to SEQ ID NO: 13, preferably wherein said polynucleotide sequence that encodes a polypeptide comprising at least 70% identity comprises TGACCT (SEQ ID NO: 31), more preferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34), and / or

[0202] • the wildtype PPO1 gene sequence of the second copy of the PPO1 gene comprises a polynucleotide sequence selected from the list consisting of:

[0203] a. SEQ ID NO: 4, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 4, preferably wherein said polynucleotide sequence with at least 70% identity comprises TAGGCA (SEQ ID NO: 32), more preferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34);b. a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 9, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity to SEQ ID NO: 9, preferably wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity comprises TAGGCA (SEQ ID NO: 32), more preferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34); or

[0204] c. a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO:

[0205] 14, or a polynucleotide sequence that encodes a polypeptide comprising at least 70% identity to SEQ ID NO: 14, preferably wherein said polynucleotide sequence that encodes a polypeptide comprising at least 70% identity comprises TAGGCA (SEQ ID NO: 32), more preferably said polynucleotide sequence with at least 70% identity comprises GAGACCA (SEQ ID NO: 34), and / or

[0206] • the wildtype PPO1 gene sequence of the third copy of the PPO1 gene comprises a polynucleotide sequence selected from the list consisting of:

[0207] a. SEQ ID NO: 5, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 5, preferably wherein said polynucleotide sequence with at least 70% identity comprises TGAGGG (SEQ ID NO: 33), more preferably said polynucleotide sequence with at least 70% identity comprises ACCCGTC (SEQ ID NO: 35);

[0208] b. a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 10, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity to SEQ ID NO: 10, preferably wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity comprises TGAGGG (SEQ ID NO: 33), more preferably said polynucleotide sequence with at least 70% identity comprises ACCCGTC (SEQ ID NO: 35); or

[0209] c. a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO:

[0210] 15, or a polynucleotide sequence that encodes a polypeptide comprising at least 70% identity to SEQ ID NO: 15, preferably wherein said polynucleotide sequence that encodes a polypeptide comprising at least 70% identity comprises TGAGGG (SEQ ID NO: 33), more preferably said polynucleotide sequence with at least 70% identity comprises ACCCGTC (SEQ ID NO: 35), optionallywherein the at least 70% identity is: at least 75%; at least 80%; at least 85%; at least 90%; at least 95%; or at least 99% identity, preferably at least 90% identity, and more preferably at least 95% identity.

[0211] In a particular embodiment, the wildtype PPO1 gene sequence comprises a polynucleotide sequence that is a PPO1 homolog of one or more of SEQ ID NOs: 1-5, preferably a PPO1 homolog of Musa acuminata. In a further embodiment, the wildtype PPO1 gene sequence comprises a polynucleotide sequence that is a PPO1 paralog of one or more of SEQ ID NOs: 1 -5, preferably a PPO1 paralog of Musa acuminata. In a further embodiment, the wildtype PPO1 gene sequence comprises a polynucleotide sequence that is a PPO1 ortholog of one or more of SEQ ID NOs: 1-5, preferably a PPO1 ortholog of Musa acuminata. Accordingly, in one embodiment the wildtype PPO1 gene sequence comprises a polynucleotide sequence of SEQ ID NOs: 1 -5, or a (PPO1) homolog, or (PPO1) paralog, or (PPO1) ortholog thereof; preferably a (PPO1) homolog, or (PPO1) paralog, or (PPO1) ortholog of Musa acuminata. The terms homolog, paralog and ortholog would be known to a person skilled in genetics.

[0212] In some embodiments, the wildtype PPO1 gene sequence comprises a polynucleotide sequence that is identifiable from accession number Ma06_g31080 and / or accession number Macma4_06_g32590. In a further embodiment, the wildtype PPO1 gene sequence comprises a polynucleotide sequence that encodes a CDS polynucleotide sequence and / or a polypeptide that is identifiable from accession number Ma06_t31080.1 and / or accession number Macma4_06_g32590.1.

[0213] As described herein, a particularly relevant element of the present invention is that the PP01 polypeptides encoded by the edited PPO1 genes are truncated. Accordingly, in some embodiments the PPO1 gene encodes a polypeptide of up to about 135 amino acids in length, preferably about 65 amino acids to about 135 amino acids in length. In a specific embodiment:

[0214] • the first PPO1 gene encodes a polypeptide of about 95 to about 100 amino acids in length, preferably about 97 amino acids in length; and / or

[0215] • the second PPO1 gene encodes a polypeptide of about 65 to about 70 amino acids in length, preferably about 67 amino acids in length; and / or

[0216] • the third PPO1 gene encodes a polypeptide of about 130 to about 135 amino acids in length, preferably about 132 amino acids in length.

[0217] In addition to the edits in the banana plant cells of the invention being described in the context of the wildtype polynucleotide sequences as outlined above, they can also be defined with reference to the edited polynucleotide sequences as outlined below.The indel described herein in which an adenine is inserted after position 3 or 4 in GAGACCA (SEQ ID NO: 34) results in the sequence of GAGAACCA (SEQ ID NO: 43), which can also be considered as the extended sequence of AGAGAACCAC (SEQ ID NO: 40). Accordingly, in some embodiments the PPO1 gene, preferably the first PPO1 gene, comprises GAGAACCA (SEQ ID NO: 43). Accordingly, in some embodiments the PPO1 gene, preferably the first PPO1 gene, comprises AGAGAACCAC (SEQ ID NO: 40). It will be appreciated that any embodiments that refer to AGAGAACCAC (SEQ ID NO: 40) may also instead include GAGAACCA (SEQ ID NO: 43), or vice versa.

[0218] The indel described herein in which GAGACCA (SEQ ID NO: 34) is deleted results in the sequence of CCACAA (SEQ ID NO: 41), which can also be considered as the extended sequence TCCACAAC (SEQ ID NO: 90). Accordingly, in some embodiments the PPO1 gene, preferably the second PPO1 gene, comprises CCACAA (SEQ ID NO: 41). Accordingly, in some embodiments the PPO1 gene, preferably the second PPO1 gene, comprises TCCACAAC (SEQ ID NO: 90). It will be appreciated that any embodiments that refer to CCACAA (SEQ ID NO: 41) may also instead include TCCACAAC (SEQ ID NO: 90), or vice versa.

[0219] The indel described herein in which a cytosine is deleted at position 4 of ACCCGTC (SEQ ID NO: 35) results in the sequence of ACCGTC (SEQ ID NO: 42) which can also be considered as the extended sequence GACCGTCC (SEQ ID NO: 91 ). Accordingly, in some embodiments the PPO1 gene, preferably the third PPO1 gene, comprises ACCGTC (SEQ ID NO: 42). Accordingly, in some embodiments the PPO1 gene, preferably the third PPO1 gene, comprises GACCGTCC (SEQ ID NO: 91). It will be appreciated that any embodiments that refer to ACCGTC (SEQ ID NO: 42) may also instead include GACCGTCC (SEQ ID NO: 91), or vice versa.

[0220] In a particular embodiment:

[0221] • the first copy of the PPO1 gene comprises AGAGAACCAC (SEQ ID NO: 40); and / or • the second copy of the PPO1 gene comprises CCACAA (SEQ ID NO: 41); and / or • the third copy of the PPO1 gene comprises ACCGTC (SEQ ID NO: 42).

[0222] In a specific embodiment:

[0223] • the first copy of the PPO1 gene comprises a polynucleotide sequence of SEQ ID NO: 83, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 83, preferably wherein said polynucleotide sequence with at least 70% identity comprises AGAGAACCAC (SEQ ID NO: 40);

[0224] • the second copy of the PPO1 gene comprises a polynucleotide sequence of SEQ ID NO: 84, or a polynucleotide sequence with at least 70% identity to SEQ ID NO:84, preferably wherein said polynucleotide sequence with at least 70% identity comprises CCACAA (SEQ ID NO: 41); and

[0225] • the third copy of the PPO1 gene comprises a polynucleotide sequence of SEQ ID NO: 85, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 85, preferably wherein said polynucleotide sequence that encodes a polypeptide comprising at least 70% identity comprises ACCGTC (SEQ ID NO: 42), optionally wherein the at least 70% identity is: at least 75%; at least 80%; at least 85%; at least 90%; at least 95%; or at least 99% identity, preferably at least 90% identity, and more preferably at least 95% identity.

[0226] In a specific embodiment:

[0227] • the first copy of the PPO1 gene comprises a polynucleotide sequence selected from the list consisting of:

[0228] a. SEQ ID NO: 20, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 20, preferably wherein said polynucleotide sequence with at least 70% identity comprises TGACCT (SEQ ID NO: 31) and AGAGAACCAC (SEQ ID NO: 40); b. a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 23, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity to SEQ ID NO: 23, preferably wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity comprises TGACCT (SEQ ID NO: 31) and AGAGAACCAC (SEQ ID NO: 40); or

[0229] c. a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO:

[0230] 26, or a polynucleotide sequence that encodes a polypeptide comprising at least 70% identity to SEQ ID NO: 26, preferably wherein said polynucleotide sequence that encodes a polypeptide comprising at least 70% identity comprises TGACCT (SEQ ID NO: 31) and AGAGAACCAC (SEQ ID NO: 40), and / or • the second copy of the PPO1 gene comprises a polynucleotide sequence selected from the list consisting of:

[0231] a. SEQ ID NO: 21, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 21, preferably wherein said polynucleotide sequence with at least 70% identity comprises TAGGCA (SEQ ID NO: 32) and CCACAA (SEQ ID NO: 41); b. a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 24, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity to SEQ ID NO: 24, preferably wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with atleast 70% identity comprises TAGGCA (SEQ ID NO: 32) and CCACAA (SEQ ID NO: 41); or

[0232] c. a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO:

[0233] 27, or a polynucleotide sequence that encodes a polypeptide comprising at least 70% identity to SEQ ID NO: 27, preferably wherein said polynucleotide sequence that encodes a polypeptide comprising at least 70% identity comprises TAGGCA (SEQ ID NO: 32) and CCACAA (SEQ ID NO: 41), and / or • the third copy of the PPO1 gene comprises a polynucleotide sequence selected from the list consisting of:

[0234] a. SEQ ID NO: 22, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 22, preferably wherein said polynucleotide sequence with at least 70% identity comprises TGAGGG (SEQ ID NO: 33) and ACCGTC (SEQ ID NO: 42); b. a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 25, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity to SEQ ID NO: 25, preferably wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity comprises TGAGGG (SEQ ID NO: 33) and ACCGTC (SEQ ID NO: 42); or

[0235] c. a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO:

[0236] 28, or a polynucleotide sequence that encodes a polypeptide comprising at least 70% identity to SEQ ID NO: 28, preferably wherein said polynucleotide sequence that encodes a polypeptide comprising at least 70% identity comprises TGAGGG (SEQ ID NO: 33) and ACCGTC (SEQ ID NO: 42), optionally wherein the at least 70% identity is: at least 75%; at least 80%; at least 85%; at least 90%; at least 95%; or at least 99% identity, preferably at least 90% identity, and more preferably at least 95% identity.

[0237] In a specific aspect, the invention provides a banana plant cell wherein the banana plant cell comprises three copies of a PPO1 gene, and wherein:

[0238] • a first copy of the PPO1 gene comprises SEQ ID NO: 20, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 20;

[0239] • a second copy of the PPO1 gene comprises SEQ ID NO: 21, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 21; and

[0240] • a third copy of the PPO1 gene comprises SEQ ID NO: 22, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 22,optionally wherein the at least 70% identity is: at least 75%; at least 80%; at least 85%; at least 90%; at least 95%; or at least 99% identity, preferably at least 90% identity, and more preferably at least 95% identity.

[0241] In a further specific aspect, the invention provides a banana plant cell wherein the banana plant cell comprises three copies of a PPO1 gene, and wherein:

[0242] • a first copy of the PPO1 gene comprises a polynucleotide sequence of SEQ ID NO:

[0243] 83, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 83;

[0244] • a second copy of the PPO1 gene comprises a polynucleotide sequence of SEQ ID NO: 84, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 84; and

[0245] • a third copy of the PPO1 gene comprises a polynucleotide sequence of SEQ ID NO: 85, or a polynucleotide sequence with at least 70% identity to SEQ ID NO: 85,

[0246] optionally wherein the at least 70% identity is: at least 75%; at least 80%; at least 85%; at least 90%; at least 95%; or at least 99% identity, preferably at least 90% identity, and more preferably at least 95% identity.

[0247] In a further specific aspect, the invention provides a banana plant cell wherein the banana plant cell comprises three copies of a PPO1 gene, and wherein:

[0248] • a first copy of the PPO1 gene comprises a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 23, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity to SEQ ID NO: 23;

[0249] • a second copy of the PPO1 gene comprises a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 24, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 70% identity to SEQ ID NO: 24; and

[0250] • a third copy of the PPO1 gene comprises a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 25, or a polynucleotide sequence that encodes a polypeptide comprising at least 70% identity to SEQ ID NO: 25,

[0251] optionally wherein the at least 70% identity is: at least 75%; at least 80%; at least 85%; at least 90%; at least 95%; or at least 99% identity, preferably at least 90% identity, and more preferably at least 95% identity.

[0252] In a further specific aspect, the invention provides a banana plant cell wherein the banana plant cell comprises three copies of a PPO1 gene, and wherein:• a first copy of the PP01 gene comprises a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 26, or a polynucleotide sequence that encodes a polypeptide comprising at least 70% identity to SEQ ID NO: 26;

[0253] • a second copy of the PPO1 gene comprises a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 27, or a polynucleotide sequence that encodes a polypeptide comprising at least 70% identity to SEQ ID NO: 27; and

[0254] • a third copy of the PPO1 gene comprises a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 28, or a polynucleotide sequence that encodes a polypeptide comprising at least 70% identity to SEQ ID NO: 28, optionally wherein the at least 70% identity is: at least 75%; at least 80%; at least 85%; at least 90%; at least 95%; or at least 99% identity, preferably at least 90% identity, and more preferably at least 95% identity.

[0255] The banana plants, and parts thereof, of the invention

[0256] As used herein, the term “banana plant cell” is a cell of a banana plant. Banana plant cells include cells of a banana fruit, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, sporophytes, microspores, embryogenic cells, somatic cells, and protoplasts. Protoplasts can be derived from any plant tissue, such as, but not limited to, roots, leaves, embryonic cell suspension, callus, or seedling tissue. According to some embodiments, a banana plant cell is a cell of an Embryogenic Cell Suspension (ECS). In a particular embodiment, the banana cell is a non-regenerable banana cell.

[0257] As used herein, the term “plant” refers to whole plants, grafted plants, ancestors and progeny of the plants, plant organs, plant tissues, and “plant parts”. “Plant parts”, as used herein, include differentiated and undifferentiated tissues including, but not limited to roots (including tubers), rootstocks, stems, scions, shoots, fruits, leaves, pollens, seeds, tumor tissue, and various forms of cells and culture (e.g. single cells, protoplasts, embryos, embryonic cells, and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture.

[0258] In particular embodiments, the plant part is a banana fruit. Banana fruit comprises tissues such as fruit flesh and fruit peel.

[0259] The term “plant organ” refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. The term “genome” refers to the entire complement of genetic material (genes and non-coding sequences) that ispresent in each cell of an organism. “Progeny” comprises any subsequent generation of a plant.

[0260] A particular aspect of the invention is a banana plant or banana fruit comprising a banana plant cell of the invention. The banana fruit, or the banana fruit from the banana plant, is characterised as having banana fruit flesh having a phenotype of reduced browning as compared to banana fruit flesh of a wildtype banana fruit.

[0261] A “transgenic plant” includes, for example, a plant which comprises within its genome a heterologous polynucleotide introduced by a transformation step. The heterologous polynucleotide can be stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct. A transgenic plant can also comprise more than one heterologous polynucleotide within its genome. Each heterologous polynucleotide may confer a different trait to the transgenic plant.

[0262] A “heterologous” polynucleotide, as used herein, comprises a sequence that originates from a foreign species.

[0263] “Transgenic” can include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. In some plants, the heterologous polynucleotide which is introduced into the plant genome can be removed through breeding. This process is not possible in banana, as described hereinabove.

[0264] According to some embodiments, the banana plant cells, banana plants, or banana plant parts (in particular, banana fruit) described herein are non-transgenic. According to some embodiments, the methods disclosed herein result in a banana plant cell, banana plant or banana plant part (in particular, banana fruit) which is non-transgenic.

[0265] The alterations of the genome by the genome editing described herein (that does not result in an insertion of a foreign polynucleotide) are not intended to be regarded as transgenic.

[0266] As used herein, the term “banana plant” refers to a plant of the genus Musa, including plantains. These include Musa acuminata (e.g. Musa acuminata banksia, Musa acuminata Calcutta, and Musa acuminata DH-Pahang), Musa balbisiana, Musa itinerans, and autotriploid Musa acuminata ‘Cavendish’ and ‘Gros Michel’. A specific cultivar of Musa acuminata ‘Cavendish’ is Grande Naine. Cultivated bananas are infertileautotriploids (AAA) derived from the progenitor species Musa acuminata (genome AA). Additionally, plantains (AAB or ABB) are infertile interspecific allotriploids derived from the hybridisation of Musa acuminata (AA) and Musa balbisiana (genome BB). The triploid nature of cultivated banana and plantain prevents them from producing viable seeds, whereas wild species are diploid and can produce viable seeds. In a preferred embodiment, the banana plant is an autotriploid banana plant, preferably the autotriploid is an autotriploid Musa acuminata, even more preferably the autotriploid is an autotriploid Musa acuminata ‘Cavendish’. In a specific embodiment, the autotriploid Musa acuminata ‘Cavendish’ is Grande Naine.

[0267] The banana plant cells of the invention can be characterised as being derived from the banana plants as described herein; for example, the banana plant cell is an autotriploid banana plant cell, preferably the autotriploid banana plant cell is an autotriploid Musa acuminata banana plant cell, even more preferably the autotriploid banana plant cell is an autotriploid Musa acuminata ‘Cavendish’ banana plant cell.

[0268] In some embodiments, the “banana plant” is, or is derived from, an elite line or purebred line, or an associated banana variety or breeding germplasm, which refers to a line of a cultivated banana having commercially valuable or agronomically desirable characteristics, as opposed to wild varieties or landraces. An “elite line” is selected for based on superior agronomic performance comprising a multitude of agronomically desirable traits. An “elite plant” is any plant from an elite line. Superior agronomic performance refers to a desired combination of agronomically desirable traits as defined herein, wherein it is desirable that the majority, preferably all of, the agronomically desirable traits are improved in the elite breeding line as compared to a non-elite breeding line.

[0269] The terms “cultivar” and “variety” are used interchangeably herein and denote a plant with has deliberately been developed for the purpose of being commercialised, e.g. used by farmers and growers, to produce agricultural products.

[0270] In a particular embodiment, the banana plant cell of the invention (or banana plant regenerated thereof) is not obtained by means of an essentially biological process.

[0271] In the context of the present invention, “browning” of banana fruit flesh can be measured by visual inspection and other methods known in the art.

[0272] As described in the Examples, the browning of banana fruit flesh can be measured using a homogenisation assay (which may also be considered an enzymatic assay). In a homogenisation assay, a known amount of the banana fruit flesh (with the banana fruit peel having been removed) is homogenised in an electrical blender or foodprocessor. Any browning of the homogenate can be observed over time (for example, at time points of 0 minutes, 30 minutes, 60 minutes, four hours, and / or 24 hours, or more) to identify the differing rates of browning between banana fruits (such as the banana fruit of the invention, and that from a wildtype plant).

[0273] Browning can also be measured based on the amount of melanin formed in the banana tissue being analysed within a set time frame. Such a quantification of browning can be performed in other simple biochemical assays (Escalante-Minakata et al., (2018) 3 Biotech, 8:30; Chi et al., (2014) BMC Plant Biology, 14:62; Sullivan et al., (2004) Plant Physiology, 136 (2) 3234-3244; DOI: 10.1104 / pp.104.047449; Escobar et al., (2008) Journal of the American Society for Horticultural Science, Volume 133: Issue 6, pages 852-858; DOI: 10.21273 / JASHS.133.6.852; and Constabel & Ryan (1998), Phytochemistry, 47: 507). In these other enzymatic browning assays, a change in the absorbance of an exogenous PPO substrate is measured after mixing with banana tissue lysate, and both the amount of product formed (melanin) and the rate of the browning reaction are proportional to PPO activity.

[0274] By “reduced browning", it is included that the rate of browning and / or the extent of browning of the fruit flesh of a banana fruit of the invention is lower than that of the fruit flesh of a wildtype banana fruit. Accordingly, in some embodiments reduced browning is characterised by the rate of browning and / or the extent of browning of the fruit flesh of a banana fruit of the invention being lower than that of the fruit flesh of a wildtype banana fruit, preferably reduced browning is characterised by the rate of browning and the extent of browning of the fruit flesh of a banana fruit of the invention being lower than that of the fruit flesh of a wildtype banana fruit. Browning may be measured as outlined above, and / or as described in the Examples.

[0275] By “rate of browning", it is included that the time it takes the fruit flesh of a banana fruit of the invention to brown is slower than that of fruit flesh of a wildtype banana fruit; for example, it takes the fruit flesh of a banana fruit of the invention longer to reach a specific stage of browning when compared to a fruit flesh of a wildtype banana fruit. For example, “rate of browning” can be characterised as it taking at least twice as long, at least three times as long, at least four times as long, at least five times as long, at least six times as long, at least seven times as long, at least eight times as long, at least nine times as long, or at least ten times as long, for fruit flesh of a banana fruit of the invention to brown when compared to fruit flesh of a wildtype banana plant. As will be appreciated by one skilled in the art, the rate of browning might preferablybe defined with reference to a specific level of browning, with the rate being the time it takes for the banana fruit flesh to reach said level.

[0276] By “extent of browning", it is included that the amount of browning and / or the darkness of the browning of the fruit flesh of a banana fruit of the invention is less than that of the fruit flesh of a wildtype banana fruit. In some embodiments, the extent of browning is a final extent of browning, after which the amount of browning and / or the darkness of the browning does not increase further.

[0277] In some embodiments, “reduced” browning means that less melanin is formed at a defined time point. For example, there may be at least a 10%, 20%, 30%, 40%, 50%, three-fold, 5-fold, 10-fold or larger reduction in melanin in fruit flesh of a banana fruit of the invention to occur as compared to fruit flesh of a wildtype banana fruit.

[0278] As will be appreciated, the rate of browning (and so specific time periods, level and extent of browning relevant to the assessment) of a particular gene-edited banana fruit might differ depending on the type of assay used to measure it; for example, the rate of browning in a homogenisation assay may be quicker than if the fruit is simply cut, because, without wanting to be bound by theory, the homogenisation assay causes more cellular damage more greatly catalysing the enzymatic reaction.

[0279] Processed banana fruit products of the invention

[0280] The harvested banana fruit of the invention, with reduced browning, is particularly useful for inclusion in processed banana products, as shown in Example 2 and 3. That is because the methods used to make such products often lead to browning of the banana fruit catalysed by the PPO genes, such as PPO1, which leads to the processed product having greatly reduced consumer appeal and generally a lower quality. As the banana fruits of the current invention having the described gene-edited PPO1 genes have reduced browning then that deleterious effect does not occur during processing, leading to a processed banana product of a much higher quality. A “processed banana product” includes a banana fruit of the invention that has been manipulated or changed when compared to the banana fruit that was harvested, such that following harvest the banana fruit has been manipulated or changed, and likely damaged, through human intervention.

[0281] Accordingly, a further aspect of the invention provides a method of producing a processed banana product, comprising processing a banana fruit of the invention.

[0282] In one embodiment, processing the banana fruit comprises one or more step from the list consisting of: peeling the banana fruit; slicing or cutting the banana fruit;mashing the banana fruit; pureeing the banana fruit; blending the banana fruit; and mixing the banana fruit with other ingredients.

[0283] An additional further aspect of the invention provides a processed banana product comprising a banana fruit of the invention. In a preferred embodiment, the processed banana product is a processed banana food product, preferably a processed banana human food product.

[0284] In one embodiment, the processed banana product comprises one or more product from the list consisting of: a peeled banana fruit; a slice of banana fruit; a puree comprising banana fruit; a liquid (such as a smoothie) comprising banana fruit; a fruit salad comprising banana fruit. In a particular embodiment, the smoothie further comprises a dairy liquid (such as milk) and / or a non-banana fruit (for example: apples; pears; berries (such as strawberries and blueberries); peaches; and grapes) and / or a vegetable and / or a herb, preferably a dairy liquid and a non-banana fruit. In a further embodiment, the milk is semi-skimmed milk. It should be noted that the processed banana product will generally be characterised by two or more of the aforementioned product definitions; for example, a slice or puree of the banana fruit will also generally be a peeled banana fruit.

[0285] In addition to the clear aesthetic and quality advantages of using banana fruit flesh of the invention when mixed with other food products (for example, mixed with other fruit in a fruit salad and / or a smoothie), doing so might also improve the retention of other fruit components in the drink that are known to have health benefits. A study has recently found that PPO activity in drinks reduces the bioavailability of flavan-3-ols, which are known to positively benefit health (Ottaviani et al., 2023, Food Funct., 14, 8217). Without being bound by theory, the inventors consider that using the banana fruit flesh of the invention when mixed with other food products might lead to processed banana products with increased bioavailability of pro-cyanidins (such as flavan-3-ols).

[0286] Accordingly, in a preferred embodiment, the aforementioned processed banana product comprising other ingredients, comprises other ingredients comprising high levels of pro-cyanidins (such as flavan-3-ols). The person skilled in nutrition would know what is considered to be a high level of pro-cyanidins, and what ingredients would be classified in such a way. In a particular embodiment, the ingredient comprising high levels of pro-cyanidins is a fruit; for example, a fruit selected from the list consisting of: apples; pears; berries (such as strawberries and blueberries); peaches; and grapes.

[0287] As shown in Example 3, the reduced browning of banana fruit flesh of the invention is particularly pronounced when combined with a preservative. Accordingly,the banana fruit of the invention might also be treated with preservatives, to further enhance the reduction in browning.

[0288] Therefore, a further aspect of the invention is a method comprising:

[0289] • providing a banana fruit of the invention (for example, a processed banana product, such as a slice of banana fruit); and

[0290] • contacting the banana fruit of the invention with a preservative.

[0291] In one embodiment, the contacting comprises dipping the banana fruit in the preservative or placing the banana fruit in the preservative.

[0292] It would be understood by the skilled person that in this context temporary is contacting the banana fruit with the preservative for a limited amount of time.

[0293] In one embodiment, the limited amount of time is about 10 minutes or less; for example: about nine minutes or less; about eight minutes or less; about seven minutes or less; about six minutes or less; about five minutes or less; about four minutes or less; about three minutes or less; about two minutes or less; about one minute or less; about 50 seconds or less; about 40 seconds or less; about 30 seconds or less; about 20 seconds or less; about 10 seconds or less; or about five seconds or less, preferably about 10 seconds or less.

[0294] Preservatives that are safe for human consumption and compatible with the banana fruit of the invention would be known to one skilled in the art. In one embodiment, the preservative is selected from the list consisting of: an ascorbate; a citrate; a sulphite; a benzoate; a sorbate; and a tocopherol, preferably an ascorbate, more preferably calcium ascorbate.

[0295] In a particular embodiment, the processed banana product comprises genomic DNA of the banana cells of the invention. From this genomic DNA it is possible to identify that the processed banana product comprises banana cells of the invention, using methods known to those skilled in genetics and as described herein.

[0296] In a further embodiment, the processed banana product comprises non-regenerable banana cells and / or non-regenerable material from banana cells.

[0297] In a particular embodiment, the only type of banana fruit in the processed banana product is a banana fruit of the invention. Having only one type of banana fruit in a processed banana product can be considered a “single source”.

[0298] Methods of making the banana plants of the invention

[0299] A further aspect of the invention provides a method of producing a banana plant of the invention.One aspect of the invention provides a method comprising:

[0300] i. providing to a banana plant cell a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease, and one or more guide RNAs, wherein said banana plant cell comprises three copies of a PPO1 gene, and wherein said CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and said one or more guide RNAs form a complex that enables the CRISPR- associated endonuclease or modified CRISPR-associated endonuclease to introduce a double-strand break in the endogenous PPO1 genes;

[0301] ii. identifying a banana plant cell comprising:

[0302] • a first copy of the PPO1 gene comprises a premature stop-codon TGA (SEQ ID NO: 29) in a nucleotide sequence of the PPO1 gene comprising TGACCT (SEQ ID NO: 31);

[0303] • a second copy of the PPO1 gene comprises a premature stop-codon TAG (SEQ ID NO: 30) in a nucleotide sequence of the PPO1 gene comprising TAGGCA (SEQ ID NO: 32); and

[0304] • a third copy of the PPO1 gene comprises a premature stop-codon TGA (SEQ ID NO: 29) in a nucleotide sequence of the PPO1 gene comprising TGAGGG (SEQ ID NO: 33).

[0305] As used herein, the term “endogenous” means native to the genome of the banana plant or banana plant cell, and at the native position within the genome.

[0306] In a preferred embodiment, the CRISPR-associated endonuclease is a Cas9 endonuclease.

[0307] As used herein, a “CRISPR-associated endonuclease” (or “Cas”) refers to an endonuclease having an RNA-guided polynucleotide-editing activity and is one of the components of the CRISPR / Cas system for genome editing, which uses at least one additional component, a “guide RNA” (gRNA). In some embodiments of the invention, the “CRISPR-associated endonuclease” is a “Cas9 endonuclease” (or “Cas9”). According to some embodiments, the “CRISPR-associated endonuclease” may be any Cas9 known in the art, such as, but not limited to, SpCas9, SaCas9, FnCas9, NmCas9, St1Cas9, BlatCas9 (Nakade et al., (2017), Bioengineered, 8:3, 265-273, and references therein). In other embodiments, the “CRISPR-associated endonuclease” may be Cpf1, such as, but not limited to, AsCpfl or LbCpfl (Nakade et al., (2017), Bioengineered, 8:3, 265-273, and references therein).

[0308] The terms “guide RNA” or “gRNA” are well known to one skilled in genetics, and as used herein may be used interchangeably and refer to a polynucleotide whichfacilitates the specific targeting of a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease to a target sequence such as a genomic or episomal sequence in a cell. According to some embodiments, gRNAs can be chimeric / uni-molecular (comprising a single RNA molecule, also referred to as single guide RNA or sgRNA) or modular (comprising more than one separate RNA molecule, typically a crRNA and tracrRNA which may be linked, for example by duplexing). According to some embodiments, a gRNA is an sgRNA.

[0309] The gRNA / Cas complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement genomic DNA. For successful binding of Cas, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence. The binding of the gRNA / Cas complex localizes the Cas to the genomic target sequence so that the Cas can cut both strands of the DNA causing a double-strand or double-stranded break, which can be repaired by HR (homologous recombination) or NHEJ (non-homologous endjoining) and are susceptible to specific sequence modification during DNA repair. A significant advantage of CRISPR / Cas is the high efficiency of this system coupled with the ability to easily create synthetic gRNAs. This creates a system that can be readily modified to target different genomic sites and / or to target different modifications at the same site.

[0310] Modified versions of the Cas enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called 'nickases'. With only one active nuclease domain, the Cas nickase cuts only one strand of the target DNA, creating a single-strand break or “nick”. A single-strand break or single-stranded break, or nick, is mostly repaired by single strand break repair mechanism involving proteins such as but not only, PARP (sensor) and XRCCl / LIG III complex (ligation). However, two proximal, opposite strand nicks introduced by a Cas nickase are treated as a double-strand break, in what is often referred to as a “double nick” CRISPR system. A double-nick, which is basically non-parallel DSB, can be repaired like other DSBs by HR or NHEJ depending on the desired effect on the gene target and the presence of a donor sequence and the cell cycle stage.

[0311] As used herein, a “modified CRISPR-associated endonuclease” (or “modified Cas”) refers to a Cas in which the catalytic domain has been altered and / or which are fused to additional domain. According to some embodiments, a “modified Cas” refers to a Cas which contains inactive catalytic domains (dead Cas, or dCas) and has no nuclease activity while still being able to bind to DNA based on gRNA specificity. According to some embodiments, a “modified Cas” refers to a Cas which has a nickaseactivity (“nCas9”), thus inducing a single strand break. In some embodiments, the modified CRISPR-associated endonuclease is a “modified Cas9 endonuclease”, possibly a catalytically inactive Cas9 (or “dCas9”) or a nickase Cas9 (“nCas9”).

[0312] In the context of the invention, modified Cas, such as dCas or nCas9, can also be used according to some embodiments together with other enzymes (possibly as a fusion protein) for base-editing. Base editing is a genome editing approach that uses components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks. DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing by-products (Rees and Liu (2018), Nature Reviews Genetics, 19(12): 770-788). According to some embodiments, the modified Cas9 is an nCas fused to a base editor enzyme such as an adenosine or cytidine deaminase. Particular base editors contemplated include APOBEC, BE1, BE2, BE3, HF-BE3, BE4, BE4max, BE4-GAM, YE1 -BE3, EE-BE3, YE-BE3, YEE-BE3, VQR-BE3, VRER-BE3, Sa-BE3, Sa-BE4, SaBE4-Gam, SaKKH-BE3, Cas12a-BE, Target-AID, Target-AID-NG, xBE3, eA3A-BE3, A3A-BE3, BE-PLUS, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, SaKKH-ABE (Rees and Liu (2018), Nature Reviews Genetics, 19(12): 770-788, and references therein).

[0313] As used herein, the “guide RNA” (or “gRNA”) is not limited to a particular sequence, provided that the sequence is either specific to the at least one polyphenol oxidase gene.

[0314] As described in the Examples, two exemplary guides were used as part of the present invention - sg857 and sg858. The guides comprise a crRNA (including a variable and constant region) connected to a tracrRNA via a Linker Loop, with the tracerRNA ending in a PolyT Terminator. The two guides only differ in the crRNA regions, which is GAGGGGTTGGTTGTGGTCTC (SEQ ID NO: 44) for sg857 and CCTCAAGGGCGAGGACGGGT (SEQ ID NO: 45) for sg858. The remaining sequences, which are the same between the two guides, are the crRNA constant region GTTTTAGAGCTA (SEQ ID NO: 46), the Linker Loop GAAA (SEQ ID NO: 47), the tracer RNA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC

[0315] (SEQ ID NO: 48) and the Poly T terminator TTTTTTT (SEQ ID NO: 49).In a particular embodiment, the guide RNA comprises a polynucleotide sequence comprising GAGGGGTTGGTTGTGGTCTC (SEQ ID NO: 44) or CCTCAAGGGCGAGGACGGGT (SEQ ID NO: 45).

[0316] In a specific embodiment, the guide RNA comprises GAGGGGTTGGTTGTGGTCTCGTTTTAGAGCTA (SEQ ID NO: 50) or CCTCAAGGGCGAGGACGGGTGTTTTAGAGCTA (SEQ ID NO: 51).

[0317] In a particular embodiment, the guide RNA comprises one or more (preferably all) of:

[0318] • GAAA (SEQ ID NO: 47);

[0319] • TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG TCGGTGC (SEQ ID NO: 48); and

[0320] • TTTTTTT (SEQ ID NO: 49).

[0321] In a specific embodiment, the guide RNA comprises GAGGGGTTGGTTGTGGTCTCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGT TATCAACTTGAAAAAGTGGCACCGAGTCGGTGC I I I I I I I (SEQ ID NO: 52) or CCTCAAGGGCGAGGACGGGTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGT TATCAACTTGAAAAAGTGGCACCGAGTCGGTGC I I I I I I I (SEQ ID NO: 53).

[0322] According to some embodiments, the Cas / modified Cas and at least one gRNA are provided to a banana cell by introducing one or more vectors which express the Cas / modified Cas and / or the at least one gRNA. The insertion vector can contain both cassettes on a single plasmid, or the cassettes are expressed from two separate plasmids. CRISPR plasmids are commercially available (such as the px330 plasmid from Addgene). The use of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) -guide RNA technology and a Cas endonuclease for modifying plant genomes are also at least disclosed by Svitashev et al. (2015), Plant Physiology, 169 (2): 931 -945; Kumar and Jain, 2015, Journal of Experimental Botany, 66: 47-57; and in U. S. Patent Application Publication No. 20150082478, which is specifically incorporated herein by reference in its entirety.

[0323] In some embodiments, the method of the invention further comprises identifying at least one banana plant cell that comprises the modification of the PPO1 gene as described herein, such as an indel as described herein.

[0324] As used herein, “identifying” can include any technique known in the art capable of detecting the modification or editing event (such as an indel) such as, but not limited to, DNA sequencing (e.g. next generation sequencing), electrophoresis, an enzymebased mismatch-detection assay, and a hybridization assay such as PCR, RT-PCR, RNaseprotection, in-situ hybridization, primer extension, Southern blot, Northern Blot and dot blot analysis. Various methods used for detection of single nucleotide polymorphisms (SNPs) can also be used, such as PCR based T7 endonuclease, Heteroduplex and Sanger sequencing. Another method of validating the presence of a DNA editing event (such as insertion-deletion events (or “indels”)) comprises a mismatch cleavage assay that makes use of a structure selective enzyme (e.g. endonuclease) that recognizes and cleaves mismatched DNA. Other methods of validating the presence of editing events are described in length in Zischewski (2017), Biotechnology Advances 1(1):95-104.

[0325] In other embodiments, said CRISPR-associated endonuclease or modified CRISPR-associated endonuclease, and / or the one or more guide RNAs, are provided to the banana plant cell in RNA form. In yet other embodiments, the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease is provided to the banana plant cell in protein form and the one or more guide RNAs are provided to said banana plant cell in RNA form. In some embodiments, the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and the one or more guide RNAs are provided to the banana plant cell as a ribonucleoprotein complex.

[0326] There are a number of methods of introducing DNA, RNA, peptides and / or proteins or combinations of nucleic acids and peptides into plant cells. These include, for example, protoplast transformation (U. S. Pat. No. 5,508,184); desiccation / inhibition-mediated DNA uptake (Potrykus et al. (1985) Mol. Gen. Genet. 199: 183-8); electroporation (U. S. Pat. No. 5,384,253); agitation with silicon carbide fibres (U. S. Pat. Nos. 5,302,523 and 5,464,765); Agrobacterium- mediated transformation (U. S. Pat. Nos.

[0327] 5,563,055, 5,591,616, 5,693,512, 5,824,877, 5,981,840, and 6,384,301); acceleration of DNA-coated particles (U. S. Pat. Nos. 5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865) and nanoparticles, nanocarriers and cell penetrating peptides (WO201126644A2; W02009046384A1; W02008148223A1 ). Other methods of transfection include the use of transfection reagents (e.g. Lipofectin, ThermoFisher), dendrimers (Kukowska-Latallo, J. F. et al. (1996), Proc. Natl. Acad. Sci. USA 93, 4897-902), cell penetrating peptides (Mae et al. (2005), Biochimica et Biophysica Acta 1669(2): 101-7) or polyamines (Zhang and Vinogradov (2010), J Control Release, 143(3):359-366).

[0328] In some embodiments of the invention, the banana plant cell is a protoplast, an embryogenic cell, and / or contained in an embryogenic cell suspension.

[0329] In some embodiments of the invention, the method further comprises regenerating a banana plant from said banana plant cell.When the PP01 gene sequences particularly relevant to the reduced browning banana plant cells of the invention had been identified, it becomes possible that an additional method of Homology-Directed Repair (HDR) can be used to produce the banana plants described herein. The use of HDR, and the genetic constructs and methods needed, would be conceivable to one skilled in genetics when provided with the relevant edited PPO1 sequences described herein.

[0330] In general terms, HDR uses gene-editing tools (such as CRISPR / Cas9) to generate a break within the genome, in a similar way to other methods described herein which generate indels (as described in the reviews: Xu et al., 2020, Front Genet. 21;11:412; Liao et al., 2024 Mol Ther Nucleic Acids;35(4):102344; and Singh et al., 2023, Plant Molecular Biology, Volume 111, pages 1-20, as well as https: / / blog.addgene.org / crispr-101-homology-directed-repair - last updated in January 2023). However, HDR uses genetic constructs that include an additional component - a so-called ‘donor’. The donor includes a specific - usually non-wildtype - sequence to be introduced into the genome, which is bordered on either side with regions of homology to the wildtype genome - so-called ‘homology arms’. Following a cut being made in the genome, the homology arms of the donor mediate a repair which introduces the non-wildtype sequence at that specific location, which essentially replaces a previous region of the genome with said, new sequence. The specific non-wildtype sequence to be introduced can be the whole or part of a gene (i.e. PPO1) including any characterised gene-edits.

[0331] As an example, the specific non-wildtype sequence could be SEQ ID NOs: 83-85 (i.e. the gene-edited PPO1 genes from the start codon to the premature stop codon) with the upstream homology arm being the 5’ Untranslated Region (UTR) and genome sequences upstream of that, with the downstream homology arm being the region of the PPO1 gene that is downstream of the premature stop codon as well as the 3’UTR. As will be appreciated, any of the sequences described herein that are relevant to the generation of the reduced-browning phenotype could be the specific non-wildtype sequence - from the very localised gene-edited regions SEQ ID NOs: 40-42 to the whole edited genes SEQ ID NOs: 21-23. The homology arms can vary in length, as would be known by one skilled in genetics.

[0332] In terms of integrating HDR into the method of making the banana plant of the invention, as an example the donor could be included in the exemplified constructs, with the same guides (sg857 and sg858) being used to generate the necessary genomic break at the correct location. However, the person skilled in genetics would be able toidentify alternative guides that could be used to generate banana plant cells of the invention, when provided with the sequence information described herein.

[0333] As used herein, “regenerating” may comprise growing banana plant cells (which include protoplasts) into whole banana plants by first growing the banana plant cells into groups that develop into a callus, followed by the regeneration of shoots (caulogenesis) from the callus using plant tissue culture methods. The growth of banana protoplasts into callus and subsequent regeneration of shoots requires the proper balance of plant growth regulators in the tissue culture medium that must be customised. Protoplasts may also be used for plant breeding, using a technique called protoplast fusion. Methods of protoplast regeneration are well known in the art. Several factors affect the isolation, culture, and regeneration of protoplasts, namely the genotype, the donor tissue and its pre-treatment, the enzyme treatment for protoplast isolation, the method of protoplast culture, the culture, the culture medium, and the physical environment (see Maheshwari et al. (1986), 3-36. Springer-Verlag, Berlin). The regenerated banana plants can be subjected to selection. The banana plant or cells thereof may be devoid of a transgene, i.e. “non-transgenic”. For example, the banana plants may be devoid of any of the DNA constructs encoding any of the CRISPR / Cas system as used in some of the embodiments of the invention. According to some embodiments, when performing genetic manipulations such as genetic editing on banana cells to be grown and regenerated into an adult plant, it is preferable not to edit genes which may be expressed in these cells and negatively affect embryo formation and / or regeneration.

[0334] In some of embodiments, the method of the invention further comprises harvesting fruit from said banana plant. Each adult banana plant produces a single bunch, which is formed by many banana fruits or “fingers” and clustered in several “hands”. In this regard, “harvesting” has the conventional meaning. For example, banana bunches may be cut by hand (usually involving 2 or 3 people) using a sharp curved knife or a machete. The harvest usually occurs when the banana fruits are still green and firm, 7 to 14 days prior to ripening.

[0335] The invention further provides a banana plant or plant part obtainable by the foregoing method.

[0336] Additional aspects of the invention defined herein

[0337] In a further aspect, the present invention provides a method for testing the browning of banana fruit flesh, comprising:• providing a banana fruit flesh sample, optionally wherein the flesh sample is of a known amount, such as a known weight and / or shape, preferably the flesh sample weighs from about 10 g to about 100 g (such as about 50 g) and / or is cut in cross sections (such as cross sections of about 1.5 cm);

[0338] • combining (and optionally mixing) the flesh sample with a solvent (such as water), optionally a solvent of a known amount, preferably the amount being from about 50 ml to about 150 ml (such as about 100 ml);

[0339] • homogenising the combined flesh sample and solvent, preferably in a blender or electric mixer;

[0340] • analysing any change of colour of the homogenised combined flesh sample and solvent over time, optionally wherein the time is a period of at least 10 minutes; and

[0341] • optionally, comparing the change of colour of the homogenised combined flesh sample and solvent (for example, from a banana fruit of the present invention) with a homogenised combined flesh sample and solvent from a wildtype banana fruit, thereby identifying if the banana fruit flesh has reduced browning when compared to the wildtype banana fruit.

[0342] In one embodiment, the period is at least 30 minutes; for example, at least 60 minutes, at least one hour, at least four hours, or at least 24 hours. As shown in the examples, and as encompassed herein, in some embodiments the change of colour can be analysed over a number of timepoints, such as those described herein; for example, 30 minutes and 60 minutes or 60 minutes, four hours, and 24 hours.

[0343] In a preferred embodiment, the homogenised combined flesh sample and solvent from a wildtype banana fruit is prepared in the same way as the homogenised combined flesh sample and solvent from a banana fruit of the invention.

[0344] The aforementioned methods of testing the browning of a banana fruit flesh are further described within the Examples, features of which are included herein as additional embodiments of these aspects of the invention.

[0345] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used, exemplary methods and / or materials are described. The materials, methods, and examples are illustrative only and are not intended to be limiting.The terms “comprises”, “comprising”, “includes”, “including”, “having”, and their conjugates mean “including but not limited to”. The term “consisting of” means “including and limited to”. The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0346] As used herein, the singular form “a”, “an”, and “the” include plural references unless the context dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof. As used herein the term “about” refers to + / - 10 %.

[0347] EXAMPLES

[0348] The following examples are illustrative and not considered to limit the scope of the invention.

[0349] The nomenclature and laboratory procedures used herein include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are explained in the literature. See, for example, " Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); " Current Protocols in Molecular Biology" Volumes l-lll Ausubel, R. M., ed. (1994); Ausubel et al., " Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, " A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., " Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) " Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U. S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994);; " Oligonucleotide Synthesis" Gait, M. J., ed. (1984); " Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); " Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); " A Practical Guide to Molecular Cloning" Perbal, B., (1984) and " Methods in Enzymology" Vol. 1-317, Academic Press; " PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990), all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout. The procedures therein are well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.Example 1 - producing and characterising banana plants of the invention

[0350] In order to generate banana plants mutated in the PP01 gene, banana embryogenic cell suspension (ECS) cultures are treated with vectors encoding the Cas9 machinery and sgRNAs targeting the PPO1 gene. To produce the ECS, an embryogenic callus is first developed from an initial explant, such as immature male flowers or shoot tip as described by Ma, Proceedings of Symposium on Tissue culture of Horticultural Crops, Taipei, Taiwan, 8-9 March 1988, pp. 181-188, and in Schoofs, H. (1997) - “The origin of embryogenic cells in Musa”, PhD thesis, KULeuven, Belgium. ECS cultures are then initiated from freshly developed highly embryogenic calli in liquid medium. Next, 80% of the medium is refreshed every 12 to 14 days until the initiated cell suspension is fully established (6 to 9 months).

[0351] The ECS is then treated with Agrobacterium tumefaciens, according to procedures such as those described by Khanna et al., (2004) Mol. Breed.; 14: 239 and Tripathi et al., (2012) In Vitro Cell Dev. Biol. -Plant; 48: 216. The plasmid - binary plasmid vector pMOL_0019 (Figure 4) - used for gene editing contain four transcriptional units within the T-DNA region. The first transcriptional unit drives transient expression of a marker gene, nptll, conferring resistance to a selection agent. The next transcriptional unit drives transient expression of human codon -optimized Cas9 adapted from Streptococcus pyogenes. The third and fourth transcriptional units each drive transient expression of an sgRNA targeted to a selected gene (the vector encodes two distinct sgRNAs). The sgRNAs included in the plasmid used for Agrobacterium- mediated transient expression are designed to target PPO1 gene. A summary of the sgRNA target sequences used, and the target gene used for designing them, is provided in Table 1 (sequences are listed in the 5’ to 3’ orientation, and each sgRNA sequence did not include the PAM sequence, which is marked in bold typeface; therefore, regions of the sgRNA sequences for sg857 are GAGGGGTTGGTTGTGGTCTC (SEQ ID NO: 44) and for sg858 are CCTCAAGGGCGAGGACGGGT (SEQ ID NO: 45)).

[0352] Table 1 - sgRNAs used to target the PPO1 gene.

[0353] Target gene used sgRNA SEQ ID sgRNA target sequence +

[0354] for designing sgRNA ID NO: PAM sequence (Bold)

[0355] PP01 - Ma06_g31080 sg857 54 GAGGGGTTGGTTGTGGTCTCTGG PP01 - Ma06_g31080 sg858 55 CCTCAAGGGCGAGGACGGGTCGG

[0356]

[0357] The two guides only differ in the crRNA regions, which is GAGGGGTTGGTTGTGGTCTC (SEQ ID NO: 44) for sg857 and CCTCAAGGGCGAGGACGGGT (SEQ ID NO: 45) for sg858. The remaining sequence, which are the same between the two guides are the crRNA constant region GTTTTAGAGCTA (SEQ ID NO: 46), the Linker Loop GAAA (SEQ ID NO: 47), the tracer RNA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 48) and the Poly T terminator TTTTTTT (SEQ ID NO: 49).

[0358] Those sgRNAs were designed with criteria to minimise the likelihood of off-target editing. Nonetheless, the Breaking-Cas tool version 1.03 (Oliveros et al., 2016, Nucleic Acids Research. 44(W1): W267-W271 ) and the Find CRISPR Sites tool in Geneious Prime® 2023.2.1 (Biomatters Ltd) were used to identify any potential off-target sites with <2 base pair mismatches with a target sequence. Such sites could then be assessed for edits with the WGS data, which is discussed below. For sg857 and sg858, no off-target sites with <2 bp mismatches to a target site were identified.

[0359] After co-cultivation of banana embryogenic cells with A. tumefaciens cells harbouring the plasmid described above, the banana cells are resuspended in 250 ml Erlenmeyer flasks in liquid proliferation medium containing the selection agent, aminoglycoside antibiotic G418 (Geneticin), and cultured for up to 5 days with gentle shaking. This selection treatment allows for the enrichment of banana cells that are transiently expressing the resistance gene, whereas non -expressing banana cells and A. tumefaciens cells are selected against. The banana cells are subsequently washed four times in liquid proliferation medium to remove the selection agent, and are then cultured on proliferation medium, followed by embryo development medium and germination medium (relevant media can be found, for example, in Strosse et al., (2003), (A. Vezina and C. Picq, eds). INIBAP Technical Guidelines 8, The International Network for the Improvement of Banana and Plantain, Montpellier, France). Young shoots are transferred to propagation medium for plantlet development.

[0360] Both polymerase chain reaction (PCR) and sequencing was used to identify the PPO1 gene edits and the absence of plasmid DNA integration.

[0361] Target gene edits in regenerated plants are identified by extracting genomic DNA from leaf samples and analysing target sites by PCR gene-specific primers listed in Table 2. PCRs were performed using Q5® High-Fidelity 2X Master Mix (New England Biolabs), in accordance with the manufacturer’s guidelines, and with a T100™ Thermal Cycler (Bio-Rad). PCR products were then purified with ExoSAP-IT™ PCR Product Cleanup Reagent (Applied Biosystems™) or a Monarch® PCR & DNA Cleanup Kit (New EnglandBiolabs) and Sanger sequenced (GENEWIZ, Azenta Life Sciences), in accordance with the manufacturer’s guidelines. The resulting Sanger sequence chromatograms were analysed in SnapGene (GSL Biotech LLC) to identify the edits with the relative heights of chromatogram peaks used to assess the allelic ratios of the identified modifications. To confirm the edits in the full molecular characterization phase, the PCR products for both gene-edited gDNA samples were also ligated into a pJET plasmid vector using a CloneJET PCR Cloning Kit (Thermo Scientific™). The plasmid products were then transformed into Escherichia coli and the bacteria were plated for incubation overnight at 37° C. Several of the resulting colonies were selected and cultured overnight at 37° C shaking before the plasmids were purified in bulk and sent for Sanger sequencing (GENEWIZ, Azenta Life Sciences). The subsequent sequencing traces were analysed in SnapGene (GSL Biotech LLC) to confirm the targeted edits identified in PPO1, as discussed below.

[0362] Table 2 - Primers used to detect gene edits the PPO1 gene.

[0363] Gene ID Primer Fwd / Rev SEQ Primer sequence 5’ - 3’ # ID NO:

[0364] PP01- G0152 Fwd 56 CGAGAACCACGGTATCGATCT Ma06_g31080

[0365] G0159 Rev 57 ATGCTGAGTGAAGTTCCGGG

[0366]

[0367] The absence of plasmid sequences in edited plant lines is assessed using the DNA samples and quantitative PCR (qPCR) reactions with primers listed in Table 3. Quantitative PCR (qPCR) reactions were performed on gDNA samples extracted from leaf material of the initial plant of the gene-edited line to test for the absence of plasmid DNA integration. These qPCRs were performed in single-plex using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems™), in accordance with the manufacturer’s guidelines, and on either a LightCycler® 96 (Roche) or CFX Opus 384 (Bio-Rad). qPCR reactions for samples were performed alongside equivalent reactions on control samples of dH₂O and / or wild-type AL acuminata gDNA. Additionally, a positive control sample was included in qPCR experiments. For the initial screen qPCRs, the positive control sample was transgenic Al. acuminata gDNA. For the full molecular characterization analyses, the positive control sample was plasmid DNA containing each of the plasmid sequences being probed for. All qPCR reactions for each sample were performed alongside a control qPCR reaction aimed to specifically amplify from the endogenousbanana gene phytoene desaturase (PDS). In the initial screen, qPCR reactions were performed to test for the presence of the U6 promoter element from the plasmid T -DNA within samples. In the full molecular characterization phase, 8 qPCR reactions were performed to test for the presence of 9 plasmid regions within samples, including the elements encoding Cas9, the sgRNA cassettes, the bacterial and plant resistance markers, and the left and right T-DNA borders (Figure 4). The qPCR conditions are highlighted in Table 4.

[0368] Table 3 - Primers used to assess absence of plasmid DNA integration.

[0369] Target qPCR SEQ ID Primer Primer Sequence (5' - Orientation

[0370] Number Target NO: Name 3')

[0371] 1 GGCCTTTTTACGGTTCC 58 G0987 Fwd

[0372] T-DNA Left TGC Border TCCCTTAATTCTCCGCTC 59 G0988 Rev

[0373] CG

[0374] 2 GACCACCAAGCGAAACA nptll 60 G0341 Fwd

[0375] TCG

[0376] Coding

[0377] ATATCACGGGTAGCCAA

[0378] Sequence 61 G0342 Rev

[0379] CGC

[0380] 3 TCCTCCCGAAAAGGAAC cas9 62 G0331 Fwd

[0381] AGC

[0382] Coding

[0383] CATTCGTTTCCGGCCGT

[0384] Sequence 63 G0332 Rev

[0385] 4 + 5 TCTGACAGTTCTGGTGC U6 64 G0991 Fwd

[0386] TCAAC

[0387] Promoters

[0388] GGGCTGCATCTCACTAT

[0389] 65 G0992 Rev

[0390] GCG

[0391] 6 T-DNA CCGTTCGTCCATTTGTA 66 G1005 Fwd

[0392] Right TGTGC Border - TCACC I I I I IAGACGGC 67 G1006 Rev

[0393] pVS1 GGC

[0394] 7 GGTGGAGAAGTTGAAG 68 G0997 Fwd

[0395] GCCG

[0396]

[0397] pVS1 Short

[0398] GCCCACGTCATAGAGCA

[0399] Fragment - 69 G0998 Rev

[0400] TCG

[0401] 1

[0402] 8 GGCGGTTTCCCATCTAA pVS1 Short 70 G0999 Fwd

[0403] CCG

[0404] Fragment - GGCCGTTCTTGGCCTTC

[0405] 2 71 G1000 Rev

[0406] TTC

[0407] 9 GTATGCGGAGTGCATCA nptlll 72 G1003 Fwd

[0408] GGC

[0409] Coding

[0410] CCTCTTCGGGC I I I I CC

[0411] Sequence 73 G1004 Rev

[0412] GTC AAACCCCGATGAGCTTT PDS 74 G1819 Fwd

[0413] CCA

[0414] (Endogeno

[0415] CAGTTCCATCGGGGTTT

[0416] us) 92 G1824 Rev

[0417] AGC

[0418]

[0419] Table 4. Thermocycler conditions for full molecular characterization qPCR assays. In the initial screen, qPCR conditions differed only be using an annealing temperature of 65° C, 40 cycles for amplification, and an altered protocol for melt curve generation.

[0420]

[0421] Following propagation to produce banana plant clones, the targeted edits and absence of plasmid DNA integration into the genome are confirmed using whole genome sequencing (WGS) and bioinformatic analyses.

[0422] WGS was performed by Future Genomics Technologies on a pooled sample of leaf tissue taken from multiple clonal gene-edited line plants alongside an equivalent wild -type M. acuminata sample and an equivalent transgenic M. acuminata sample transformed with pMOL_0019. gDNA was extracted from all the samples for Nextera (RTM) DNA Flex (Illumina) library preparation. Next-generation sequencing (NGS) was then performed with the Illumina NovaSeq 6000 platform, with 2 x 150 bp paired reads, to a coverage of greater than 200x across the 523 Mb banana genome.NGS reads for WGS were analysed bioinformatically by Tropic Biosciences. The sequencing reads were first aligned to the reference banana genome which gave precise in-depth sequence coverage across the genomes of the gene-edited line, the wild-type control banana and the transgenic control banana. To validate the edits in PPO1 identified in the reduced browning banana, the NGS reads aligning to the targeted region were visualised with the Integrative Genomics Viewer software (IGV, Robinson et al., 2011 Nature Biotechnology 29, 24-26). The target sites were then assessed for variants to confirm the expected edits and the alleles that they are present in. To assess for off-target edits using the WGS data, potential off-target sites were first identified using the Breaking-Cas tool version 1.03 (Oliveros et al., 2016, Nucleic Acids Research.

[0423] 44(W1): W267-W271) and the Find CRISPR Sites tool in Geneious Prime® 2023.2.1 (Biomatters Ltd). For any off-target site with two or fewer mismatches with a target sequence, WGS reads aligning to the region were visualised in IGV (Robinson et al., 2011 Nature Biotechnology 29, 24-26) to identify any nucleotide variation not present in the wild-type sample data. To confirm the absence of plasmid DNA integration using the WGS data, the sequencing reads were mapped to the pMOL_0019 plasmid map. The coverage of those reads was then visualized with IGV (Robinson et al., 2011 Nature Biotechnology 29, 24-26) and compared between the wild-type banana, gene-edited and transgenic banana samples.

[0424] The above procedure was successfully used to obtain banana plants that contain targeted edits in the PPO1 gene and that lack foreign plasmid sequences integrated into the genome.

[0425] Banana embryogenic cells were treated with A. tumefaciens carrying plasmid pMOL_0019, containing sgRNAs sg857 and sg858 (see Table 1), which both target the first exon of PPO1. The pMOL_0019-treated cells were transiently selected and regenerated into shoots that were screened and then characterised by sequencing and qPCRs (as described above) before WGS was used to confirm the edits and absence of plasmid DNA integration.

[0426] The PCR and WGS analysis showed that the PPO1 -edited plants contain a single base pair insertion in one of three PPO1 alleles (A insertion between positions 151 -152), a seven base pair deletion and a single base pair deletion in the second PPO1 allele (GAGACCA (SEQ ID NO: 34) deletion at positions 149 - 155 and C deletion at position 386), and a single base pair deletion in the third PPO1 allele (C deletion at position 386). These edits lead to premature stop codons in all three alleles of PPO1 in the edited banana plants (Figure 5).The resulting protein, coding, and gene sequences are provided in SEQ ID NOs: 20-22, 23-25, and 26-28 respectively. The target region of the PPO1 gene is depicted in Figure 6, and the double-stranded break (DSB) sites, sgRNA target sequences, and genotyping primers are highlighted. The edited PPO1 alleles are shown alongside a wildtype consensus sequence in Figure 7, with the protein sequences encoded by the edited PPO1 alleles being shown alongside that encoded by the wild-type consensus sequence in Figure 8.

[0427] As shown in Table 5, in the qPCR analysis plasmid-specific primers failed to amplify target sequences from genomic DNA extracted from the PPO1 -edited banana line. This was also the case for DNA from a negative control wild-type plant and a H₂O control sample, whereas these primers did amplify sequences from the plasmid DNA positive control. As an internal control, an endogenous banana genomic region was amplified in all samples derived from plants. These analyses, therefore, confirm that plasmid sequences are absent from the genome of the PPO1 -edited banana plants.

[0428] Table 5. Quantitative PCR results from assays assessing for the presence of plasmid DNA in the edited plant genome.

[0429] *-* indicates that no fluorescence greater than the threshold was detected within 30 cycles and that the target sequence is likely absent in the plant genome. ‘gDNA’ indicates genomic DNA.■ ■ ■ ■

[0430] q C Values

[0431] p p 7. VS1 8. VS1

[0432] g 6. Riht

[0433] 3. cas9 Border MaP 1. Left 4.5. U6 Short Short +p Samle

[0434] gg Bopp ntll ntlllDS 2. 9. - ■ ■ ■ ■ pVS1rder Promoter Frament Frament - - 12

[0435] Reduced

[0436] g Brownin Banana 24.62

[0437] gpDNA Samle 1 - Reduced

[0438] ■ ■ ■ ■g Brownin Banana 24.74

[0439] gpDNA Samle 2 - Plasmid DNA - 21.32 22.63 20.62 22.22 21.78 21.56 21.11

[0440] Control

[0441] yp Wildte Banana- 22.61 gDNA Control - ■ ■ ■ ■

[0442] z dHO Control -

[0443] ■ ■ ■ ■

[0444] ■ ■ ■ ■

[0445] ■ ■ ■ ■

[0446] ■ ■ ■ ■

[0447] ■ ■

[0448]

[0449] Using WGS to test for the integration of plasmid DNA into the genome of the gene-edited line, bioinformatic analyses were used to map the NGS reads to the reference banana genome and the pMOL_0019 plasmid map (Table 6). The coverage of those reads was assessed and visualized with IGV (Robinson et al., 2011, Nature Biotechnology 29, 24-26), as displayed for the plasmid map in Figure 9. No reads aligned to the plasmid mapfrom the wild-type control sample. For the transgenic control sample, 27685 reads aligned to the plasmid map with a mean coverage of 301.04. In contrast, for the gene-edited line sample, only 10 reads aligned to the plasmid map with a mean coverage of 0.12 and a maximum coverage of 2. This highly limited number of reads aligning for the gene-edited line gave a low coverage depth relative to the coverage of the reference genome, a low continuity of coverage, and included reads with mismatches or soft-clipping. Such properties together strongly indicate that these reads are a product of background noise or artefacts that can occur with NGS (Stoler and Nekrutenko, 2021, NAR genomics and bioinformatics, 3(1), lqab019). Therefore, these data demonstrate the absence of plasmid DNA integration into the genome of the gene-edited line. From the qPCR and WGS analyses, the absence of plasmid DNA integration into the genome of the gene-edited line was validated across multiple clonal individuals and generations.

[0450] Table 6. Whole genome sequencing data for the gene-edited line demonstrating the absence of plasmid DNA integration.

[0451] Data is presented for the total number of whole genome sequencing reads and the reads mapped to the reference of the M. acuminata genome or the pMOL_0019 plasmid map.o' §

[0452] s o < 2 a.

[0453] § 5 c

[0454]

[0455] Example 2 - growing and testing the banana plants of the invention

[0456] The banana plants produced in Example 1 were grown at a private and highly secure field trial site, under very strict confidentiality controls.

[0457] Homogenisation method

[0458] The banana fruit harvested from the banana plants produced in the earlier Examples were tested using a “homogenisation” method, which tests the reduced -browning phenotype in the banana fruit flesh.

[0459] The ends of the banana fruit were removed, and the banana fruit was peeled. A 50 ± 1g section of banana fruit flesh was weighed and cut into smaller cross-section slices (to form circular discs) of -1.5 cm in width, using a scalpel. The 50 ± 1g of slicedfruit was then added to a blender (Smoothie 10 Speed Blender; cat. No. 50167; Hamilton Beach) containing 100 ml deionised water. The blender was set to “High” and activated on “Puree” mode for 30 seconds, to homogenise the banana fruit sample. 25 ml of the homogenised banana sample was transferred from the blender to a 94 mm petri dish (Greiner Bio-One, cat. No. 633181), using an electronic pipettor (StarLab, S7166-0010) and pipette (Greiner Bio-One, cat. No. 760180).

[0460] The 94 mm petri dish containing the homogenised banana fruit flesh was then immediately transferred for photographing. The photographs were taken in a light box (Duculus Light Box 2 LEDs 5500K; Amazon ASIN: B0BKB7TZTZ; manufacture reference: DUNW5060) with a camera (Canon EOS M100) positioned at a height of -24 cm using a camera stand (Pro Copy Stand A; Amazon ASIN: B00BVR4BNI; model number: Copy A). Within the frame of the photograph of the tested banana fruit flesh was a chart of seven colours from light yellow increasing in intensity to brown (Table 7, as shown in representative greyscale in Figure 10 and 12). The homogenised samples were stored at room temperature between timepoints.

[0461] Table 7 - The RGB colour model values for the seven colour gradient used to assess banana browning.

[0462] Standard R G B

[0463] 1 127 96 0

[0464] 2 189 142 0

[0465] 3 251 187 0

[0466] 4 255 204 58

[0467] 5 255 219 120

[0468] 6 255 235 182

[0469] 7 255 252 244

[0470]

[0471] The photographs containing the homogenised flesh sample, the sample label and the reference colour chart were processed in ImageJ software as follows. The mean grey value [ = sum of pixels’ grey values in the selected area of the image / number of pixels ] is measured across the entire area of the homogenised flesh sample, as well as across the darkest (‘standard 1’) and brightest (‘standard 7’) colours in the reference colour chart. RGB browning intensity [ = 255 - mean grey value ] of the sample isnormalised to that of the reference colours using the formula [ = sample * 160 / (standard1 - standard7) - standard1 * 160 / (standard1 - standard7) + 181 ].

[0472] The output of the ImageJ analysis for the gene-edited and wildtype plants was plotted as shown in Figure 11 and 13, with the statistical significance (as a P-value) calculated by linear mixed modelling of repeated measures followed by pairwise comparisons between test line and controls using T-tests.

[0473] The homogenisation assay was undertaken over two time-periods. The first was for up to an hour, and the second was up to 24 hours.

[0474] For the one-hour test, measurements were taken at 0 minutes, 30 minutes and 60 minutes. The number of gene-edited plants assayed were 9, with 27 banana fingers tested. These were compared to 28 wildtype (Grand Naine) plants, for which 86 banana fingers were tested. The results of this are shown in Figure 10 and 11.

[0475] For the 24-hour test, measurements were taken at 60 minutes, four hours, and 24 hours. The results of this are shown in Figure 12 and 13. The tested number of gene-edited banana fingers were 11 and wildtype (Grand Naine) banana fingers were 14.

[0476] Cutting assay

[0477] In addition to the homogenisation assay, an assay was undertaken in which gene-edited and wildtype (Grand Naine) banana fruit was peeled and cut into slices. These were then left alongside each other at room temperature for 12 hours to compare how browning occurred. The results of this are shown in Figure 14. In a further experiment, the gene-edited and wildtype (Grand Naine) banana fruit was peeled and cut into slices and then left under refrigeration (4°C) for up to 12 days. Images of the banana fruit taken every other day are shown in Figure 15.

[0478] Results and conclusions

[0479] As shown in Figure 11 and 13, the exemplary gene-edited line of the invention shows a statistically significant reduction in browning, when compared to the wildtype control for up to 24 hours.

[0480] As shown in Figure 14 and 15, the visually apparent level of browning of the gene-edited banana fruit is much lower than that of the wildtype, stored both at room temperature and under refrigeration (at 4°C).

[0481] This demonstrates that a gene-edited line, with the genetic profile as described herein, has an impressive and highly useful reduced browning phenotype in the banana fruit flesh. The stability of the reduced browning of the banana of the invention seemsto be maintained when the banana fruit is stored in different ways - either at room temperature or under refrigeration.

[0482] Example 3 - processed and preserved products, containing banana fruit of the invention

[0483] The banana fruit of the invention, as developed, grown, and cut as described in Example 1 and 2, was also assessed to see how the reduced browning of the flesh of the banana of the invention behaves in processed banana products - specifically, juice, smoothies and fruit salads - and when combined with a preservative.

[0484] Fruit salad preparation

[0485] Banana flesh slices (approximately 8 mm thick), from the middle portion of the banana finger) were prepared in a similar manner to that described in Example 2. These were then added to a plastic container with cut pieces of kiwi, strawberry, cantaloupe melon, and whole blueberries, to create a fruit salad. The fruit salad was stored under refrigeration (at 4°C) for up to 12 days. Images of the banana fruit taken every other day are shown in Figure 16.

[0486] Juice preparation

[0487] Banana juice was prepared from 50 grams (g) of banana fruit flesh from the middle portion of the banana finger which was sliced into approximately 3mm thick sections, and homogenised for 1 minute in 75 millilitre (ml) of deionised water (ThermoScientific) using a blender. The banana juice was then poured into plastic cups and stored under refrigeration (at 4°C) for up to 48 hours. Images were taken at 12, 24, and 48 hours and are shown in Figure 17.

[0488] Smoothie preparation

[0489] A banana-strawberry smoothie was prepared. 60g of banana fruit flesh from the middle portion of the banana finger was sliced into approximately 3mm thick sections.

[0490] 60g ripe strawberries were diced into 5 mm cubes. The fruit material was homogenised for 2 minutes in 120 ml semi-skimmed milk using a blender. The banana-strawberry smoothie was then stored under refrigeration (at 4°C) for up to 48 hours. Images were taken at 12, 24, and 48 hours and are shown in Figure 18.Calcium ascorbate preservation

[0491] Banana flesh slices (approximately 8 mm thick), from the middle portion of the banana finger) were prepared in a similar manner to that described in Example 2. The banana slices were pre-treated by being immediately placed in a 20% w / v calcium ascorbate solution - 5 seconds on each side - before being removed. The 20% w / v calcium ascorbate solution was freshly prepared by dissolving 20 g calcium L-ascorbate dihydrate (Merck, 359645) in 100 ml deionised water (ThermoScientific) at room temperature (21 °C).

[0492] The banana slices were then either put in a plastic container or into a fruit salad (containing kiwi, strawberry, cantaloupe melon, and whole blueberries). The banana slices alone and banana slices in a fruit salad were stored under refrigeration (at 4°C) for up to 12 days. Images were taken every other day and are shown in Figure 19 and 20.

[0493] Results and conclusions

[0494] The results show that when the exemplary banana fruit of the invention, with the genetic profile as described herein, retains its impressive and highly useful reduced browning phenotype when included in a wide-range of processed banana products.

[0495] Specifically, the cut banana fruit flesh of the invention shows a phenotype with minimal browning when combined with other fruit in a fruit salad, when compared to the wildtype control (Figure 16). This demonstrates that the reduction in browning of the exemplary banana fruit of the invention is not altered by the chemicals / components of other fruits when they are mixed.

[0496] The banana fruit of the invention also, and surprisingly, shows minimal browning over a long period of time when it is included in a banana juice drink, when compared to the wildtype control (Figure 17). This reduction in browning is despite the homogenisation process used to produce the juice causing extensive damage to the banana fruit flesh which usually results in high levels of browning, as shown in the control.

[0497] The banana fruit of the invention further, and surprisingly, shows minimal browning over a long period of time even when homogenised and mixed with other food products (for example, milk and strawberry), with the banana-strawberry smoothie showing minimal discolouration when compared to a smoothie made with wildtype banana fruit (Figure 18). This reduction in browning is particularly impressive asproducing a smoothie exposes the banana fruit flesh to both extensive damage and chemicals / components of other food products.

[0498] Lastly, the banana fruit flesh shows a further improved reduced browning when treated with the preservative, calcium ascorbate. It showed very little browning when either stored alone or in a fruit salad, when compared to the wildtype control (Figure 19 and 20).

[0499] Accordingly, the inventors have demonstrated that using the banana fruit flesh of the invention greatly increases the aesthetics and quality of processed banana products; in particular banana products which are of high commercial value, such as fruit salads, juices, and smoothies.

[0500] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0501] All publications, patents and patent applications mentioned herein are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

CLAIMS1. A banana plant cell,wherein the banana plant cell comprises three copies of a PPO1 gene, and wherein:• a first copy of the PPO1 gene comprises a premature stop-codon TGA (SEQ ID NO: 29) in a nucleotide sequence of the PPO1 gene comprising TGACCT (SEQ ID NO: 31);• a second copy of the PPO1 gene comprises a premature stop-codon TAG (SEQ ID NO: 30) in a nucleotide sequence of the PPO1 gene comprising TAGGCA (SEQ ID NO: 32); and• a third copy of the PPO1 gene comprises a premature stop-codon TGA (SEQ ID NO: 29) in a nucleotide sequence of the PPO1 gene comprising TGAGGG (SEQ ID NO: 33).

2. The banana plant cell of Claim 1,wherein each copy of the PPO1 gene comprises one or more indel, and wherein the indel causes a frameshift in the wildtype PPO1 gene sequence leading to each premature stop-codon.

3. The banana plant cell of Claim 2, wherein the indel is a deletion of 1, 2, 4, 5 or 7 nucleotides or an addition of 1, 2, 4, 5 or 7 nucleotides.

4. The banana plant cell of Claim 2 or 3, wherein the one or more indel is at a polynucleotide sequence of GAGACCA (SEQ ID NO: 34) of the PPO1 gene and / or is at a polynucleotide sequence of ACCCGTC (SEQ ID NO: 35) of the PPO1 gene.

5. The banana plant cell of any one of Claims 2-4, wherein the one or more indel is selected from the group consisting of:• an insertion of any one nucleotide in GAGACCA (SEQ ID NO: 34) of the PPO1 gene, preferably the insertion of any one nucleotide after position 3 or 4 in GAGACCA (SEQ ID NO: 34), most preferably the insertion of an adenine;• a deletion of GAGACCA (SEQ ID NO: 34) of the PPO1 gene; or• a deletion of any one nucleotide from ACCCGTC (SEQ ID NO: 35) of the PPO1 gene, preferably the deletion of a cytosine, most preferably the cytosine at position 4 from ACCCGTC (SEQ ID NO: 35).

6. The banana plant cell of any one of Claims 1 -5, wherein:• the first PPO1 gene comprises an insertion of any one nucleotide in GAGACCA (SEQ ID NO: 34), preferably the insertion of any one nucleotide after position 3 or 4 in GAGACCA (SEQ ID NO: 34), most preferably the insertion of an adenine; and / or• the second PPO1 gene comprises a deletion of GAGACCA (SEQ ID NO: 34); and / or • the third PPO1 gene comprises a deletion of any one nucleotide from ACCCGTC (SEQ ID NO: 35), preferably the deletion of a cytosine, most preferably the cytosine at position 4 of ACCCGTC (SEQ ID NO: 35).

7. A banana plant cell,wherein the banana plant cell comprises three copies of a PPO1 gene, and wherein:• a first PPO1 gene comprises: (1) an insertion of any one nucleotide in a nucleotide sequence GAGACCA (SEQ ID NO: 34) of the PPO1 gene, preferably the insertion of any one nucleotide after position 3 or 4 in GAGACCA (SEQ ID NO: 34), most preferably the insertion of an adenine; and (2) a premature stop-codon TGA (SEQ ID NO: 29) in a nucleotide sequence of the PPO1 gene comprising TGACCT (SEQ ID NO: 31); and / or• a second PPO1 gene comprises: (1 ) a deletion of a nucleotide sequence GAGACCA (SEQ ID NO: 34) of the PPO1 gene; and (2) a premature stop-codon TAG (SEQ ID NO: 30) in a nucleotide sequence of the PPO1 gene comprising TAGGCA (SEQ ID NO: 32); and / or• a third PPO1 gene comprises: (1) a deletion of any one nucleotide from in a nucleotide sequence ACCCGTC (SEQ ID NO: 35) of the PPO1 gene, preferably the deletion of a cytosine, most preferably the cytosine at position 4 from ACCCGTC (SEQ ID NO: 35); and (2) a premature stop-codon TGA (SEQ ID NO: 29) in a nucleotide sequence of the PPO1 gene comprising TGAGGG (SEQ ID NO: 33), wherein said insertions and / or deletions cause a frameshift in the wildtype PPO1 gene sequence leading to each premature stop-codon.

8. The banana plant cell of any one of Claims 4-7, wherein:• GAGACCA (SEQ ID NO: 34) is in a nucleotide sequence of the PPO1 gene comprising GAGACCACAACCAACCCCTCGT (SEQ ID NO: 36) or CCAGAGACCACAA (SEQ ID NO: 37); and / or• ACCCGTC (SEQ ID NO: 35) is in a nucleotide sequence of the PPO1 gene comprising ACCCGTCCTCGCCCTTGAGG (SEQ ID NO: 38) or CCGACCCGTCCTC (SEQ ID NO: 39).

9. The banana plant cell of any one of Claims 2-8,wherein the wildtype PPO1 gene sequence comprises a polynucleotide sequence selected from the list consisting of:• SEQ ID NO: 16, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 16 wherein said polynucleotide sequence with at least 90% identity comprises TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33);• a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 18, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity to SEQ ID NO: 18 wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity comprises TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33); or• a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO:19, or a polynucleotide sequence that encodes a polypeptide comprising at least 90% identity to SEQ ID NO: 19 wherein said polynucleotide sequence that encodes a polypeptide comprising at least 90% identity comprises TGACCT (SEQ ID NO: 31), TAGGCA (SEQ ID NO: 32), and TGAGGG (SEQ ID NO: 33).

10. The banana plant cell of any one of Claims 1-9, wherein:• the first PPO1 gene encodes a polypeptide of about 95 to about 100 amino acids in length, preferably about 97 amino acids in length; and / or• the second PPO1 gene encodes a polypeptide of about 65 to about 70 amino acids in length, preferably about 67 amino acids in length; and / or• the third PPO1 gene encodes a polypeptide of about 130 to about 135 amino acids in length, preferably about 132 amino acids in length.

11. The banana plant cell of any one of Claims 1-10, wherein:• the first copy of the PPO1 gene comprises AGAGAACCAC (SEQ ID NO: 40); and / or • the second copy of the PPO1 gene comprises CCACAA (SEQ ID NO: 41); and / or • the third copy of the PPO1 gene comprises ACCGTC (SEQ ID NO: 42).

12. The banana plant cell of any one of Claims 1-11, wherein:• the first copy of the PPO1 gene comprises a polynucleotide sequence of SEQ ID NO: 83, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 83 wherein said polynucleotide sequence with at least 90% identity comprises AGAGAACCAC (SEQ ID NO: 40);• the second copy of the PPO1 gene comprises a polynucleotide sequence of SEQ ID NO: 84, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 84 wherein said polynucleotide sequence with at least 90% identity comprises CCACAA (SEQ ID NO: 41); and• the third copy of the PPO1 gene comprises a polynucleotide sequence of SEQ ID NO: 85, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 85 wherein said polynucleotide sequence that encodes a polypeptide comprising at least 90% identity comprises ACCGTC (SEQ ID NO: 42).

13. The banana plant cell of any one of Claims 1-12, wherein:• the first copy of the PPO1 gene comprises a polynucleotide sequence selected from the list consisting of:a. SEQ ID NO: 20, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 20 wherein said polynucleotide sequence with at least 90% identity comprises TGACCT (SEQ ID NO: 31) and AGAGAACCAC (SEQ ID NO: 40);b. a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 23, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity to SEQ ID NO: 23 wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity comprises TGACCT (SEQ ID NO: 31) and AGAGAACCAC (SEQ ID NO: 40); orc. a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 26, or a polynucleotide sequence that encodes a polypeptide comprising at least 90% identity to SEQ ID NO: 26 wherein saidpolynucleotide sequence that encodes a polypeptide comprising at least 90% identity comprises TGACCT (SEQ ID NO: 31) and AGAGAACCAC (SEQ ID NO: 40), and / or• the second copy of the PPO1 gene comprises a polynucleotide sequence selected from the list consisting of:a. SEQ ID NO: 21, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 21 wherein said polynucleotide sequence with at least 90% identity comprises TAGGCA (SEQ ID NO: 32) and CCACAA (SEQ ID NO: 41); b. a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 24, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity to SEQ ID NO: 24 wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity comprises TAGGCA (SEQ ID NO: 32) and CCACAA (SEQ ID NO: 41); orc. a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 27, or a polynucleotide sequence that encodes a polypeptide comprising at least 90% identity to SEQ ID NO: 27 wherein said polynucleotide sequence that encodes a polypeptide comprising at least 90% identity comprises TAGGCA (SEQ ID NO: 32) and CCACAA (SEQ ID NO: 41), and / or• the third copy of the PPO1 gene comprises a polynucleotide sequence selected from the list consisting of:a. SEQ ID NO: 22, or a polynucleotide sequence with at least 90% identity to SEQ ID NO: 22 wherein said polynucleotide sequence with at least 90% identity comprises TGAGGG (SEQ ID NO: 33) and ACCGTC (SEQ ID NO: 42); b. a polynucleotide sequence that encodes a CDS polynucleotide sequence of SEQ ID NO: 25, or a polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity to SEQ ID NO: 25 wherein said polynucleotide sequence that encodes a CDS polynucleotide sequence with at least 90% identity comprises TGAGGG (SEQ ID NO: 33) and ACCGTC (SEQ ID NO: 42); orc. a polynucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 28, or a polynucleotide sequence that encodes a polypeptide comprising at least 90% identity to SEQ ID NO: 28 wherein said polynucleotide sequence that encodes a polypeptide comprising at least90% identity comprises TGAGGG (SEQ ID NO: 33) and ACCGTC (SEQ ID NO: 42).

14. The banana plant cell of any one of Claims 1-13, wherein the second PPO1 gene comprises a deletion of any one nucleotide from ACCCGTC (SEQ ID NO: 35), preferably the deletion of a cytosine, most preferably the cytosine at position 4 from ACCCGTC (SEQ ID NO: 35).

15. A banana plant or a banana fruit comprising a banana plant cell as defined in any one of Claims 1-14.

16. The banana plant cell of any one of Claims 1-14 or the banana plant or banana fruit of Claim 15, wherein the banana plant cell, banana plant or banana fruit is an autotriploid, preferably an autotriploid Musa acuminata.

17. A processed banana product comprising a banana fruit as defined in Claim 15 or 16.

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