Delaying or preventing browning in banana fruit

Abstract

The present invention relates to compositions and methods for slowing or preventing browning in banana fruits by genetically editing one or more genes encoding polyphenol oxidase (PPO).

Description

[Technical field]

[0001] The present invention, according to some embodiments, relates to compositions and methods for delaying or preventing browning in banana fruits. In some embodiments of the present invention, delaying or preventing browning in banana fruits is achieved by genetically editing one or more genes encoding polyphenol oxidase (PPO). [Background technology]

[0002] Cultivated bananas and plantains are large herbaceous plants of the Musa genus. They are sterile and parthenocarpic, meaning that the fruit develops without seeds. Most cultivated hybrids and species are triploid, although some are diploid or tetraploid. Most have been bred from mutants found in the wild.

[0003] Bananas belong to the climacteric type of fruit. After harvesting, green bananas must undergo climacteric changes through a ripening process (including internal ethylene production, starch and protopectin hydrolysis) until the flesh becomes soft, sweeter, more aromatic, and therefore more valuable as a food. Traditionally, bananas are harvested before they are ripe, so the duration of transportation and storage is affected by the length of ripening progression. Banana fruits can often ripen during the transportation process due to the production of ethylene. In addition, the fruit may become overripe and begin to brown, rotting and reducing its market value. Therefore, there is a need to control the browning of banana fruits. In particular, the ability to delay or prevent fruit browning would facilitate the transportation of banana fruits and improve the shelf life of banana fruits. One factor that may affect fruit browning is polyphenol oxidase (PPO).

[0004] Polyphenol oxidase (PPO) is an enzyme found throughout the plant and animal kingdoms, including most fruits and vegetables such as bananas. PPO is important in the food industry because it catalyzes enzymatic browning when tissues are damaged by bruising or pressure, reducing the marketability of agricultural products and resulting in economic losses. Enzymatic browning due to the action of PPO can also lead to a loss of nutrient content in agricultural products, further reducing their value. The substrates for the PPO reaction are located within the vacuole of the plant cell, where PPO initiates a series of browning reactions. Exposure to oxygen during slicing or straining of fruits also triggers enzymatic browning by PPO. PPO is known to accept monophenols and / or o-diphenols as substrates and acts by catalyzing the o-hydroxylation of monophenolic molecules whose benzene ring contains one hydroxyl substituent to o-diphenols (phenolic molecules containing two hydroxyl substituents at the 1,2 positions and no carbon between them). The enzyme can also catalyze the oxidation of o-diphenols to produce o-quinones. PPO catalyzes the rapid polymerization of o-quinones to produce black, brown, or red pigments (polyphenols) that cause fruit browning. Thus, in an effort to slow or prevent fruit browning, for example in bananas, it is desirable to find ways to reduce the level or activity of PPO active in the fruit.

[0005] Reducing the expression of PPO genes to reduce the levels or activity of PPO is one way to prevent fruit browning (as described in US Pat. No. 9,580,723 for apples, the so-called "Arctic apple"). Typical approaches to improve agricultural productivity (such as improving yield or engineering pest resistance) have so far relied on introducing genes into the genome of crop species by mutation breeding or transformation. However, these processes are inherently non-specific and relatively inefficient. For example, plant transformation methods deliver foreign DNA and integrate it into the genome at random locations. Furthermore, the random nature of these integrations makes it difficult to predict whether pleiotropic effects have occurred due to unintended genome disruptions. Recent advances in genome editing technology, such as the use of CRISPR-Cas9 gene editing, have made it possible to modify DNA sequences in living cells more precisely than traditional breeding or standard genetic engineering.

[0006] However, unlike most other food crops, bananas are difficult to genetically improve. This is in part because banana species are parthenogenetic (do not produce viable seeds), making it impossible to remove genetically inserted sequences by sexual reproduction (e.g., removal of transfer DNA, T-DNA, inserted using agrobacteria to introduce Cas9 sequences). Furthermore, backcrossing of bananas is not possible because nearly all banana cultivars and landraces are triploid, with high rates of male and female sterility, thus precluding the possibility of introgressing new traits into current cultivars. Furthermore, the banana genome is incompletely annotated and expression data is limited, providing insufficient depth of information on the optimal genes to target.

[0007] The present invention is based in part on the identification of nine different PPO genes in bananas (designated PPO1-PPO9), and in particular the characterization that certain PPOs, such as PPO1, PPO2, PPO8, PPO9, and PPO4, are expressed in banana fruit but are characterized by low or no expression in other tissues. According to some embodiments of the present invention, the identified PPO genes, in particular PPO1, PPO2, PPO8, PPO9, and / or PPO4, are targeted herein to delay or prevent browning in banana fruit. Previously, Gooding et al., "Molecular cloning and characterisation of banana fruit polyphenol oxidase", Planta, Sep 2001;123(5):748-57, used degenerate primers to identify banana PPOs, which do not match any of the PPOs herein, but are most closely correlated with either PPO4 and PPO5. The same degenerate primers were used in WO 9637617, WO 9729193, and WO 9853080 as in Gooding et al. (2001) supra. China Patent No. 104404007 discloses the discovery of a PPO gene in banana, referred to herein as PPO8. Summary of the Invention

[0008] The present invention provides a method for reducing the level or activity of at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase encoded by a PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene in a banana plant or banana plant cell. Optionally, the method further comprises regenerating a banana plant from the banana plant cell. Optionally, the method further comprises harvesting a fruit from the banana plant. According to some embodiments, reducing the level or activity of at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase comprises introducing a modification into a PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene encoding the at least one PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase.

[0009] The present invention further provides banana plant cells obtainable by the above-mentioned methods of the invention, banana plants or plant parts obtainable by the above-mentioned methods of the invention, and fruits harvested from banana plants obtainable by the above-mentioned methods of the invention, wherein the pulp and / or peel are characterized by a phenotype of delayed and / or reduced browning compared to the pulp and / or peel of banana plants not having reduced levels or activity of the at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase.

[0010] 1. A method for producing a banana plant characterized by a phenotype of delayed and / or reduced browning of the flesh and / or skin compared to a wild-type banana plant, comprising: (a) providing to a banana plant cell a CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and one or more guide RNAs, wherein the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and the one or more guide RNAs are selected from the group consisting of: (A) SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; SEQ ID NO:6 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; SEQ ID NO:12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; SEQ ID NO:13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:13; and SEQ ID NO:8 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8. (B) encodes a polyphenol oxidase selected from the group consisting of SEQ ID NO:40 or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; SEQ ID NO:41 or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; SEQ ID NO:48 or a polypeptide having at least 75% sequence identity to SEQ ID NO:48; and SEQ ID NO:43 or a polypeptide having at least 75% sequence identity to SEQ ID NO:43; or (C) a polynucleotide having at least 75% sequence identity to SEQ ID NO:151 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; SEQ ID NO:152 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; SEQ ID NO:158 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:158; SEQ ID NO:159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:159;and SEQ ID NO: 154, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 154; (b) identifying at least one banana plant cell comprising an alteration in the at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene, wherein the alteration is selected from the group consisting of at least one nucleotide insertion; at least one nucleotide deletion; at least one nucleotide substitution; or any combination of the foregoing; and regenerating a banana plant from the banana plant cell, wherein the banana plant is characterized by a phenotype of delayed and / or reduced browning of the flesh and / or skin compared to a wild-type banana plant.

[0011] The present invention further relates to a banana plant or plant part comprising at least one modified endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene in its genome, wherein the modification results in a reduction or decreased function of at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase encoded by the modified endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene, and the modification is selected from the group consisting of: (A) SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; SEQ ID NO:6 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; SEQ ID NO:12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; SEQ ID NO:13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:13; and SEQ ID NO:8 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8. (B) encodes a polyphenol oxidase selected from the group consisting of SEQ ID NO:40 or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; SEQ ID NO:41 or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; SEQ ID NO:48 or a polypeptide having at least 75% sequence identity to SEQ ID NO:48; and SEQ ID NO:43 or a polypeptide having at least 75% sequence identity to SEQ ID NO:43; or (C) a polynucleotide having at least 75% sequence identity to SEQ ID NO:151 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; SEQ ID NO:152 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; SEQ ID NO:158 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:158; SEQ ID NO:159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:159;and SEQ ID NO: 154, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 154, located in at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene.

[0012] The present invention further provides a banana fruit harvested from a banana plant of the present invention, characterized by a phenotype of delayed and / or reduced browning compared to fruit from a banana plant not having a reduced level or activity of the at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase. Further provided by the present invention is a method for obtaining a banana fruit food product, the method comprising processing a banana fruit of the present invention.

[0013] A DNA sequence comprising a banana PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase polynucleotide, comprising: (A) a coding sequence selected from the group consisting of SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; SEQ ID NO:6 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; SEQ ID NO:12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; SEQ ID NO:13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:13; and SEQ ID NO:8 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8; (B) SEQ ID NO:40 or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; SEQ ID NO:41 or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47. Further provided by the present invention is a DNA sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:151 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; SEQ ID NO:152 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; SEQ ID NO:158 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:158; SEQ ID NO:159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:159; and SEQ ID NO:154 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:154.

[0014] The present invention further relates to a polypeptide comprising a coding sequence selected from the group consisting of: (A) SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; SEQ ID NO:6 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; SEQ ID NO:12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; SEQ ID NO:13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:13; and SEQ ID NO:8 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8; (B) SEQ ID NO:40 or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; SEQ ID NO:41 or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; SEQ ID NO:48 or a polypeptide having at least 75% sequence identity to SEQ ID NO:48. or (C) a polynucleotide sequence selected from the group consisting of SEQ ID NO:151 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; SEQ ID NO:152 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; SEQ ID NO:158 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:158; SEQ ID NO:159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:159; and SEQ ID NO:154 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:154. Further provided by the present invention is a plant cell transformed with a vector of the present invention, which is optionally a banana plant cell.

[0015] The invention further provides polyphenol oxidase proteins that are encoded by, or encoded by a polynucleotide having at least 75% sequence identity to, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:8; that comprise, or comprise a sequence with at least 75% sequence identity to, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:43; or that are encoded by, or encoded by a polynucleotide having at least 75% sequence identity to, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:158, SEQ ID NO:159, or SEQ ID NO:154.

[0016] A method for expressing a polyphenol oxidase in a plant cell, comprising: introducing into the plant cell (A) a coding sequence selected from the group consisting of SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; SEQ ID NO:6 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; SEQ ID NO:12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; SEQ ID NO:13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:13; and SEQ ID NO:8 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8; (B) SEQ ID NO:40 or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; SEQ ID NO:41 or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47, operably linked to a promoter active in the plant cell. Further provided by the present invention is a method comprising introducing a banana polyphenol oxidase polynucleotide comprising a polynucleotide sequence selected from the group consisting of: (A) a polynucleotide encoding a polyphenol oxidase selected from the group consisting of: a polypeptide having at least 75% sequence identity to SEQ ID NO:48; SEQ ID NO:48; and SEQ ID NO:43;

[0017] The present invention further provides synthetic banana polyphenol oxidase guide RNAs comprising a variable region selected from the group consisting of SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:35; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:38; SEQ ID NO:39; SEQ ID NO:62; SEQ ID NO:76; and SEQ ID NO:77.

[0018] The present invention further relates to a recombinant DNA construct comprising a promoter operably linked to a nucleotide sequence expressing at least one banana PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase guide RNA, the guide RNA being capable of forming a complex with a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease, the complex comprising a coding sequence selected from the group consisting of: SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; SEQ ID NO:6 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; SEQ ID NO:12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; SEQ ID NO:13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:13; and SEQ ID NO:8 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8; (B) a coding sequence in the banana genome selected from the group consisting of: SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; or (C) a polynucleotide having at least 75% sequence identity to SEQ ID NO:151 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; SEQ ID NO:152 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; SEQ ID NO:158 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:158; SEQ ID NO:159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:159;and SEQ ID NO: 154, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 154, wherein the recombinant DNA construct is capable of binding to at least one endogenous banana PPO1, PPO2, PPO8, or PPO9 polyphenol oxidase gene and creating a double-stranded or single-stranded break; [Brief description of the drawings]

[0019] The present disclosure can be better understood from the following description of the accompanying drawings and sequence listing, which form a part of this application. [Figure 1] Percent identity matrix of selected Musa acuminata PPO proteins. The PPOs have 35%-97% homology to each other. [Diagram 2] Percent identity matrix of selected PPO proteins from Malus domestica and Malus domestica. The PPOs have approximately 39% to 97% homology to the query sequence. [Diagram 3] Protein alignment of the DWL domains of selected PPOs targeted for gene editing. The query sequence from Apple is shown in the box. [Figure 4] Expression profiles showing the average expression level of each PPO in each tissue tested. Units are arbitrary, derived by visual quantification, expressing the quantification as a number, and linearly converting the number onto a scale from - to +, ++, +++. [Diagram 5]Bar graph showing RNA-seq expression of PPO1, PPO2, and PPO4-PPO9 in banana based on comparison of total RNA-seq data from various tissues and measuring expression with TMM normalization (trimmed mean of M-values). Expression was observed in banana fruit and / or flesh for PPO1, PPO2, PPO8, and PPO9. TMM normalization was performed by edgeR to eliminate inter- and intra-sample compositional bias. TMM normalization is a method to estimate relative RNA production levels from RNA-seq data, especially in situations where the underlying distribution of expressed transcripts between samples differs significantly. The TMM method estimates a scale factor between samples. PPO3 was not detected by RNA-seq. [Figure 6] Generation of edits in PPO2 with the gene shown in Figure 6A and the deletion shown in Figure 6B. [Figure 7](A) Partial genomic sequence of the first exon of the banana PPO1 gene. Black type indicates nucleotides of the coding sequence (depicting nucleotides 1 to 695 from the adenosine start codon) and grey type indicates nucleotides of the upstream untranslated region (depicting nucleotides -166 to -1 from the adenosine start codon). The DNA double-strand break (DSB) site associated with CRISPR / Cas9 is indicated by a dotted line. Two sgRNAs (857, 858) targeting the PPO1 gene are shaded, along with their protospacer adjacent motif (PAM) sequences. Bold and underlined further indicate the nucleotide (cytosine, bp 371) that is deleted in banana plants with reduced browning. Shading further indicates the primers used for PCR to amplify the PPO1 target site region for sequencing and confirmation of editing. (B) Partial alignment of PPO1 protein sequences produced from unedited and edited PPO1 genes. Boxes indicate changes in protein sequence caused by a single base pair deletion in the PPO1 gene induced by sgRNA 858-guided CRISPR / Cas9 DSB. The full length of the unedited PPO1 protein is 578 amino acids long, while the edited PPO1 protein is truncated (127 amino acids long) due to the presence of a premature stop codon (indicated by an asterisk) resulting from a frameshift caused by the single base pair deletion. [Figure 8]RNA-seq expression analysis performed on Grand Nine bananas over the course of natural ripening without exogenous ethylene application. Peel and flesh samples were taken from five ripening stages: all green (immature), green-yellow (first turning point), all yellow (mature), yellow-brown (second turning point), and all brown (overripe). High-quality RNA was obtained from all flesh samples and from the peel at the all-green stage. Tissue samples were also taken from leaves and roots of in vitro Grand Nine plants, as well as from in vitro cultures of embryos and embryogenic cells. Relative mRNA abundance was quantified as above using TMM normalization. PPO1, PPO4, and PPO9 account for >90% of PPO expression in Grand Nine banana peel at the immature green stage, with PPO1 being the dominant PPO gene expressed in the flesh of Grand Nine bananas, with the exception of the overripe brown stage where PPO8 is more highly expressed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] array SEQ ID NO:1 is a PPO peptide sequence from Arctic apple. SEQ ID NO:2 is a PPO peptide sequence from Arctic apple. SEQ ID NO:3 is a PPO peptide sequence from Arctic apple. SEQ ID NO:4 is a PPO peptide sequence from Arctic apple. SEQ ID NO:5 is the PPO1 gene coding sequence from banana (Accession number Ma06_31080). SEQ ID NO:6 is the PPO2 gene coding sequence from banana (Accession number Ma07_03540). SEQ ID NO:7 is the PPO3 gene coding sequence from banana (Accession number Ma07_03650). SEQ ID NO:8 is the PPO4 gene coding sequence from banana (Accession number Ma08_09150). SEQ ID NO:9 is the PPO5 gene coding sequence from banana (Accession number Ma08_09160). SEQ ID NO: 10 is the PPO6 gene coding sequence from banana (accession number Ma08_09170). SEQ ID NO:11 is the PPO7 gene coding sequence from banana (Accession number Ma08_09180). SEQ ID NO: 12 is the PPO8 gene coding sequence from banana (accession number Ma08_34740). SEQ ID NO: 13 is the PPO9 gene coding sequence from banana (accession number Ma10_20510). SEQ ID NO:14 is the forward oligonucleotide used to identify expression of the mRNA encoding PPO1. SEQ ID NO:15 is the reverse oligonucleotide used to identify expression of mRNA encoding PPO1. SEQ ID NO:16 is the forward oligonucleotide used to identify expression of the mRNA encoding PPO2. SEQ ID NO:17 is the reverse oligonucleotide used to identify expression of mRNA encoding PPO2. SEQ ID NO:18 is the forward oligonucleotide used to identify expression of the mRNA encoding PPO3. SEQ ID NO:19 is the reverse oligonucleotide used to identify expression of mRNA encoding PPO3. SEQ ID NO:20 is the forward oligonucleotide used to identify expression of the mRNA encoding PPO4. SEQ ID NO:21 is the reverse oligonucleotide used to identify expression of mRNA encoding PPO4. SEQ ID NO:22 is the forward oligonucleotide used to identify expression of the mRNA encoding PPO5. SEQ ID NO:23 is the reverse oligonucleotide used to identify expression of mRNA encoding PPO5. SEQ ID NO:24 is the forward oligonucleotide used to identify expression of mRNA encoding PPO6. SEQ ID NO:25 is the reverse oligonucleotide used to identify expression of mRNA encoding PPO6. SEQ ID NO:26 is a forward oligonucleotide used to identify expression of mRNA encoding PPO7. SEQ ID NO:27 is the reverse oligonucleotide used to identify expression of mRNA encoding PPO7. SEQ ID NO:28 is a forward oligonucleotide used to identify expression of mRNA encoding PPO8. SEQ ID NO:29 is the reverse oligonucleotide used to identify expression of mRNA encoding PPO8. SEQ ID NO:30 is a forward oligonucleotide used to identify expression of mRNA encoding PPO9. SEQ ID NO:31 is the reverse oligonucleotide used to identify expression of mRNA encoding PPO9. SEQ ID NO:32 is the variable sequence of PPO1 sgRNA1 sg2019. SEQ ID NO:33 is the variable sequence of PPO1 sgRNA2 sg858. SEQ ID NO:34 is the variable sequence of PPO2 sgRNA1 sg854. SEQ ID NO:35 is the variable sequence of PPO2 sgRNA2 sg855. SEQ ID NO:36 is the variable sequence of PPO3 sgRNA1 sg1435. SEQ ID NO:37 is the variable sequence of PPO3 sgRNA2 sg1436. SEQ ID NO:38 is the variable sequence of PPO9 sgRNA1 sg850. SEQ ID NO:39 is the variable sequence of PPO9 sgRNA2 sg851. SEQ ID NO: 40 is the PPO1 polypeptide sequence from banana (Accession No. Ma06_31080). SEQ ID NO:41 is a PPO2 polypeptide sequence from banana (Accession No. Ma07_03540). SEQ ID NO: 42 is a PPO3 polypeptide sequence from banana (Accession No. Ma07_03650). SEQ ID NO: 43 is a PPO4 polypeptide sequence from banana (Accession No. Ma08_09150). SEQ ID NO: 44 is a PPO5 polypeptide sequence from banana (Accession No. Ma08_09160). SEQ ID NO: 45 is a PPO6 polypeptide sequence from banana (Accession No. Ma08_09170). SEQ ID NO: 46 is a PPO7 polypeptide sequence from banana (Accession No. Ma08_09180). SEQ ID NO: 47 is the PPO8 polypeptide sequence from banana (Accession No. Ma08_34740). SEQ ID NO: 48 is a PPO9 polypeptide sequence from banana (Accession No. Ma10_20510). SEQ ID NO:49 is the variable sequence of PPO1 sgRNA sg2019 containing the PAM site. SEQ ID NO:50 is the variable sequence of PPO1 sgRNA sg858 containing the PAM site. SEQ ID NO:51 is the variable sequence of PPO2 sgRNA sg854 containing the PAM site. SEQ ID NO:52 is the variable sequence of PPO2 sgRNA sg855 containing the PAM site. SEQ ID NO:53 is the variable sequence of PPO3 sgRNA sg1435 containing the PAM site. SEQ ID NO:54 is the variable sequence of PPO3 sgRNA sg1436 containing the PAM site. SEQ ID NO:55 is the variable sequence of PPO9 sgRNA sg850 containing the PAM site. SEQ ID NO:56 is the variable sequence of PPO9 sgRNA sg851 containing the PAM site. SEQ ID NO:57 is the variable sequence of PPO8 sgRNA1 sg852. SEQ ID NO:58 is the variable sequence of PPO8 sgRNA2 sg853. SEQ ID NO:59 is another PPO1 sgRNA variable sequence. SEQ ID NO: 60 is another PPO1 sgRNA variable sequence. SEQ ID NO:61 is another PPO1 sgRNA variable sequence. SEQ ID NO:62 is a variable sequence of another PPO1 sgRNA (sg857). SEQ ID NO:63 is another PPO1 sgRNA variable sequence. SEQ ID NO:64 is a variable sequence of another PPO2 sgRNA. SEQ ID NO:65 is a variable sequence of another PPO2 sgRNA. SEQ ID NO:66 is a variable sequence of another PPO2 sgRNA. SEQ ID NO:67 is a variable sequence of another PPO2 sgRNA. SEQ ID NO:68 is a variable sequence of another PPO2 sgRNA. SEQ ID NO:69 is a variable sequence of another PPO2 sgRNA. SEQ ID NO: 70 is a variable sequence of another PPO2 sgRNA. SEQ ID NO:71 is another PPO3 sgRNA variable sequence. SEQ ID NO:72 is a variable sequence of another PPO3 sgRNA. SEQ ID NO:73 is a variable sequence of another PPO3 sgRNA. SEQ ID NO:74 is another PPO3 sgRNA variable sequence. SEQ ID NO: 75 is another PPO3 sgRNA variable sequence. SEQ ID NO: 76 is a variable sequence of another PPO4 sgRNA. SEQ ID NO:77 is a variable sequence of another PPO4 sgRNA. SEQ ID NO:78 is a variable sequence of another PPO5 sgRNA. SEQ ID NO:79 is another PPO5 sgRNA variable sequence. SEQ ID NO:80 is another PPO6 sgRNA variable sequence. SEQ ID NO:81 is another PPO6 sgRNA variable sequence. SEQ ID NO:82 is another PPO7 sgRNA variable sequence. SEQ ID NO: 83 is another PPO7 sgRNA variable sequence. SEQ ID NO:84 is another PPO7 sgRNA variable sequence. SEQ ID NO:85 is another PPO7 sgRNA variable sequence. SEQ ID NO: 86 is another PPO7 sgRNA variable sequence. SEQ ID NO: 87 is another PPO7 sgRNA variable sequence. SEQ ID NO: 88 is another PPO7 sgRNA variable sequence. SEQ ID NO:89 is another PPO7 sgRNA variable sequence. SEQ ID NO: 90 is another PPO8 sgRNA variable sequence. SEQ ID NO: 91 is another PPO8 sgRNA variable sequence. SEQ ID NO: 92 is another PPO8 sgRNA variable sequence. SEQ ID NO: 93 is another PPO8 sgRNA variable sequence. SEQ ID NO: 94 is another PPO9 sgRNA variable sequence. SEQ ID NO: 95 is another PPO9 sgRNA variable sequence. SEQ ID NO: 96 is another PPO9 sgRNA variable sequence. SEQ ID NO: 97 is another PPO9 sgRNA variable sequence. SEQ ID NO: 98 is the scaffold used with the variable sequence of the sgRNA. SEQ ID NO: 99 is the variable sequence of PPO8 sgRNA sg852 containing the PAM site. SEQ ID NO:100 is the variable sequence of PPO8 sgRNA sg853 containing the PAM site. SEQ ID NO: 101 is a variable sequence of another PPO1 sgRNA containing a PAM site. SEQ ID NO: 102 is a variable sequence of another PPO1 sgRNA containing a PAM site. SEQ ID NO: 103 is a variable sequence of another PPO1 sgRNA containing a PAM site. SEQ ID NO: 104 is a variable sequence of another PPO2 sgRNA containing a PAM site. SEQ ID NO: 105 is a variable sequence of another PPO2 sgRNA containing a PAM site. SEQ ID NO: 106 is a variable sequence of another PPO2 sgRNA containing a PAM site. SEQ ID NO: 107 is a variable sequence of another PPO3 sgRNA containing a PAM site. SEQ ID NO: 108 is a variable sequence of another PPO3 sgRNA containing a PAM site. SEQ ID NO: 109 is a variable sequence of the PPO7 sgRNA containing the PAM site. SEQ ID NO:110 is a variable sequence of the PPO7 sgRNA containing the PAM site. SEQ ID NO:111 is the variable sequence of the PPO7 sgRNA containing the PAM site. SEQ ID NO: 112 is the variable sequence of the PPO7 sgRNA containing the PAM site. SEQ ID NO: 113 is a variable sequence of the PPO7 sgRNA containing the PAM site. SEQ ID NO: 114 is a variable sequence of the PPO7 sgRNA containing the PAM site. SEQ ID NO: 115 is a variable sequence of another PPO8 sgRNA containing a PAM site. SEQ ID NO: 116 is a variable sequence of another PPO8 sgRNA containing a PAM site. SEQ ID NO: 117 is a variable sequence of another PPO8 sgRNA containing a PAM site. SEQ ID NO: 118 is a variable sequence of another PPO8 sgRNA containing a PAM site. SEQ ID NO: 119 is a variable sequence of another PPO9 sgRNA containing a PAM site. SEQ ID NO: 120 is a variable sequence of another PPO9 sgRNA containing a PAM site. SEQ ID NO: 121 is a variable sequence of another PPO9 sgRNA containing a PAM site. SEQ ID NO: 122 is a variable sequence of another PPO9 sgRNA containing a PAM site. SEQ ID NO: 123 is a forward primer for detecting gene editing events in PPO1. SEQ ID NO: 124 is a reverse primer for detecting gene editing events in PPO1. SEQ ID NO: 125 is a forward primer for detecting gene editing events in PPO2. SEQ ID NO: 126 is a reverse primer for detecting gene editing events in PPO2. SEQ ID NO: 127 is a forward primer for detecting gene editing events in PPO3. SEQ ID NO: 128 is a reverse primer for detecting gene editing events in PPO3. SEQ ID NO: 129 is a forward primer for detecting a gene editing event in PPO8. SEQ ID NO: 130 is a reverse primer for detecting gene editing events in PPO8. SEQ ID NO: 131 is a forward primer for detecting a gene editing event in PPO9. SEQ ID NO: 132 is a reverse primer for detecting gene editing events in PPO9. SEQ ID NO: 133 is primer 1684 used to confirm the absence of Cas9. SEQ ID NO: 134 is primer 1685 used to confirm the absence of Cas9. SEQ ID NO: 135 is primer 1686 used to confirm the absence of Cas9. SEQ ID NO: 136 is primer 1687 used to confirm the absence of Cas9. SEQ ID NO:137 is primer 1563 used to confirm the absence of the backbone. SEQ ID NO:138 is primer 1564 used to confirm the absence of the backbone. SEQ ID NO:139 is primer 1565 used to confirm the absence of the backbone. SEQ ID NO:140 is primer 1566 used to confirm the absence of the backbone. SEQ ID NO:141 is primer 1567 used to confirm the absence of the backbone. SEQ ID NO:142 is primer 1568 used to confirm the absence of the backbone. SEQ ID NO:143 is primer 1569 used to confirm the absence of the backbone. SEQ ID NO:144 is primer 1570 used to confirm the absence of the backbone. SEQ ID NO:145 is primer 1571 used to confirm the absence of the backbone. SEQ ID NO:146 is primer 1572 used to confirm the absence of the backbone. SEQ ID NO:147 is primer 1573 used to confirm the absence of the backbone. SEQ ID NO:148 is primer 1574 used to confirm the absence of the backbone. SEQ ID NO:149 is primer 1575 used to confirm the absence of the backbone. SEQ ID NO:150 is primer 1576 used to confirm the absence of the backbone. SEQ ID NO: 151 is the PPO1 gene sequence from banana (accession number Ma06_31080). SEQ ID NO: 152 is the PPO2 gene sequence from banana (accession number Ma07_03540). SEQ ID NO: 153 is the PPO3 gene sequence from banana (accession number Ma07_03650). SEQ ID NO: 154 is the PPO4 gene sequence from banana (accession number Ma08_09150). SEQ ID NO: 155 is the PPO5 gene sequence from banana (accession number Ma08_09160). SEQ ID NO: 156 is the PPO6 gene sequence from banana (accession number Ma08_09170). SEQ ID NO: 157 is the PPO7 gene sequence from banana (accession number Ma08_09180). SEQ ID NO: 158 is the PPO8 gene sequence from banana (accession number Ma08_34740). SEQ ID NO: 159 is the PPO9 gene sequence from banana (accession number Ma10_20510). SEQ ID NO:160 is the wheat TaU6 promoter. SEQ ID NO: 161 is Ma10_p20510-PPO9 from Figure 3. SEQ ID NO: 162 is Ma08_p09180-PPO7 from Figure 3. SEQ ID NO: 163 is Ma08_p09170-PPO6 from Figure 3. SEQ ID NO: 164 is Ma08_p09150-PPO4 from Figure 3. SEQ ID NO: 165 is Ma08_p09160-PPO5 from Figure 3. Sequence number 166 is GPO3_21_US9580723B2_21 from Figure 3. SEQ ID NO: 167 is PPO3_BAA21676 from Figure 3. SEQ ID NO: 168 is APO5_AAA69902 from Figure 3. SEQ ID NO: 169 is PPO7_BAA21677 from Figure 3. SEQ ID NO: 170 is Ma06_p31080-PPO1 from Figure 3. SEQ ID NO: 171 is Ma07_p03650-PPO3 from Figure 3. SEQ ID NO: 172 is Ma07_p03540-PPO2 from Figure 3. SEQ ID NO: 173 is Ma08_p34740-PPO8 from Figure 3. SEQ ID NO: 174 is Ma07_g03540-PPO2-WT from Figure 6. SEQ ID NO: 175 is Ma07_g03450-PPO2-GE-pooled-embryo. SEQ ID NO: 176 is the variable sequence of PPO1 sgRNA sg857 containing the PAM site. SEQ ID NO: 177 is the PPO1 variant protein sequence. SEQ ID NO: 178 is a PPO1 variant coding sequence. SEQ ID NO: 179 is the PPO1 variant gene sequence. SEQ ID NO:180 to SEQ ID NO:225 are sgRNAs provided in Table 8 that do not contain a PAM site.

[0021] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is based in part on the discovery that there are nine polyphenol oxidase (PPO) genes present in bananas, which the inventors have named PPO1 through PPO9, most of which have not previously been identified as PPO genes. The invention is further based in part on the discovery that some of the identified PPOs, in particular polyphenol oxidase 1 (PPO1), polyphenol oxidase 2 (PPO2), polyphenol oxidase 8 (PPO8), polyphenol oxidase 9 (PPO9), and polyphenol oxidase 4 (PPO4), are expressed in the flesh and / or peel of the banana fruit but are characterized by low or no expression in other tissues, in particular embryonic tissues or embryonic cell suspensions (ECS). Without wishing to be bound by theory or mechanism, according to some embodiments of the invention, reducing the levels or activity of PPO1, PPO2, PPO8, PPO9, and / or PPO4 in banana plants delays or prevents browning of the flesh and / or peel of banana fruits without causing undesirable effects in other plant parts. According to some embodiments, the invention is directed to various methods of reducing the levels or activity of PPO, particularly PPO1, PPO2, PPO8, PPO9, and / or PPO4, in banana plants or banana plant cells to delay or prevent browning of banana fruits. Products of such methods are also provided by the invention.

[0022] In particular, the present invention provides a method of reducing the level or activity of at least one endogenous polyphenol oxidase encoded by a polyphenol oxidase gene or polynucleotide in a banana plant or banana plant cell. The present invention further provides a method of reducing and / or delaying browning of at least one of a banana fruit and a banana peel. According to some embodiments, provided herein is a method of reducing and / or delaying browning of at least one of the flesh and peel of a banana fruit, comprising reducing or inducing loss of function of at least one endogenous PPO in the banana plant cell or banana plant from which the banana fruit arises (e.g., by reducing or inducing loss of expression of a gene encoding at least one endogenous PPO). Each possibility represents a separate embodiment of the present invention. According to some embodiments, the endogenous PPO is PPO1, PPO2, PPO8, PPO9, and / or PPO4. According to some embodiments, the PPO is selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9.

[0023] As used herein, the term "reduced level or activity" of one or more endogenous polyphenol oxidases means (a) a delayed or reduced browning phenotype conferred by one or more endogenous polyphenol oxidases in a banana plant or banana plant cell or a function of one or more endogenous polyphenol oxidases in a banana plant or banana plant cell is reduced compared to a "wild type" banana plant or plant cell; and / or (b) a reduced expression level of genes encoding one or more endogenous polyphenol oxidases in a banana plant or banana plant cell compared to a "wild type" banana plant or plant cell. The expression level may be mRNA or protein. Such a reduction may be at least 50%, 60%, 70%, 80%, 90%, or preferably 100%. According to some embodiments, the reduction is a complete loss of function. In this context, "wild type" means the same genetic background and the equivalent developmental stage. In some embodiments, the method of the invention reduces the function of at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase in the banana plant or banana plant cell. In other embodiments, the method abolishes the function of at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase in the banana plant or banana plant cell. In some embodiments, the method of the invention reduces the function of at least one endogenous polyphenol oxidase in the banana plant or banana plant cell, wherein the at least one polyphenol oxidase is selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, PPO9, and combinations thereof. In other embodiments, the method comprises eliminating the function of at least one endogenous polyphenol oxidase in the banana plant or banana plant cell, wherein the at least one polyphenol oxidase is selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, PPO9, and combinations thereof.

[0024] As used herein, the term "endogenous" means native to the genome of a banana plant or banana plant cell and in its native location within the genome.

[0025] As used herein, the term "polyphenol oxidase" or "PPO" refers to an enzyme that belongs to a group of enzymes found throughout the plant and animal kingdoms, including most fruits and vegetables, such as bananas. Correspondingly, the term "polyphenol oxidase gene" or "PPO gene" as used herein refers to a gene encoding a polyphenol oxidase or PPO. PPO catalyzes enzymatic browning, including when tissue is damaged by bruising or squeezing. Exposure to oxygen, such as when fruits are sliced ​​or pureed, also causes enzymatic browning by PPO. PPO is known to accept monophenols and / or o-diphenols as substrates, and acts by catalyzing the o-hydroxylation of monophenol molecules, whose benzene ring contains one hydroxyl substituent, to o-diphenols (phenol molecules that contain two hydroxyl substituents at the 1,2 positions and no carbon between them). The enzyme can also catalyze the oxidation of o-diphenols to produce o-quinones. PPO catalyzes the rapid polymerization of o-quinones to produce black, brown, or red pigments (polyphenols) that cause fruit browning.

[0026] Although many plants contain several PPO members, the banana PPO family has not been characterized to date. The present inventors have identified nine endogenous PPOs in banana (Musa acuminata DH-Pahang) and named them "PPO1", "PPO2", "PPO3", "PPO4", "PPO5", "PPO6", "PPO7", "PPO8", and "PPO9". As described herein, these PPOs were identified through multiple iterations of complex phylogenetic analyses on the genomes of various species, including: Coffea canephora, date palm (Phoenix dactylifera), Musa acuminata banksia, Musa acuminata Calcutta, Musa acuminata DH-Pahang, Musa balbisiana, Musa itinerans, Arabidopsis thaliana, soybean (Glycine max), apple (Malus domestica), bell pepper (Capsicum annuum), Nicotiana benthamiana, tomato (Solanum lycopersicum), and rice (Oryza sativa).

[0027] In some embodiments, the level or activity of endogenous PPO1 is decreased. In some embodiments, the activity of endogenous PPO2 is decreased. In some embodiments, the level or activity of endogenous PPO3 is decreased. In some embodiments, the level or activity of endogenous PPO4 is decreased. In some embodiments, the level or activity of endogenous PPO5 is decreased. In some embodiments, the level or activity of endogenous PPO6 is decreased. In some embodiments, the level or activity of endogenous PPO7 is decreased. In some embodiments, the level or activity of endogenous PPO8 is decreased. In some embodiments, the level or activity of endogenous PPO9 is decreased. In some embodiments, the level or activity of two or more of endogenous PPOs selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9 is decreased. In certain embodiments, the level or activity of endogenous PPO1 and PPO2 is decreased. In any of these embodiments, the term "reduced level or activity" of one or more endogenous polyphenol oxidases selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9 means that the phenotype conferred by the one or more endogenous polyphenol oxidases selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9 in a banana plant or banana plant cell, or the function of one or more endogenous polyphenol oxidases selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9 in a banana plant or banana plant cell is reduced or completely lost compared to a wild-type banana plant or plant cell.

[0028] An endogenous PPO gene in any of the embodiments herein may be described as having "sequence identity" or homology to a polynucleotide sequence. The amount of homology or sequence identity may vary, and may range from about 1-20 bp, about 20-50 bp, about 50-100 bp, about 75-150 bp, about 100-250 bp, about 150-300 bp, about 200-400 bp, about 250-500 bp, about 300-600 bp, about 350-750 bp, about 400-800 bp, about 450-900 bp, about 500-1000 bp, about 600-125 bp, about 750-800 bp, about 800-900 bp, about 900-1000 bp, about 1000-1250 bp, about 1500-1500 bp, about 1000-1500 bp, about 15 ... The ranges include full lengths and / or regions having unit integer values ​​of about 0 bp, about 700 to 1500 bp, about 800 to 1750 bp, about 900 to 2000 bp, about 1 to 2.5 kb, about 1.5 to 3 kb, about 2 to 4 kb, about 2.5 to 5 kb, about 3 to 6 kb, about 3.5 to 7 kb, about 4 to 8 kb, about 5 to 10 kb, or the full length of the gene sequence or polynucleotide sequence of an endogenous PPO. These ranges include all integers within the ranges, and for example, the range of 1 to 20 bp includes 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, and 20 bp. The amount of homology or sequence identity can also be described by the percent sequence identity over the aligned lengths of two genes or two polynucleotides, including 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, percent global sequence identity, and, optionally, conserved regions of contiguous nucleotides or percent local sequence identity. For example, sufficient homology can be described as a 75-150 bp region having at least 80% sequence identity with a region of the gene sequence or polynucleotide sequence of an endogenous PPO. Sufficient homology can also be described by the predicted ability of two genes or polynucleotides to specifically hybridize under high stringency conditions.See, e.g., 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).

[0029] In certain embodiments of the invention, a PPO1 gene refers to a polynucleotide sequence comprising a coding sequence having 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% sequence identity to SEQ ID NO:5; a PPO2 gene refers to a polynucleotide sequence comprising a coding sequence having 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% sequence identity to SEQ ID NO:6. 7, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7; a PPO3 gene refers to a polynucleotide sequence comprising a coding sequence having 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% sequence identity to SEQ ID NO:7. PPO4 gene refers to a polynucleotide sequence comprising a coding sequence having 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% sequence identity to SEQ ID NO:8; PPO5 gene refers to a polynucleotide sequence comprising a coding sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% sequence identity to SEQ ID NO:9. , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:10; PPO6 gene refers to a polynucleotide sequence comprising a coding sequence having 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% sequence identity to SEQ ID NO:10;A PPO7 gene refers to a polynucleotide sequence that contains a coding sequence having 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% sequence identity to SEQ ID NO:11; a PPO8 gene refers to a polynucleotide sequence that contains a coding sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 100% sequence identity to SEQ ID NO:12. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:13; or PPO9 gene refers to a polynucleotide sequence comprising a coding sequence having 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% sequence identity to SEQ ID NO:13. In certain embodiments of the invention, the PPO1, PPO2, PPO8, or PPO9 polyphenol oxidase gene of the invention comprises a coding sequence selected from the group consisting of SEQ ID NO:5 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; SEQ ID NO:6 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; SEQ ID NO:12 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; and SEQ ID NO:13 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:13.

[0030] In certain embodiments of the invention, the PPO1 gene refers to a polynucleotide sequence encoding a polyphenol oxidase having 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% sequence identity to SEQ ID NO:40; the PPO2 gene refers to a polynucleotide sequence encoding a polyphenol oxidase having 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% sequence identity to SEQ ID NO:41. , 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% sequence identity to SEQ ID NO:42; a PPO3 gene refers to a polynucleotide sequence encoding a polyphenol oxidase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, A PPO4 gene refers to a polynucleotide sequence encoding a polyphenol oxidase having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:43; a PPO4 gene refers to a polynucleotide sequence encoding a polyphenol oxidase having 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% sequence identity to SEQ ID NO:43. PPO5 gene refers to a polynucleotide sequence encoding a polyphenol oxidase having 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% sequence identity to SEQ ID NO:44;PPO6 gene refers to a polynucleotide sequence encoding a polyphenol oxidase having 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% sequence identity to SEQ ID NO:45; PPO7 gene refers to a polynucleotide sequence encoding a polyphenol oxidase having 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% sequence identity to SEQ ID NO:46; P PO8 gene refers to a polynucleotide sequence encoding a polyphenol oxidase having 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% sequence identity to SEQ ID NO:47; or A PPO9 gene refers to a polynucleotide sequence encoding a polyphenol oxidase having 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% sequence identity to SEQ ID NO: 48. In certain embodiments of the invention, a PPO1, PPO2, PPO8, or PPO9 polyphenol oxidase gene of the invention is a polypeptide having at least 75% sequence identity to SEQ ID NO: 40 (PPO1) or a polypeptide having at least 75% sequence identity to SEQ ID NO: 40; SEQ ID NO: 41 (PPO2) or a polypeptide having at least 75% sequence identity to SEQ ID NO: 41; SEQ ID NO: 47 (PPO8) or a polypeptide having at least 75% sequence identity to SEQ ID NO: 47;and SEQ ID NO: 48 (PPO9), or a polypeptide having at least 75% sequence identity to SEQ ID NO: 48;

[0031] In certain embodiments of the invention, a PPO1 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 151; a PPO2 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 152; A polynucleotide sequence that contains a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 153; a PPO3 gene contains a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 154; PPO4 gene refers to a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 154. PP05 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 155;PPO6 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO:156; PPO7 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 157; P PO8 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO:158; or The PPO9 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO:159. In certain embodiments of the invention, the PPO1, PPO2, PPO8, or PPO9 polyphenol oxidase gene of the invention comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO: 151 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 151; SEQ ID NO: 152 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 152; SEQ ID NO: 158 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; and SEQ ID NO: 159 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159.

[0032] References herein to particular sequences may also include sequences that substantially correspond to their complementary sequences, including minor sequence variations resulting from, for example, sequencing errors, cloning errors, or other modifications that result in base substitutions, deletions, or additions, provided that the frequency of such variations is less than 1 nucleotide in 50 nucleotides, alternatively less than 1 nucleotide in 100 nucleotides, alternatively less than 1 nucleotide in 200 nucleotides, alternatively less than 1 nucleotide in 500 nucleotides, alternatively less than 1 nucleotide in 1000 nucleotides, alternatively less than 1 nucleotide in 5,000 nucleotides, or alternatively less than 1 nucleotide in 10,000 nucleotides.

[0033] Any sequence identification number (SEQ ID NO) disclosed herein may refer to either a DNA sequence or an RNA sequence depending on the context in which the SEQ ID NO is mentioned, even if the SEQ ID NO is expressed only in DNA sequence format or RNA sequence format. For example, a given SEQ ID NO is expressed in DNA sequence format (e.g., T for thymine is written) but may refer to either the DNA sequence corresponding to the given nucleic acid sequence or the RNA sequence of the nucleic acid sequence of an RNA molecule. Similarly, some sequences are expressed in RNA sequence format (e.g., U for uracil is written) but may refer to either the sequence of an RNA molecule that constitutes a double-stranded RNA (dsRNA) or the sequence of a DNA molecule that corresponds to the depicted RNA sequence depending on the actual type of molecule being described. In any event, both DNA and RNA molecules having the disclosed sequences with any substitutions are envisioned.

[0034] When using percent sequence identity in the context of proteins, non-identical residue positions often differ by conservative amino acid substitutions, where amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) so that the functional properties of the molecule are not altered. When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are considered to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches to increase the percent sequence identity. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions are given a score between 0 and 1. Scoring of conservative substitutions is calculated, for example, according to the algorithm of Henikoff S and Henikoff JG., Proc. Natl. Acad. Sci. USA 1992, 89(22):10915-9. Identity can be determined using any homology comparison software, including, for example, the "BlastN" software from NCBI (National Center of Biotechnology Information), such as by using default parameters. In some embodiments, identity is global identity, i.e., identity over the entire nucleic acid sequence and not over a portion thereof.

[0035] As used herein, the term "plant" refers to whole plants, grafted plants, plant ancestors and progeny, plant organs, plant tissues, and "plant parts." As used herein, "plant parts" include differentiated and undifferentiated tissues, including but not limited to roots (including tubers), rootstocks, stems, shoots, fruits, leaves, pollen, seeds, tumor tissue, and various forms of cells and cultures (e.g., single cells, protoplasts, embryos, embryonic cells, and callus tissue). Plant tissues may be in whole plants, or plant organs, tissues, or cell cultures.

[0036] In certain embodiments, the plant part is a fruit. Fruit includes tissues such as flesh and pericarp. In other embodiments, the plant part is a seed. As used herein, the term "seed" refers to a reproductive unit of a flowering plant that can develop into another such plant. The term "plant organ" refers to a tissue or group of tissues of a plant that constitutes a morphologically and functionally distinct part of the plant. The term "genome" refers to the entire complement of genetic material (genes and non-coding sequences) present in each cell of an organism, or virus, or organelle, and / or the complete set of chromosomes inherited as a (haploid) unit from one parent. "Progeny" includes any subsequent generation of a plant.

[0037] A "transgenic plant" includes a plant that contains a heterologous polynucleotide in its genome, for example, introduced by a transformation process. The heterologous polynucleotide can be stably integrated into the genome, such that the polynucleotide is passed on to subsequent generations. The heterologous polynucleotide can be integrated into the genome alone or as part of a recombinant DNA construct. A transgenic plant can also contain more than one heterologous polynucleotide in its genome. Each heterologous polynucleotide can confer a different trait to the transgenic plant.

[0038] As used herein, a "heterologous" polynucleotide contains sequences derived from a foreign species.

[0039] "Transgenic" may include any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered by the presence of a heterologous nucleic acid, including not only transgenics originally so altered, but also those produced from the original transgenics by sexual or asexual breeding. In some plants, heterologous polynucleotides introduced into the plant genome can be removed through breeding. This process is not possible in bananas, as discussed above.

[0040] According to some embodiments, the banana cells, banana plants, or banana plant parts described herein are non-transgenic. According to some embodiments, the methods disclosed herein result in banana cells, banana plants, or banana plant cells that are non-transgenic.

[0041] Modification of the genome (chromosomal or extrachromosomal) by traditional plant breeding methods, by genome editing procedures described herein (which do not result in the insertion of an exogenous polynucleotide), or by naturally occurring events such as random crossing, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation are not intended to be considered transgenic.

[0042] In certain embodiments, a fertile plant is a plant that produces viable male and female gametes and is self-fertile. Such self-fertile plants can produce progeny plants without contribution from any other plant's gametes and genetic material contained therein. Other embodiments may include the use of plants that are not self-fertile because they do not produce viable or otherwise fertile male or female gametes, or both. As used herein, a "male-sterile plant" is a plant that does not produce viable or otherwise fertile male gametes. As used herein, a "female-sterile plant" is a plant that does not produce viable or otherwise fertile female gametes. Male-sterile and female-sterile plants can be female-fertile and male-fertile, respectively. Male fertile (but female sterile) plants can produce viable progeny when crossed with female fertile plants, and female fertile (but male sterile) plants can produce viable progeny when crossed with male fertile plants.

[0043] As used herein, the term "banana plant" refers to plants of the Musa genus, including plantains. These include Musa acuminata (e.g., Musa acuminata banksia, Musa acuminata calcutta, and Musa acuminata DH bahan), Musa chinensis, Musa itinerans, and autotriploid Musa acuminata "Cavendish" and "Gros Michelle". According to a particular embodiment, the banana is an autotriploid Musa acuminata "Cavendish". Cultivated bananas are incombustible autotriploids (AAA) derived from the ancestral species Musa acuminata (genome AA). Furthermore, plantains (AAB or ABB) are sterile interspecific autotriploids derived from hybridization of Musa acuminata (AA) and Musa chinensis (genome BB). Cultivated bananas and plantains are triploids and therefore cannot produce viable seeds, whereas wild species are diploids and therefore can produce viable seeds. In certain embodiments, the banana plant is triploid. Other polyploids are contemplated, including diploids and tetraploids.

[0044] In some embodiments, the "banana plant" is a banana breeding line, such as an elite line or pure line, or a banana variety or breeding germplasm. As used herein, the term "breeding line" refers to a line of cultivated banana that has commercially valuable or agronomically desirable characteristics, as opposed to wild varieties or landraces. The term includes reference to "elite breeding lines" or "elite lines", which refer to essentially homozygous, usually inbred, plant lines used to produce commercial F1 hybrids. An "elite breeding line" is obtained by breeding and selection for superior agronomic performance, including many agronomically desirable traits. An "elite plant" is any plant of an elite line. Superior agronomic performance refers to a desirable combination of agronomically desirable traits, as defined herein, and it is desirable for an elite breeding line to have improved most, preferably all, of the agronomically desirable traits compared to non-elite breeding lines. An elite breeding line is essentially homozygous, and preferably inbred. As used herein, the term "elite line" refers to any line that is the result of breeding and selection for superior agronomic performance.

[0045] As used herein, the terms "cultivar" and "variety" are used interchangeably and refer to plants that have been purposefully developed by breeding, e.g., crossing and selection, for the purpose of commercialization, e.g., for use by farmers and producers to produce agricultural products for home consumption or commercialization. The term "breeding germplasm" refers to plants having a biological state other than the "wild" state, which refers to the original, uncultivated, or natural state of the plant or genetic line.

[0046] The term "breeding germplasm" includes, but is not limited to, semi-natural, semi-wild, weedy, traditional cultivars, landraces, breeding material, research material, breeder's lines, synthetic populations, hybrids, founder strains / foundation populations, inbreds (parents of hybrid cultivars), segregating populations, mutants / genetic material, market classes, and advanced / improved cultivars. As used herein, the terms "pure breed," "pure inbred," or "inbred" are interchangeable and refer to substantially homozygous plants or plant lines obtained by repeated selfing and / or backcrossing.

[0047] As used herein, the term "banana plant cell" refers to a cell of a banana plant. Banana plant cells include cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, microspores, embryogenic cells, somatic cells, and protoplasts, etc. Protoplasts can be derived from any plant tissue, such as, but not limited to, roots, leaves, embryonic cell suspensions, callus, or seedling tissue. According to some embodiments, the banana plant cells are cells of an embryonic cell suspension (ECS).

[0048] In some embodiments, the methods of the invention delay browning of the pulp and / or skin of a banana plant compared to the pulp and / or skin of a banana plant that does not have a reduced level or activity of the at least one endogenous polyphenol oxidase (selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9); and / or reduce browning of the pulp and / or skin of a banana plant compared to the pulp and / or skin of a banana plant that does not have a reduced level or activity of the at least one endogenous polyphenol oxidase (selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9). In some embodiments, the methods of the invention delay and / or reduce browning of the flesh and / or skin of banana fruits from banana plants having reduced or lost levels or activity of at least one endogenous polyphenol oxidase selected from PPO1, PPO2, PPO8, PPO9, and PPO4. In some embodiments, the methods of the invention delay and / or reduce browning of the flesh and / or skin of banana fruits from banana plants having reduced or lost expression of at least one endogenous polyphenol oxidase gene selected from PPO1, PPO2, PPO8, PPO9, and PPO4.

[0049] In this context, "browning" of the flesh of a banana fruit and / or the skin of a banana fruit can be measured by visual inspection and other methods known in the art.

[0050] In some embodiments, "browning" is measured based on the color of the skin. In such embodiments, a browning index is constructed such that a particular color (e.g., green, partially green, yellow, brown, etc.) of the skin of a wild-type banana fruit can be correlated with the number of days since the appearance of the flower. With such an index, a visual assessment of the skin color on a particular day can provide an indication of delayed browning (e.g., where a wild-type banana turns brown a particular number of days after the appearance of the flower, but a banana genetically edited for the PPO gene is yellow the same number of days after the appearance of the flower) ("Dole Retail Banana Ripening Guide", https: / / www.dolenz.co.nz / uploads / media / 59236082048a9 / banana-trade-section-web.pdf and Gooding et al., "Molecular cloning and characterisation of banana fruit polyphenol oxidase", Planta, Sep 2001;123(5):748-57). This is based on the fact that the fruit matures approximately 60-90 days after the emergence of the flowers. Banana fruits are harvested as early as 40 days until they are dark brown (stage 10). Stage 10 is expected to be reached approximately 90 days after the emergence of the flowers. Fruit color is visually assessed on all 10 scales based on color descriptions defined, for example, in the "Dole Retail Banana Ripening Guide." Stage 1 is when all the fingers of the bunch have a green skin, whereas at stage 7, all the fingers of the bunch have yellow spots and brown skin, and at stage 10, all the fingers of the bunch have a dark brown skin. Colorimetric coordinates are taken on a Minolta Chroma Meter CR 400 or Minolta CR-300 Chroma Meter with a DP-301 data processor to standardize the color scale.The CR-300 measurement head uses diffuse illumination / 0° viewing geometry (including specular components) to provide measurements of a wide variety of surfaces that correlate well with color when viewed under diffuse lighting conditions, as described in Bruno Bonnet, C., Hubert, O., Mbeguie-A-Mbeguie, D. et al., Effect of physiological harvest stages on the composition of bioactive compounds in Cavendish bananas. J. Zhejiang Univ. Sci. B14, 270-278 (2013). This allows the measurement of reflected color at each fruit development stage (1-10), which is used to correlate skin color with fruit ripening and browning.

[0051] In some embodiments, "browning" is measured based on the Banana Browning Guide, which utilizes visual evaluation of sliced ​​and strained banana flesh over time (0-180 hours). In such embodiments, three fingers are taken from banana bunches representing stages 3 to 10 (see above) and the peel is gently washed with 0.2% sodium hypochlorite for 5 minutes (to avoid mechanical damage that may cause browning). A banana puree is then prepared from each individual banana finger after peeling, cutting into small pieces, and homogenizing in an electric mixer or food processor. The resulting puree is poured into a Petri dish and images are taken after 0, 15, 30, 60, and 120 minutes, and after 24, 48, and 72 hours. Additionally, bananas are sliced ​​and placed in Petri dishes and images are taken after 0, 12, 24, 36, 48, and 72 hours for banana bunches at color stages 3 and 4. For banana pulp at stages 5 to 10, images of banana slices are taken every 8 hours from 0 to 180 hours (approximately 22 time points) (Chi, M., Bhagwat, B., Lane, W D et al. Reduced polyphenol oxidase gene expression and enzymatic browning in potato (Solanum tuberosum L.) with artificial microRNAs. BMC Plant Biol 14, 62 (2014). https: / / doi.org / 10.1186 / 1471-2229-14-62 and Escalante-Minakata, P., Ibarra-Junquera, V., Ornelas-Paz, J de et al., Comparative study of the banana pulp browning process of 'Giant Dwarf' and FHIA-23 during fruit ripening based on image analysis and the polyphenol oxidase and peroxidase biochemical properties. 3 Biotech 8,30 (2018)). The images are processed to correlate the color of the images with the browning.The color is expected to vary anywhere from the color of freshly cut banana slices or banana puree (yellow / off-white) to brown banana (dark brown).

[0052] In some embodiments, "browning" is measured based on assessment of flesh firmness and skin firmness. In such embodiments, flesh firmness and skin firmness are measured using a TA-XT2 penetrometer as described in Bruno Bonnet, C., Hubert, O., Mbeguie-A-Mbeguie, D. et al., Effect of physiological harvest stages on the composition of bioactive compounds in Cavendish bananas. J. Zhejiang Univ. Sci. B14, 270-278 (2013). Three fingerlets are harvested from bunches representing stages 3 to 10 (proven based on skin color; see above). A 4.9 mm cylindrical metal perforator is used to penetrate clean, fresh, unpeeled fruit to a depth of 10 mm at a constant speed (2 mm / s). The maximum force applied to break the skin represents the skin hardness, and the slope of the force / time curve represents the fruit firmness.

[0053] In some embodiments, "browning" is measured based on the correlation between skin color (and firmness) and flesh color / texture. Such embodiments utilize a catalog of color (visual and colorimetric), skin firmness, and flesh firmness in wild-type plants versus banana ripeness stage and skin and flesh browning over time. This provides a benchmark against which reduction of browning in banana skin and flesh can be assessed.

[0054] In some embodiments, "browning" is measured based on the amount of melanin formed in banana tissues analyzed within a set time frame. Quantification of such browning can be performed with a simple biochemical assay (Michael L. Sullivan et al., Cloning and Characterization of Red Clover Polyphenol Oxidase cDNAs and Expression of Active Protein in Escherichia coli and Transgenic Alfalfa. Plant Physiology Oct 2004, 136(2)3234-3244; DOI:10.1104 / pp.104.047449; Matthew A. Escobar et al., Characterization of Polyphenol Oxidase from Walnut. Journal of the American Society for Horticultural Science Nov 2008, Volume 133: Issue 6, pages 852-858; DOI:10.21273 / JASHS.133.6.852). In these enzymatic browning assays, the change in absorbance of an exogenous PPO substrate was measured after mixing with banana tissue lysate, and both the amount of product (melanin) formed and the rate of the browning reaction are proportional to PPO activity.

[0055] In some embodiments, for example when considering browning associated with ripening, "delayed" browning means that the onset of browning of the flesh and / or skin is delayed by at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month, compared to the flesh and / or skin of a banana plant that does not have a reduced level or activity of at least one endogenous polyphenol oxidase (selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9). According to certain embodiments, "delayed" refers to a delay of at least 3 days. Browning can be measured as outlined above.

[0056] In some embodiments, for example when considering bruising-related or slicing-related browning, "delayed" browning means that the onset of browning of the pulp and / or peel is delayed by at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, or at least 10 hours compared to the pulp and / or peel of a banana plant that does not have a reduced level or activity of at least one endogenous polyphenol oxidase (selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9). Browning can be measured as outlined above.

[0057] In some embodiments, "reduced" browning means that the pulp and / or peel takes 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times longer to brown compared to the pulp and / or peel of a banana plant that does not have a reduced level or activity of at least one endogenous polyphenol oxidase (selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9). Browning can be measured as outlined above.

[0058] In some embodiments, "reduced" browning means that less melanin is formed at a given time point. For example, the melanin produced in the pulp and / or peel may be at least 10%, 20%, 30%, 40%, 50%, 3-fold, 5-fold, 10-fold or more reduced compared to the pulp and / or peel of a control banana plant that does not have a reduced level or activity of at least one endogenous polyphenol oxidase (selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9). Melanin measurements may be performed at any time point, as outlined above. For example, when considering ripening-related browning, melanin may be measured when the control banana reaches stage 7, 8, 9, or 10 as defined in Example 1 below, and the banana of the present invention is of the same age in terms of days since flowering. For example, there may be more than two times less melanin in bananas of the present invention at stages 7, 8, 9, or 10; there may be more than three times less melanin in bananas of the present invention at stages 7, 8, 9, or 10; or there may be more than five times less melanin in bananas of the present invention at stages 7, 8, 9, or 10.

[0059] In some embodiments, the method of the invention comprises providing a silencing RNA to a banana plant cell or part of a banana plant that targets a transcript of at least one endogenous polyphenol oxidase gene selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9. In some embodiments, the transcript of the endogenous PPO1 gene is targeted. In some embodiments, the transcript of the endogenous PPO2 gene is targeted. In some embodiments, the transcript of the endogenous PPO3 gene is targeted. In some embodiments, the transcript of the endogenous PPO4 gene is targeted. In some embodiments, the transcript of the endogenous PPO5 gene is targeted. In some embodiments, the transcript of the endogenous PPO6 gene is targeted. In some embodiments, the transcript of the endogenous PPO7 gene is targeted. In some embodiments, the transcript of the endogenous PPO8 gene is targeted. In some embodiments, the transcript of endogenous PPO9 gene is targeted. In some embodiments, two or more of endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9 transcripts are targeted. In certain embodiments, endogenous PPO1 and PPO2 transcripts are targeted.

[0060] The silencing RNA can be provided to a banana plant cell or part of a banana plant by a method selected from the group consisting of: introduction of an exogenous silencing RNA; introduction of a sequence expressing the silencing RNA into the cell (i.e., creating a transgene); or transiently editing an endogenous gene encoding an existing non-coding RNA (such as a silencing RNA) to redirect its silencing specificity towards (and optionally activate) a target gene encoding at least one polyphenol oxidase, optionally PPO1, PPO2, PPO8, PPO9, and / or PPO4 (see WO 2019 / 058255, incorporated herein by reference). Potential silencing RNAs that can be introduced, expressed, or redirected include, but are not limited to, small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), phased small interfering RNA (phasiRNA), trans-acting siRNA (tasiRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and autonomous and non-autonomous translocating RNA.

[0061] In other embodiments, the method of the present invention comprises introducing a modification into at least one endogenous polyphenol oxidase gene selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9. In some embodiments, the modification is introduced into the endogenous PPO1 gene. In some embodiments, the modification is introduced into the endogenous PPO2 gene. In some embodiments, the modification is introduced into the endogenous PPO3 gene. In some embodiments, the modification is introduced into the endogenous PPO4 gene. In some embodiments, the modification is introduced into the endogenous PPO5 gene. In some embodiments, the modification is introduced into the endogenous PPO6 gene. In some embodiments, the modification is introduced into the endogenous PPO7 gene. In some embodiments, the modification is introduced into the endogenous PPO8 gene. In some embodiments, the modification is introduced into the endogenous PPO9 gene. In some embodiments, the modification is introduced into two or more of endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9. In certain embodiments, the modification is introduced into endogenous PPO1 and PPO2. Thus, according to some embodiments, provided herein is a method for reducing the level or activity of at least one endogenous polyphenol oxidase encoded by a polyphenol oxidase gene selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9, comprising introducing a modification into at least one endogenous polyphenol oxidase gene selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9.

[0062] In this context, "introducing a modification" means introducing at least one mutation into at least one allele of one or more endogenous polyphenol oxidase genes. According to some embodiments, a mutation is introduced into one allele of one or more endogenous polyphenol oxidase genes, while the other two alleles do not contain a mutation. According to some embodiments, a mutation is introduced into two alleles of one or more endogenous polyphenol oxidase genes, while the third allele does not contain a mutation. According to some embodiments, a mutation is introduced into each allele of one or more endogenous polyphenol oxidase genes. In any embodiment, the mutation may be homozygous or heterozygous. As used herein, "modification" may mean an insertion of at least one nucleotide, a deletion of at least one nucleotide, an insertion-deletion (indel), an inversion, a substitution of at least one nucleotide, or any combination of the above, so long as it reduces the level or activity of at least one endogenous PPO. The modification may result in a frameshift, missense mutation, loss of function mutation, or nonsense mutation in one or more corresponding endogenous polyphenol oxidase genes, resulting in one or more endogenous polyphenol oxidase genes being abolished or expressed at reduced levels. In any embodiment, the size of the modification may be less than 1 kb, or even less than 0.1 kb. In some embodiments, the loss of function mutation is in the 5' region of the respective PPO gene (e.g., in exon 1) to inhibit the production of any expression product. However, the loss of function mutation may be in any part of the respective PPO gene, such as, but not limited to, the regulatory elements of the gene (e.g., its promoter).

[0063] In some embodiments, a modification is provided to the banana plant cell and introduced into one or more polyphenol oxidase genes by an endonuclease capable of targeting the at least one polyphenol oxidase gene, such as at least one of the PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase genes.

[0064] Endonucleases are enzymes that cleave phosphodiester bonds in polynucleotide chains, including restriction endonucleases, which cleave DNA at specific sites without damaging bases. Restriction enzymes include type I, type II, type III, and type IV endonucleases, which further include subtypes. In type I and type III systems, both methylase and restriction activities are contained in a single complex. Endonucleases also include meganucleases, also known as homing endonucleases (HEases), which bind and cleave at specific recognition sites, similar to restriction endonucleases. Endonucleases allow precise genetic engineering of eukaryotic genomes, such as plant genomes.

[0065] In some embodiments, the endonuclease is selected from the group consisting of meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), homing endonuclease, CRISPR-associated endonuclease, and modified CRISPR-associated endonuclease. According to some embodiments, the endonuclease is a CRISPR-associated endonuclease, and optionally, the CRISPR-associated endonuclease is Cas9. Each possibility represents a separate embodiment of the invention. In some embodiments, the endonuclease is selected from the group consisting of meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and homing endonuclease.

[0066] According to some embodiments, provided herein is a method of reducing the level or activity of at least one endogenous polyphenol oxidase (such as a PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase encoded by a PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene) in a banana plant or banana plant cell, wherein reducing the level or activity of the at least one endogenous polyphenol oxidase comprises introducing a modification into a polyphenol oxidase gene encoding the at least one polyphenol oxidase; the modification is introduced by providing an endonuclease that enables the at least one polyphenol oxidase gene to target the banana plant or banana plant cell; and the endonuclease is a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease. According to some embodiments, the endonuclease is provided to the banana cells with at least one "targeting molecule" that allows the endonuclease to specifically target a selected polyphenol oxidase gene. According to some embodiments, the targeting molecule is a guide RNA that allows the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease to cleave a selected sequence within the polyphenol oxidase gene.

[0067] As used herein, "meganucleases" are generally classified into four families: LAGLIDADG family, GIY-YIG family, His-Cys box family, and HNH family. These families are characterized by structural motifs that affect catalytic activity and recognition sequence. For example, members of the LAGLIDADG family are characterized by having one or two copies of the conserved LAGLIDADG motif. The four families of meganucleases are significantly different from each other in terms of conserved structural elements and thus specificity of DNA recognition sequence and catalytic activity. Meganucleases are commonly found in microbial species and have the unique property of having very long recognition sequences (>14bp), which naturally give them a very high specificity for cleavage at the desired position. This can be used to create site-specific double-strand breaks in genome editing. Those skilled in the art can use these naturally occurring meganucleases, but the number of such naturally occurring meganucleases is limited. To overcome this challenge, variants of meganucleases that recognize unique sequences have been created using mutagenesis and high-throughput screening methods. For example, various meganucleases have been fused to create hybrid enzymes that recognize new sequences. Alternatively, sequence-specific meganucleases can be designed by modifying the DNA-interacting amino acids of meganucleases (see, for example, U.S. Patent No. 8,021,867). Meganucleases can be designed using methods described, for example, in Certo, MT et al. Nature Methods (2012) 9:073-975, U.S. Patent Nos. 8,304,222, 8,021,867, 8,119,381, 8,124,369, 8,129,134, 8,133,697, 8,143,015, 8,143,016, 8,148,098, or 8,163,514.Alternatively, meganucleases with site-specific cleavage properties can be obtained using commercially available technologies, such as Precision Biosciences' Directed Nuclease Editor™ genome editing technology.

[0068] "Zinc finger nucleases" (or "ZFNs") and "transcription activator-like effector nucleases" (or "TALENs"), as used herein, have proven effective in generating targeted double-strand breaks (see Christian M, Cermak T, Doyle EL, et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010;186(2):757-761. doi:10.1534 / genetics.110.120717). In essence, ZFN and TALEN restriction endonuclease technologies utilize a non-specific DNA cleavage enzyme linked to a specific DNA binding domain (either a series of zinc finger domains or TALE repeats, respectively). Typically, a restriction enzyme is selected whose DNA recognition site and cleavage site are distant from each other. The cut portion is separated and then linked to a DNA binding domain, thereby resulting in an endonuclease with very high specificity for the desired sequence. An exemplary restriction enzyme with such properties is FokI. Furthermore, FokI has the advantage that it requires dimerization to become nuclease active, which means that each nuclease partner recognizes a unique DNA sequence, increasing specificity. To enhance this effect, FokI nucleases are engineered to only function as heterodimers and have high catalytic activity. Such nucleases avoid the possibility of unwanted homodimer activity and increase the specificity of double-stranded breaks. Thus, to target specific sites, ZFNs and TALENs are constructed as nuclease pairs, with each member of the pair designed to bind to adjacent sequences in the target site. When transiently expressed in cells, the nuclease binds to its target site and the FokI domain heterodimerizes to create a double-stranded break. Repair of these double-stranded breaks through the "non-homologous end joining" (or "NHEJ") pathway often results in small deletions or small sequence insertions.Since each repair performed by NHEJ is unique, a single nuclease pair can be used to create a set of alleles with various deletions at the target site. Typically, the length of the deletions ranges anywhere from a few base pairs to hundreds of base pairs, but two pairs of nucleases have been used simultaneously to successfully create larger deletions in cell culture (Carlson DF, Fahrenkrug SC, Hackett PB. Targeting DNA With Fingers and TALENs. Mol Ther Nucleic Acids. 2012; 1(1): e3. Published 2012 Jan 24. doi: 10.1038 / mtna.2011.5). Furthermore, when a DNA fragment with homology to the target region is introduced together with a nuclease pair, the double-strand break can be repaired via homologous recombination (HR) to generate a specific modification (Urnov, F., Miller, J., Lee, Y. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646-651 (2005). https: / / doi.org / 10.1038 / nature03556). Although the nuclease moieties of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases lies in their DNA recognition peptides. ZFNs rely on Cys2-His2 zinc fingers, and TALENs rely on TALEs. Both of these DNA recognition peptide domains have the characteristic that they are naturally found in combination in their proteins. Cys2-His2 zinc fingers are typically found in repeats spaced 3 bp apart and in a variety of combinations in various nucleic acid interacting proteins. TALEs, on the other hand, are found in repeats with a one-to-one recognition ratio between the amino acid and the recognized nucleotide pair. Because both zinc fingers and TALEs occur in a repeating pattern, different combinations can be tried to generate a wide variety of sequence specificities.Approaches for generating site-specific zinc finger endonucleases include, among others, modular assembly (arrangement and binding of triplet-sequence-correlated zinc fingers to cover the required sequence), OPEN (low stringency selection of peptide domains versus triplet nucleotides, followed by high stringency selection of peptide combinations versus final targets in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries. ZFNs have been designed, for example, at Sangamo Biosciences™ (Richmond, Calif.), and some are commercially available. Methods for designing and obtaining TALENs are described in Reyon et al. Nature Biotechnology (2012) 30(5): 460-465; Miller et al. Nature Biotechnology (2011) 29: 143-148; Cermak et al. Nucleic Acids Research (2011) 39(12): e82, and Zhang et al. Nature Biotechnology (2011) 29(2): 149-153. A recently developed web-based program named "Mojo Hand" has been introduced by Mayo Clinic to design TAL and TALEN constructs for genome editing applications (accessible through www.talendesign.org).

[0069] As used herein, a "homing endonuclease" is a double-stranded DNase with a large asymmetric recognition site (12-40 base pairs (bp)) and a coding sequence that is usually embedded in either an intron or an intein (Belfort, M. and Roberts, RJ (1997) Nucleic Acids Research, 25, 3379-3388). Introns are spliced ​​out of precursor RNAs, whereas inteins are spliced ​​out of precursor proteins (Dujon, B. et al. (1989) Gene, 82, 115-118; Perler, F B et al. (1994) Nucleic Acids Research, 22, 1125-1127). Homing endonucleases are named using a convention similar to that of restriction endonucleases, with intron-encoded endonucleases having the prefix "I-" and intein endonucleases having the prefix "PI-" (Belfort, M. and Roberts, RJ (1997) Nucleic Acids Research, 25, 3379-3388; Roberts, RJ et al. (2003) Nucleic Acids Research, 31, 1805-1812). Homing endonuclease recognition sites are rare. For example, an 18 base pair (bp) recognition sequence is 7×10 times more likely to be identified than a random sequence. 10 Only one occurs per base pair. However, unlike restriction endonucleases, homing endonucleases tolerate some sequence degeneracy within their recognition sequences (Gimble, FS and Wang, J. (1996) Journal of Molecular Biology, 263, 163-180; Argast, M Get al. (1998) Journal of Molecular Biology, 280, 345-353). That is, a single base change does not abolish cleavage, but reduces its efficiency to various degrees. As a result, the sequence specificity observed is typically in the range of 10-12 base pairs.

[0070] In some embodiments, the method of the present invention comprises providing a banana plant cell with a CRISPR-associated endonuclease or modified CRISPR-associated endonuclease.In some embodiments, the method of the present invention comprises providing a banana plant cell with a CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and one or more guide RNAs specific to at least one polyphenol oxidase gene, wherein the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and the one or more guide RNAs form a complex that allows the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease to introduce a double-stranded or single-stranded break in at least one endogenous polyphenol oxidase gene.In some of these embodiments, the CRISPR-associated endonuclease is a Cas9 endonuclease. In some of these embodiments, the at least one endogenous polyphenol oxidase gene or polynucleotide is selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO1 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO2 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO3 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO4 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO5 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO6 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO7 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO8 gene.In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO9 gene. In some embodiments, the endogenous polyphenol oxidase gene is two or more of the PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9 genes. In some embodiments, the endogenous polyphenol oxidase genes are the PPO1 and PPO2 genes.

[0071] In certain embodiments of the method, the PPO1 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:5; the PPO2 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:6. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7; the PPO4 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:8; the PPO5 gene comprises a coding sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, the PPO6 gene comprises a coding sequence having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:10; the PPO6 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:10;The PPO7 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:11; the PPO8 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:12. or the PPO9 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:13. In certain embodiments of the method, the PPO1, PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene comprises a coding sequence selected from the group consisting of SEQ ID NO:5 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; SEQ ID NO:6 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; SEQ ID NO:12 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; SEQ ID NO:13 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:13; and SEQ ID NO:8 (PPO4) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8.

[0072] In certain embodiments of the method, the PPO1 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:40; the PPO2 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:41. %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:42; the PPO3 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:42. the PPO4 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:43; the PPO5 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:44; %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:45; the PPO6 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:45;The PPO7 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:46; the PPO8 gene encodes a polyphenol oxidase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% sequence identity to SEQ ID NO:47. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:48; or the PPO9 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:48. In certain embodiments of the method, the PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene encodes a polyphenol oxidase selected from the group consisting of SEQ ID NO:40 (PPO1) or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; SEQ ID NO:41 (PPO2) or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; SEQ ID NO:47 (PPO8) or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; SEQ ID NO:48 (PPO9) or a polypeptide having at least 75% sequence identity to SEQ ID NO:48; and SEQ ID NO:43 (PPO4) or a polypeptide having at least 75% sequence identity to SEQ ID NO:43.

[0073] In certain embodiments of the method, the PPO1 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 151; the PPO2 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 152. , 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 153; PPO3 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 153. 154、55%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:154; a PPO4 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO:154; or 100% sequence identity to SEQ ID NO:155; PPO5 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO:155;PPO6 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO:156; PPO7 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 157; P PO8 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO:158; or The PPO9 gene refers to a polynucleotide sequence comprising a polynucleotide sequence having 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% sequence identity to SEQ ID NO:159. In certain embodiments of the method, the polyphenol oxidase gene of PPO1, PPO2, PPO8, PPO9, or PPO4 is selected from the group consisting of SEQ ID NO:151 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; SEQ ID NO:152 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; SEQ ID NO:158 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:158; SEQ ID NO:159 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:159;and SEQ ID NO: 154 (PPO4), or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 154;

[0074] As used herein, "CRISPR-associated endonuclease" (or "Cas") refers to an endonuclease with RNA-guided polynucleotide editing activity, and is one of the components of the CRISPR / Cas system for genome editing using at least one additional component, the "guide RNA" (gRNA). In some embodiments of the present 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 (Shota Nakade, Takashi Yamamoto & Tetsushi Sakuma (2017), Cas9, Cpf1 and C2c1 / 2 / 3--What's next?, Bioengineered, 8:3, 265-273, and references therein). In other embodiments, the "CRISPR-associated endonuclease" may be Cpf1, such as, but not limited to, AsCpf1 or LbCpf1 (Shota Nakade, Takashi Yamamoto & Tetsushi Sakuma (2017), Cas9, Cpf1 and C2c1 / 2 / 3--What's next?, Bioengineered, 8:3, 265-273, and references therein).

[0075] As used herein, the terms "guide RNA" or "gRNA" may be used interchangeably and refer to a polynucleotide that facilitates specific targeting of a CRISPR-associated endonuclease or modified CRISPR-associated endonuclease to a target sequence, such as a genomic or episomal sequence in a cell. According to some embodiments, the gRNA may be chimeric / unimolecular (comprising a single RNA molecule, also called single guide RNA or sgRNA) or modular (comprising more than one separate RNA molecule, typically crRNA and tracrRNA, which may be linked, for example, by duplex formation). According to some embodiments, the gRNA is an sgRNA.

[0076] sgRNA is an RNA molecule that contains both tracrRNA and crRNA (and a linking loop). sgRNA contains a sequence of nucleotides that encodes a target homologous sequence (crRNA) and an endogenous bacterial RNA (tracrRNA) that links the crRNA to the Cas9 nuclease in a single chimeric transcript. This region of the crRNA, known as the variable region, confers cleavage specificity for the associated endonuclease and is typically 20 nucleotides long, but can be about 17-20 nucleotides long. The gRNA / Cas complex is recruited to the target sequence by base pairing between the sgRNA sequence and the complementary 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. Upon binding of the gRNA / Cas complex, Cas localizes to the genomic target sequence such that Cas can cleave both strands of DNA to create a double-stranded break. Similar to ZFNs and TALENs, double-stranded breaks generated by CRISPR / Cas can be repaired by HR (homologous recombination) or NHEJ (non-homologous end joining) and are susceptible to specific sequence modifications during DNA repair. Cas nuclease has two functional domains: RuvC and HNH, each of which cleaves a different DNA strand. When both of these domains are active, Cas will cause a double-stranded break in the genomic DNA. A major advantage of CRISPR / Cas is the combination of the high efficiency of the system with the ability to easily generate synthetic gRNAs. This results in a system that can be easily modified to target different genomic sites and / or target different modifications at the same site. Furthermore, protocols have been established that allow multiple genes to be targeted simultaneously. The majority of cells carrying mutations have biallelic mutations in the targeted genes. However, the apparent flexibility of the base-pairing interactions between the gRNA sequence and the genomic DNA target sequence allows for cleavage by Cas even when the match to the target sequence is imperfect.

[0077] Engineered versions of Cas enzymes that contain a single inactive catalytic domain of either RuvC or HNH are called "nickases." With only one active nuclease domain, Cas nickases cleave only one strand of the target DNA, creating a single-strand break or "nick." Single-strand breaks or nicks are repaired by single-strand break repair mechanisms that mostly involve proteins such as, but not limited to, PARP (sensor) and the XRCCl / LIG III complex (ligation). When single-strand breaks (SSBs) are generated by topoisomerase I poisons or by drugs that trap PARP1 at naturally occurring SSBs, these can persist and become single-ended DSBs that can only be repaired by HR when cells enter S phase and a replication fork encounters such an SSB. However, nicks in two adjacent opposing strands introduced by Cas nickases are treated as double-strand breaks and are often referred to as "double-nick" CRISPR systems. Double nicks are essentially non-parallel DSBs and, like other DSBs, can be repaired by HR or NHEJ depending on the desired effect on the gene target and the presence of donor sequences and the stage of the cell cycle (HR is much less abundant and can only occur in the S and G2 phases of the cell cycle). Thus, where specificity and reduced off-target effects are important, the use of Cas nickases to create double nicks by designing two gRNAs with target sequences that are in close proximity and present on opposite strands of the genomic DNA will reduce off-target effects, as they create nicks that are unlikely, if not impossible, to alter the genomic DNA with either gRNA alone.

[0078] As used herein, "modified CRISPR-associated endonuclease" (or "modified Cas" refers to a Cas in which the catalytic domain has been modified and / or fused to additional domains. In some embodiments, a "modified Cas" refers to a Cas in which the catalytic domain has been modified and / or fused to additional domains. "Modified Cas" refers to Cas that contains a nickase activity ("nCas9") and does not have nuclease activity, but can still bind to DNA based on gRNA specificity. According to some embodiments, "modified Cas" refers to Cas that has nickase activity ("nCas9") and thus induces single-strand breaks. In some embodiments, the modified CRISPR-associated endonuclease is a "modified Cas9 endonuclease", optionally a catalytically inactive Cas9 (or "dCas9") or a nickase Cas9 ("nCas9"). dCas can be utilized as a platform for DNA transcription regulators to activate or repress gene expression by fusing the inactive enzyme to a known regulatory domain. For example, dCas alone can bind to a target sequence in genomic DNA and interfere with the transcription of the gene. In addition to publicly available tools available to aid in the selection and / or design of target sequences, there are numerous published lists of gRNAs specific to different genes in different species that have been determined based on bioinformatics, such as the Target Cas9 library from the Feng Zhang lab. Finder, Target Finder “E-CRISP” from the Michael Boutros lab, RGEN Tools: “Cas-OFFinder”, and CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes and the CRISPR Optimal Target Finder.

[0079] In the context of the present invention, modified Cas such as dCas or nCas9 can also be used in accordance with some embodiments together with other enzymes for base editing (optionally as fusion proteins). Base editing is a genome editing approach that uses components of the CRISPR system together with other enzymes to directly introduce point mutations into cellular DNA or RNA without creating double-stranded DNA breaks. DNA base editors include catalytically inactive nucleases fused to nucleobase deaminase enzymes and, optionally, DNA glycosylase inhibitors. RNA base editors achieve similar changes using RNA-targeting components. Base editors directly convert one base or base pair to another, allowing for efficient introduction of point mutations in non-dividing cells without generating excessive undesired editing by-products (Rees and Liu (2018), “Base Editing: Precision Chemistry on the Genome and Transcriptome of Living Cells”, Nature Reviews Genetics, 19(12):770-788). In some embodiments, the modified Cas9 is a nCas fused to a base editor enzyme, such as an adenosine or cytidine deaminase.Specific 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, and SaKKH-ABE (Rees and Liu (2018), “Base Editing: Precision Chemistry on the Genome and Transcriptome of Living Cells”, Nature Reviews Genetics, 19(12):770-788, and references therein).

[0080] As used herein, a "guide RNA" (or "gRNA") is not limited to a particular sequence, so long as the sequence is specific to at least one polyphenol oxidase gene or targets a genomic sequence encoding a silencing RNA whose sequence has been modified so that the encoded silencing RNA silences a polyphenol oxidase gene as defined herein. In some embodiments of the invention, one or more guide RNAs comprise a variable region having a sequence selected from the group consisting of SEQ ID NOs: 32-39 and 57-97; and any combination of the foregoing. In some embodiments of the invention, one or more guide RNAs comprise a variable region having a sequence selected from the group consisting of SEQ ID NOs: 180-196, 199-202, and 207-225; and any combination of the foregoing. In some embodiments of the invention, one or more guide RNAs comprise a variable region having a sequence selected from the group consisting of SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 62; SEQ ID NO: 76; SEQ ID NO: 77; and any combination of the foregoing.

[0081] In some embodiments of the invention, the one or more guide RNAs are a pair of guide RNAs comprising variable regions selected from the group consisting of SEQ ID NOs: 32 and 33; SEQ ID NOs: 34 and 35; SEQ ID NOs: 32 and 34; SEQ ID NOs: 32 and 35; SEQ ID NOs: 33 and 34; SEQ ID NOs: 33 and 35; SEQ ID NOs: 57 and 58; SEQ ID NOs: 38 and 39; SEQ ID NOs: 62 and 33; and SEQ ID NOs: 76 and 77. According to some embodiments, the gRNA comprises a variable region sequence as described above followed by a constant scaffold sequence of SEQ ID NO: 98.

[0082] Preferably, the present invention relates to a method for reducing the level or activity of at least one endogenous PPO1 polyphenol oxidase in a banana plant or banana plant cell, the PPO1 polyphenol oxidase gene being encoded by: (A) a coding sequence of SEQ ID NO:5 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (B) a coding sequence of SEQ ID NO:40 (PPO1) or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; or (C) a polynucleotide sequence of SEQ ID NO:151 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; the method comprising providing to the banana plant cell a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease and one or more guide RNAs specific to the at least one endogenous PPO1 polyphenol oxidase gene, wherein the CRISPR-associated endonuclease or the modified CRISPR-associated endonuclease and the one or more guide RNAs form a complex that enables the CRISPR-associated endonuclease or the modified CRISPR-associated endonuclease to introduce a double-stranded or single-stranded break in the at least one endogenous PPO1 polyphenol oxidase gene; Optionally, the method is provided wherein the one or more guide RNAs comprise a variable region having the sequence of SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:62.

[0083] Preferably, the present invention relates to a method for reducing the level or activity of at least one endogenous PPO2 polyphenol oxidase in a banana plant or banana plant cell, the PPO2 polyphenol oxidase gene comprising: (A) a coding sequence of SEQ ID NO:6 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (B) a coding sequence of SEQ ID NO:41 (PPO2) or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; or (C) a polynucleotide sequence of SEQ ID NO:152 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; the method comprising providing to the banana plant cell a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease and one or more guide RNAs specific to the at least one endogenous PPO2 polyphenol oxidase gene, wherein the CRISPR-associated endonuclease or the modified CRISPR-associated endonuclease and the one or more guide RNAs form a complex that enables the CRISPR-associated endonuclease or the modified CRISPR-associated endonuclease to introduce a double-stranded or single-stranded break in the at least one endogenous PPO2 polyphenol oxidase gene; Optionally, the one or more guide RNAs comprise a variable region having the sequence of SEQ ID NO:34 or SEQ ID NO:35.

[0084] Preferably, the present invention relates to a method for reducing the level or activity of at least one endogenous PPO5 polyphenol oxidase in a banana plant or banana plant cell, the PPO5 polyphenol oxidase gene comprising: (A) a coding sequence of SEQ ID NO:9 (PPO5) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:9; (B) a coding sequence of SEQ ID NO:44 (PPO5) or a polypeptide having at least 75% sequence identity to SEQ ID NO:44; or (C) a polynucleotide sequence of SEQ ID NO:155 (PPO5) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:155; The method comprises providing to the banana plant cell a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease and one or more guide RNAs specific to the at least one endogenous PPO5 polyphenol oxidase gene, wherein the CRISPR-associated endonuclease or the modified CRISPR-associated endonuclease and the one or more guide RNAs form a complex that enables the CRISPR-associated endonuclease or the modified CRISPR-associated endonuclease to introduce a double-stranded or single-stranded break in the at least one endogenous PPO5 polyphenol oxidase gene; Optionally, the one or more guide RNAs comprise a variable region having the sequence of SEQ ID NO:78 or SEQ ID NO:79.

[0085] Preferably, the present invention relates to a method for reducing the level or activity of at least one endogenous PPO8 polyphenol oxidase in a banana plant or banana plant cell, the PPO8 polyphenol oxidase gene comprising: (A) a coding sequence of SEQ ID NO: 12 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 12; (B) a coding sequence of a polyphenol oxidase of SEQ ID NO: 47 (PPO8) or a polypeptide having at least 75% sequence identity to SEQ ID NO: 47; or (C) a polynucleotide sequence of SEQ ID NO: 158 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; The method comprises providing to the banana plant cell a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease and one or more guide RNAs specific to the at least one endogenous PPO8 polyphenol oxidase gene, wherein the CRISPR-associated endonuclease or the modified CRISPR-associated endonuclease and the one or more guide RNAs form a complex that enables the CRISPR-associated endonuclease or the modified CRISPR-associated endonuclease to introduce a double-stranded or single-stranded break in the at least one endogenous PPO8 polyphenol oxidase gene; Optionally, the one or more guide RNAs comprise a variable region having the sequence of SEQ ID NO:57 or SEQ ID NO:58.

[0086] Preferably, the present invention relates to a method for reducing the level or activity of at least one endogenous PPO9 polyphenol oxidase in a banana plant or banana plant cell, the PPO9 polyphenol oxidase gene being encoded by: (A) a coding sequence of SEQ ID NO: 13 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13; (B) a coding sequence of a polyphenol oxidase of SEQ ID NO: 48 (PPO9) or a polypeptide having at least 75% sequence identity to SEQ ID NO: 48; or (C) a polynucleotide sequence of SEQ ID NO: 159 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159; The method comprises providing to the banana plant cell a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease and one or more guide RNAs specific to the at least one endogenous PPO9 polyphenol oxidase gene, wherein the CRISPR-associated endonuclease or the modified CRISPR-associated endonuclease and the one or more guide RNAs form a complex that enables the CRISPR-associated endonuclease or the modified CRISPR-associated endonuclease to introduce a double-stranded or single-stranded break in the at least one endogenous PPO9 polyphenol oxidase gene; Optionally, the one or more guide RNAs comprise a variable region having the sequence of SEQ ID NO:38 or SEQ ID NO:39.

[0087] Preferably, the present invention relates to a method for reducing the level or activity of at least one endogenous PPO4 polyphenol oxidase in a banana plant or banana plant cell, the PPO4 polyphenol oxidase gene comprising: (A) a coding sequence of SEQ ID NO:8 (PPO4) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8; (B) a coding sequence of SEQ ID NO:43 (PPO4) or a polypeptide having at least 75% sequence identity to SEQ ID NO:43; or (C) a polynucleotide sequence of SEQ ID NO:154 (PPO4) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:154; The method comprises providing to the banana plant cell a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease and one or more guide RNAs specific to the at least one endogenous PPO4 polyphenol oxidase gene, wherein the CRISPR-associated endonuclease or the modified CRISPR-associated endonuclease and the one or more guide RNAs form a complex that enables the CRISPR-associated endonuclease or the modified CRISPR-associated endonuclease to introduce a double-stranded or single-stranded break in the at least one endogenous PPO4 polyphenol oxidase gene; Optionally, the method is provided, wherein the one or more guide RNAs comprise a variable region having the sequence of SEQ ID NO: 76 or SEQ ID NO: 77. To use the CRISPR / Cas system, both the gRNA and Cas must be in the target cell or delivered as a ribonucleoprotein complex. According to some embodiments, the Cas / modified Cas and at least one gRNA are provided to the banana cell by introducing one or more vectors expressing the Cas / modified Cas and / or at least one gRNA. The insertion vector may contain both cassettes on one 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 Cas endonucleases to modify plant genomes has been disclosed at least in Svitashev et al. (2015), Plant Physiology, 169(2):931-945; Kumar and Jain, 2015, Journal of Experimental Botany, 66:47-57; and U.S. Patent Application Publication No. 20150082478, which are specifically incorporated by reference herein in their entireties.

[0088] In some embodiments, the method of the present invention further comprises identifying at least one banana plant cell comprising a modification of at least one endogenous polyphenol oxidase gene. In some embodiments, the method of the present invention further comprises identifying at least one banana plant cell comprising a modification of at least one endogenous polyphenol oxidase gene, the modification being selected from the group consisting of at least one nucleotide insertion; at least one nucleotide deletion; at least one nucleotide substitution; and any combination of the foregoing. In some of these embodiments, the at least one endogenous polyphenol oxidase gene is selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO1 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO2 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO3 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO4 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO5 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO6 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO7 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO8 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO9 gene. In some embodiments, the endogenous polyphenol oxidase gene is two or more of the PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9 genes. In some embodiments, the endogenous polyphenol oxidase genes are the PPO1 and PPO2 genes.

[0089] As used herein, "identification" may include any technique known in the art that can detect modification or editing events selected from the group consisting of at least one nucleotide insertion; at least one nucleotide detection; at least one nucleotide substitution; and any combination of the foregoing, including but not limited to DNA sequencing (e.g., next-generation sequencing), electrophoresis, enzyme-based mismatch detection assays, and hybridization assays such as PCR, RT-PCR, RNase protection, in situ hybridization, primer extension, Southern blot, Northern blot, and dot blot analysis. Also, various methods used to detect single nucleotide polymorphisms (SNPs), such as PCR-based T7 endonuclease, heteroduplex, and Sanger sequencing, may be used. Another method for verifying the presence of DNA editing events (such as insertion-deletion events (i.e., "indels")) includes mismatch cleavage assays that utilize structure-selective enzymes (e.g., endonucleases) that recognize and cleave mismatched DNA. The mismatch cleavage assay is a typical procedure for detecting mutations induced by genome editing, as it is a simple and cost-effective method for detecting indels. This assay uses an enzyme that cleaves heteroduplex DNA at mismatches and extrahelical loops formed by multiple nucleotides, generating two or more small fragments. PCR products of approximately 300-1000 bp are generated, with the predicted nuclease cleavage site off-center, so that the resulting fragments differ in size and can be easily separated by conventional gel electrophoresis or high performance liquid chromatography (HPLC). End-labeled digestion products can also be analyzed by automated gel or capillary electrophoresis. The frequency of indels at a locus can be estimated by measuring the integrated intensity of the PCR amplicon and the cleaved DNA band. The digestion step takes 15-60 min, and with the addition of the DNA preparation and PCR steps, the entire assay can be completed within 3 h. Two separate enzymes are usually used for this assay.T7 endonuclease 1 (T7E1) is a resolvase that recognizes and cleaves imperfectly matched DNA at the first, second, or third phosphodiester bond upstream of the mismatch. The sensitivity of T7E1-based assays is 0.5-5%. In contrast, Surveyor™ nuclease (Transgenomic Inc., Omaha, NE, USA) is a member of the CEL family of mismatch-specific nucleases derived from celery. It recognizes and cleaves mismatches resulting from the presence of single nucleotide polymorphisms (SNPs) or small indels, cleaving both DNA strands downstream of the mismatch. It can detect indels of up to 12 nucleotides and is sensitive to mutations present at a frequency as low as about 3%, i.e., 1 in 32 copies. Yet another method to verify the presence of editing events involves high-resolution melting analysis. High-resolution melting analysis (HRMA) involves amplifying DNA sequences (90-200 bp) spanning a genomic target by real-time PCR incorporating a fluorescent dye, followed by analysis of the melting curves of the amplicons. HRMA is based on the disappearance of fluorescence as the intercalating dye is released from double-stranded DNA during thermal denaturation. The temperature-dependent denaturation profile of the amplicon is recorded to detect whether one or more molecular species are involved in the melting process. Yet another method is the heteroduplex mobility assay. Mutations can also be detected by analyzing rehybridized PCR fragments directly by native polyacrylamide gel electrophoresis (PAGE). This method exploits the difference in migration between heteroduplex and homoduplex DNA in polyacrylamide gels. The angle between the matched and mismatched DNA strands caused by indels means that heteroduplex DNA migrates at a significantly slower rate than homoduplex DNA under native conditions, and the two can be easily distinguished based on their mobility. Fragments of 140-170 bp can be separated in a 15% polyacrylamide gel. The sensitivity of such an assay can approach 0.5% under optimal conditions, which is similar to that of T7E1. After reannealing of the PCR products, the electrophoresis step of the assay takes approximately 2 hours.Other methods for verifying the presence of editing events are described in detail in Zischewski (2017), Biotechnology Advances 1(1):95-104.

[0090] In some embodiments of the present invention, one or more guide RNAs (gRNAs) are provided to banana plant cells in one or more recombinant DNA constructs that encode the one or more guide RNAs operably linked to one or more promoters. The DNA constructs useful in embodiments of the present invention can be constructed using recombinant DNA techniques well known to those skilled in the art. Such DNA constructs can be commercially available and suitable for transformation into plants and for expressing a gene of interest in transformed cells.

[0091] As used herein, a "promoter" is expressible in a plant, i.e., capable of inducing, conferring, activating, or enhancing expression in a cell, tissue, or organ of a plant. Examples of promoters useful in the methods of the invention include, but are not limited to, actin, CANV 35S, CaMV19S, GOS2. Promoters active at various tissues or developmental stages can also be used. In any of the embodiments herein, the PPO polynucleotide sequence may be optimized for plant expression. Examples of such sequence modifications include, but are not limited to, changing the G / C content to more closely resemble that normally found in bananas and generally removing codons not normally found in the plant species (referred to as codon optimization). Banana plant cells may be stably or transiently transformed with the DNA constructs of the embodiments of the invention. In stable transformation, the PPO polynucleotide is integrated into the genome of the plant, thus representing a stable and heritable trait. In transient transformation, the PPO polynucleotide is expressed in the transformed cell but is not integrated into the genome (thus representing a transient trait). In some embodiments, the promoter in the DNA construct comprises a Pol3 promoter. Examples of Pol3 promoters include, but are not limited to, AtU6-29, AtU626, AtU3B, AtU3d, and TaU6. In some embodiments, the promoter in the DNA construct comprises a Pol2 promoter. Examples of Pol2 promoters include, but are not limited to, CaMV 35S, CaMV 19S, ubiquitin, and CVMV. In some embodiments, the promoter in the DNA construct comprises a 35S promoter. In some embodiments, the promoter in the DNA construct comprises a U6 promoter. In some embodiments, the promoter in the DNA construct comprises a Pol3 promoter (such as U6) operably linked to a nucleic acid agent encoding at least one gRNA and / or a Pol2 promoter (such as CamV35S) operably linked to a nucleic acid sequence encoding a CRISPR-associated endonuclease and / or a selection marker gene.This DNA construct may be useful for transient expression by Agrobacterium-mediated transformation (Helens et al. (2005), Plant Methods 1:13). In some embodiments, the nucleic acid sequences contained in the DNA construct do not contain sequences homologous to the genome of a banana plant cell (other than any guide sequences) to avoid integration into the banana genome. In some embodiments, the DNA construct is a non-integrating construct, such as when the nucleic acid sequence encoding the selection marker is also non-integrating. As used herein, "non-integrating" refers to a DNA construct or sequence that is not actively designed to facilitate integration of the construct or sequence into the genome of a plant of interest. For example, a functional T-DNA vector system for Agrobacterium-mediated genetic transformation is not a non-integrating vector system, since it is actively designed to integrate into the plant genome. Similarly, a selection marker gene sequence that has flanking sequences homologous to the genome of a plant of interest to facilitate homologous recombination of the selection marker gene sequence into the banana genome is not a non-integrating selection marker sequence.

[0092] In the context of the present invention, various cloning kits can be used. As used herein, a "DNA construct" can be a binary vector. Examples of binary vectors are pBIN19, pBI101, pBinAR, pGPTV, pCAMBIA, pBIB-HYG, pBecks, pGreen, or pPZP (Hajukiewicz, P. et al., Plant Molecular Biology, 25, 989 (1994) and Hellens et al. Trends in Plant Science 5, 446 (2000)). Examples of other vectors that can be used in the context of the present invention in other methods of DNA delivery (e.g., transfection, electroporation, biolistics, and viral inoculation) are pGE-sgRNA (Zhang et al. Nature Communications 2016 7:12697), pJIT163-Ubi-Cas9 (Wang et al. Nature Biotechnology 2004,32,947-951), pICH47742::2x35S-5'UTR-hCas9(STOP)-NOST (Belhan et al. Plant Methods 2013,11;9(1):39).

[0093] In other embodiments, the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and / or one or more guide RNAs are provided to the banana plant cell in the form of RNA. In yet other embodiments, the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease is provided to the banana plant cell in the form of protein, and one or more guide RNAs are provided to the banana plant cell in the form of RNA. In some embodiments, the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and one or more guide RNAs are provided to the banana plant cell as a ribonucleoprotein complex.

[0094] There are many methods for 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 fibers (U.S. Pat. Nos. 5,302,523 and 5,464,765); Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055; 5,591,616; 5,693,512; 5,824,877; 5,981,840). , and 6,384,301); acceleration of particles coated with DNA (U.S. Pat. Nos. 5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865), as well as nanoparticles, nanocarriers, and cell-penetrating peptides (WO201126644A2; WO2009046384A1; WO2008148223A1). Other methods of transfection include the use of transfection reagents (e.g., Lipofectin, ThermoFisher), dendrimers (Kukowska-Latallo, JF et al. (1996), Proc. Natl. Acad. Sci. USA 93, 4897-902), cell-penetrating peptides (Mae et al. (2005), “Internalisation of cell-penetrating peptides into tobacco protoplasts”, Biochimica et Biophysica Acta 1669(2):101-7), or polyamines (Zhang and Vinogradov (2010), “Short biodegradable polyamines for gene delivery and transfection of brain capillary endothelial cells”, J Control Release, 143(3):359-366).

[0095] In some embodiments of the invention, the endonuclease, one or more guide RNAs, and / or one or more recombinant DNA constructs are provided to the banana plant cell using a biolistics or gene gun. In other embodiments, the endonuclease, one or more guide RNAs, and / or one or more recombinant DNA constructs are provided to the banana plant cell using Agrobacterium transformation. In still other embodiments, the endonuclease, one or more guide RNAs, and / or one or more recombinant DNA constructs are provided to the banana plant cell using protoplast transfection. In still other embodiments, the endonuclease, one or more guide RNAs, and / or one or more recombinant DNA constructs are provided to the banana plant cell using electroporation. In still other embodiments, the endonuclease, one or more guide RNAs, and / or one or more recombinant DNA constructs are provided to the banana plant cell using nanoparticle-mediated transfection. In some embodiments, the endonuclease is provided to the banana plant cell as a polynucleotide encoding an endonuclease polypeptide. In some of these embodiments, the endonuclease is a Cas9 endonuclease and the polynucleotide is a Cas9 polynucleotide encoding a Cas9 polypeptide. In some embodiments, the one or more guide RNAs and the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease are provided to a banana plant cell via Agrobacterium transformation of one or more plasmids encoding the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease, at least one selectable marker gene, and one or more guide RNAs.

[0096] In any of the embodiments of the present invention, one or more guide RNAs comprise a variable region having a sequence selected from the group consisting of SEQ ID NOs: 32-39 and 57-97; and any combination of the foregoing. In some embodiments of the present invention, one or more guide RNAs comprise a variable region having a sequence selected from the group consisting of SEQ ID NOs: 180-196, 199-202, and 207-225; and any combination of the foregoing. In any of the embodiments of the present invention, one or more guide RNAs comprise a variable region having a sequence selected from the group consisting of SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 62; SEQ ID NO: 76; SEQ ID NO: 77; and any combination of the foregoing. In some embodiments of the invention, the one or more guide RNAs are a pair of guide RNAs comprising variable regions selected from the group consisting of SEQ ID NOs: 32 and 33; SEQ ID NOs: 34 and 35; SEQ ID NOs: 32 and 34; SEQ ID NOs: 32 and 35; SEQ ID NOs: 33 and 34; SEQ ID NOs: 33 and 35; SEQ ID NOs: 57 and 58; SEQ ID NOs: 38 and 39; SEQ ID NOs: 62 and 33; and SEQ ID NOs: 76 and 77.

[0097] In some embodiments of the invention, the banana plant cells are protoplasts, are embryogenic cells, and / or are included in an embryogenic cell suspension.

[0098] The present invention further provides a banana plant cell obtainable by any one of the methods of the present invention described above.

[0099] In some embodiments of the invention, the method further comprises regenerating a banana plant from the banana plant cell.

[0100] As used herein, "regeneration" can include growing banana plant cells (including protoplasts) into whole banana plants by first growing the banana plant cells (including protoplasts) into clusters that develop into callus, followed by regenerating shoots from the callus using plant tissue culture techniques (caulogenesis). Growing banana protoplasts into callus and then regenerating shoots requires the right balance of plant growth regulators in the tissue culture medium, which must be customized. Protoplasts can also be used for plant breeding, using a technique called protoplast fusion. Fusion of protoplasts from different species is induced by using an electric field or a solution of polyethylene glycol. This technique can be used to generate somatic hybrids in tissue culture. Methods for regenerating protoplasts are well known in the art. Several factors influence the isolation, culture, and regeneration of protoplasts, namely genotype, donor tissue and its pretreatment, enzymatic treatment to isolate protoplasts, protoplast culture method, culture, culture medium, and physical environment (see Maheshwari et al. (1986), “Differentiation of Protoplasts and of Transformed Plant Cells”: 3-36. Springer-Verlag, Berlin). Regenerated bananas may be subjected to selection. The banana plant or cells thereof may be free of transgenes, i.e. “non-transgenic”. For example, the banana plant may be free of any DNA constructs encoding any of the CRISPR / Cas systems as used in some of the embodiments of the present invention. According to some embodiments, when genetically modifying, such as by gene editing, banana cells that are to be grown and regenerated into adult plants, it is preferred not to edit genes that are expressed in these cells and that may adversely affect embryogenesis and / or regeneration. Without wishing to be bound by theory or mechanism, the discovery of a PPO gene, e.g., PPO1, PPO2, PPO8, PPO9, or PPO4, that is expressed in the flesh and / or skin but not in embryonic cells, would allow for the editing of embryonic cells without affecting plant regeneration.

[0101] In some embodiments, the method further comprises harvesting the fruit from the banana plant. Each adult banana plant produces one bunch, which is formed by many banana fruits or "fingers" that are grouped into several "tiers" of the bunch. In this context, "harvesting" has its conventional meaning, for example, cutting the bunch by hand (usually 2-3 people) using a sharp curved knife or machete. Harvesting usually occurs when the banana fruit is still green and firm, 7-14 days before it ripens.

[0102] The present invention further provides a banana plant or plant part obtainable by the above-mentioned method.

[0103] Further provided by the present invention is a fruit harvested from a banana plant obtainable by the aforementioned method of the present invention, wherein the pulp and / or peel of the fruit is characterized by a phenotype of delayed and / or reduced browning compared to the pulp and / or peel of a banana plant not having a reduced level or activity of the at least one endogenous polyphenol oxidase (selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9).

[0104] According to some embodiments, provided herein is a fruit harvested from a banana plant obtainable by the aforementioned method of the invention, wherein the pulp and / or peel of the fruit is characterized by a phenotype of delayed and / or reduced browning compared to the pulp and / or peel of a banana plant not having a reduced level or activity of the at least one endogenous polyphenol oxidase (selected from the group consisting of PPO1, PPO2, PPO8, PPO9, and PPO4).

[0105] The present invention further provides a method for producing a banana plant characterized by a phenotype of delayed and / or reduced browning of the flesh and / or skin compared to a wild-type banana plant, comprising providing a banana plant cell with a CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and one or more guide RNAs, wherein the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and one or more guide RNAs induce a single strand break in at least one endogenous polyphenol oxidase gene selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9. or a double-stranded break; identifying at least one banana plant cell comprising an alteration of at least one endogenous polyphenol oxidase gene selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9, wherein the alteration is selected from the group consisting of at least one nucleotide insertion; at least one nucleotide deletion; at least one nucleotide substitution; or any combination of the foregoing; and regenerating a banana plant from the banana plant cell, wherein the banana plant is characterized by a phenotype of delayed and / or reduced browning of the flesh and / or skin compared to a wild-type banana plant. In some embodiments, the method further comprises harvesting fruit from the banana plant, wherein the fruit is characterized by a phenotype of delayed and / or reduced browning compared to fruit from a banana plant that does not have a reduced or lost level or activity of at least one endogenous polyphenol oxidase.

[0106] The present invention further provides a banana plant or plant part comprising in its genome at least one modified endogenous polyphenol oxidase gene, the modification resulting in a reduction or loss of function of at least one endogenous polyphenol oxidase encoded by the modified endogenous polyphenol oxidase gene, the modification being located in the at least one endogenous polyphenol oxidase gene.

[0107] In some embodiments of the aforementioned methods, banana plants, or banana plant parts, the at least one endogenous polyphenol oxidase gene is a PPO1 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO2 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO3 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO4 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO5 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO6 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO7 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO8 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO9 gene. In some embodiments, the endogenous polyphenol oxidase genes are two or more of the PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9 genes. In some embodiments, the endogenous polyphenol oxidase genes are the PPO1 and PPO2 genes. In certain embodiments of the aforementioned methods, banana plants, or banana plant parts, the PPO1 gene is 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% identical to SEQ ID NO:5. the PPO2 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:6;The PPO3 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:7; the PPO4 gene comprises a coding sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, a coding sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:9; a PPO5 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:9; The PO6 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:10; the PPO7 gene comprises a coding sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, a coding sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; a PPO8 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:12;Or the PPO9 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:13. In certain embodiments of the aforementioned methods, banana plants, or banana plant parts, the PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene polynucleotide comprises a coding sequence selected from the group consisting of SEQ ID NO:5 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; SEQ ID NO:6 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; SEQ ID NO:12 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; SEQ ID NO:13 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:13; and SEQ ID NO:8 (PPO4) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8.

[0108] In certain embodiments of the aforementioned methods, banana plants, or banana plant parts, the PPO1 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:40; the PPO2 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:40; 1, the PPO3 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:42; the PPO4 gene encodes a polyphenol oxidase having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:43; %, 98%, 99%, or 100% sequence identity to SEQ ID NO:44; the PPO5 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:44;the PPO6 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:45; The PPO7 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:46; the PO8 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:47; or The PPO9 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:48. In certain embodiments of the aforementioned methods, banana plants, or banana plant parts, the PPO1, PPO2, PPO8, or PPO9 polyphenol oxidase gene is selected from the group consisting of SEQ ID NO:40 (PPO1) or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; SEQ ID NO:41 (PPO2) or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; SEQ ID NO:47 (PPO8) or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; SEQ ID NO:48 (PPO9) or a polypeptide having at least 75% sequence identity to SEQ ID NO:48;and SEQ ID NO: 43 (PPO4), or a polypeptide having at least 75% sequence identity to SEQ ID NO: 43;

[0109] In certain embodiments of the aforementioned methods, banana plants, or banana plant parts, the PPO1 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 151; the PPO2 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 153; the PPO3 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 153. the PPO4 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO:154; the PPO5 gene comprises a polynucleotide sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, the PPO6 gene comprises a polynucleotide sequence having 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:156; the PPO6 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO:156;The PPO7 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO:157; the PPO8 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO:158. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 159; or the PPO9 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 159. In certain embodiments of the aforementioned methods, banana plants, or banana plant parts, the PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:151 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; SEQ ID NO:152 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; SEQ ID NO:158 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:158; SEQ ID NO:159 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:159; and SEQ ID NO:154 (PPO4) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:154.

[0110] In some embodiments, the banana plants or plant parts of the present invention are non-transgenic. For example, the banana plants or plant parts of the present invention may not have any DNA constructs encoding any of the CRISPR / Cas systems as used in some of the embodiments of the present invention.

[0111] Further provided by the present invention is a banana fruit harvested from the banana plant of any of the embodiments of the present invention, wherein the fruit is characterized by a phenotype of delayed and / or reduced browning compared to fruit of a banana plant that does not have a reduced level or activity of the at least one endogenous polyphenol oxidase selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9.

[0112] The present invention further provides a method for obtaining a banana fruit food product comprising processing the banana fruit of the present invention. In some embodiments, the banana fruit food product is a thickener, colorant, or flavoring. Also contemplated are livestock feed, natural fiber, and a source of natural bioactive compounds and biofertilizers.

[0113] The present invention further provides DNA sequences comprising banana polyphenol oxidase polynucleotides.

[0114] Further provided by the present invention is a DNA construct or vector comprising a banana polyphenol oxidase polynucleotide.

[0115] The present invention further provides a plant cell transformed with a vector comprising a banana polyphenol oxidase polynucleotide, hi some of these embodiments, the plant cell is a banana plant cell.

[0116] Further provided by the present invention is a polyphenol oxidase protein. In some embodiments, the polyphenol oxidase protein is selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:5. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:6. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:7. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:8.In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:9. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:10. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:11. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:12. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:13.In some embodiments, the polyphenol oxidase protein comprises a sequence having 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% sequence identity to SEQ ID NO:40. In some embodiments, the polyphenol oxidase protein comprises a sequence having 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% sequence identity to SEQ ID NO:41. In some embodiments, the polyphenol oxidase protein comprises a sequence having 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% sequence identity to SEQ ID NO:42. In some embodiments, the polyphenol oxidase protein comprises a sequence having 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% sequence identity to SEQ ID NO:43. In some embodiments, the polyphenol oxidase protein comprises a sequence having 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% sequence identity to SEQ ID NO:44.In some embodiments, the polyphenol oxidase protein comprises a sequence having 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% sequence identity to SEQ ID NO:45. In some embodiments, the polyphenol oxidase protein comprises a sequence having 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% sequence identity to SEQ ID NO:46. In some embodiments, the polyphenol oxidase protein comprises a sequence having 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% sequence identity to SEQ ID NO:47. In some embodiments, the polyphenol oxidase protein comprises a sequence having 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% sequence identity to SEQ ID NO:48. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:151.In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:152. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:153. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:154. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:155. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:156.In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:157. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:158. In some embodiments, the polyphenol oxidase protein is encoded by a polynucleotide sequence having 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% sequence identity to SEQ ID NO:159.

[0117] The present invention further provides a method for expressing polyphenol oxidase in a plant cell, comprising introducing into the plant cell a banana polyphenol oxidase polynucleotide operably linked to a promoter active in the plant cell.

[0118] In some embodiments of the DNA sequences, DNA constructs, vectors, plant cells transformed with vectors, banana plant cells transformed with vectors, and methods of expressing polyphenol oxidase in plant cells, the polyphenol oxidase polynucleotide is selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9. In some embodiments of the DNA sequences, DNA constructs, vectors, plant cells transformed with vectors, banana plant cells transformed with vectors, and methods of expressing polyphenol oxidase in plant cells, the at least one endogenous polyphenol oxidase polynucleotide is a PPO1 polynucleotide. In some embodiments, the polyphenol oxidase polynucleotide is a PPO2 polynucleotide. In some embodiments, the polyphenol oxidase polynucleotide is a PPO3 polynucleotide. In some embodiments, the polyphenol oxidase polynucleotide is a PPO4 polynucleotide. In some embodiments, the polyphenol oxidase polynucleotide is a PPO5 polynucleotide. In some embodiments, the polyphenol oxidase polynucleotide is a PPO6 polynucleotide. In some embodiments, the polyphenol oxidase polynucleotide is a PPO7 polynucleotide. In some embodiments, the polyphenol oxidase polynucleotide is a PPO8 polynucleotide. In some embodiments, the polyphenol oxidase polynucleotide is a PPO9 polynucleotide. In some embodiments, the polyphenol oxidase polynucleotide comprises two or more of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9 polynucleotides. In some embodiments of the DNA sequences, DNA constructs, vectors, plant cells transformed with vectors, banana plant cells transformed with vectors, and methods of expressing polyphenol oxidase in plant cells, the polyphenol oxidase polynucleotide isIn certain embodiments of the DNA sequences, DNA constructs, vectors, plant cells transformed with vectors, banana plant cells transformed with vectors, and methods of expressing polyphenol oxidase in plant cells, the PPO1 polynucleotide has 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% sequence identity to SEQ ID NO:5. PPO2 polynucleotides include coding sequences having 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% sequence identity to SEQ ID NO:6; PPO3 polynucleotides include coding sequences having 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% sequence identity to SEQ ID NO:7. 8, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8; PPO4 polynucleotides contain coding sequences having 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% sequence identity to SEQ ID NO:8; PPO5 polynucleotides contain coding sequences having 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% sequence identity to SEQ ID NO:8; a coding sequence having 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% sequence identity to SEQ ID NO:9; a PPO6 polynucleotide having 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% sequence identity to SEQ ID NO:10;a PPO7 polynucleotide comprises a coding sequence having 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% sequence identity to SEQ ID NO:11; a PPO8 polynucleotide comprises a coding sequence having 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% sequence identity to SEQ ID NO:12; or the PPO9 polynucleotide comprises a coding sequence having 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% sequence identity to SEQ ID NO:13. In certain embodiments of the DNA sequences, DNA constructs, vectors, plant cells transformed with the vectors, banana plant cells transformed with the vectors, and methods of expressing polyphenol oxidase in plant cells, the PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase polynucleotide comprises a coding sequence selected from the group consisting of SEQ ID NO:5 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; SEQ ID NO:6 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; SEQ ID NO:12 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; SEQ ID NO:13 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:13; and SEQ ID NO:8 (PPO4) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8.

[0119] In certain embodiments of the DNA sequences, DNA constructs, vectors, plant cells transformed with the vectors, banana plant cells transformed with the vectors, and methods of expressing polyphenol oxidase in plant cells, the PPO1 polynucleotide has a sequence similar to SEQ ID NO:40 but is 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 similar to SEQ ID NO:40. The PPO2 polynucleotide encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:41; the PPO3 polynucleotide encodes a polyphenol oxidase having at least 75% sequence identity to SEQ ID NO:42. , 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% sequence identity to SEQ ID NO:43; , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44; the PPO5 polynucleotide encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:44;The PPO6 polynucleotide encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:45; The PPO7 polynucleotide encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:46; The PO8 polynucleotide encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:47; or The PPO9 polynucleotide encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:48. In certain embodiments of the DNA sequences, DNA constructs, vectors, plant cells transformed with vectors, banana plant cells transformed with vectors, and methods of expressing polyphenol oxidase in plant cells, the PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase polynucleotide is selected from the group consisting of SEQ ID NO:40 (PPO1) or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; SEQ ID NO:41 (PPO2) or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; SEQ ID NO:47 (PPO8) or a polypeptide having at least 75% sequence identity to SEQ ID NO:47;The polyphenol oxidase is selected from the group consisting of SEQ ID NO:48 (PPO9) or a polypeptide having at least 75% sequence identity to SEQ ID NO:48; and SEQ ID NO:43 (PPO4) or a polypeptide having at least 75% sequence identity to SEQ ID NO:43;

[0120] In certain embodiments of the DNA sequences, DNA constructs, vectors, plant cells transformed with the vectors, banana plant cells transformed with the vectors, and methods of expressing polyphenol oxidase in plant cells, the PPO1 polynucleotide has a sequence similar to SEQ ID NO: 151, but is 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, %, 99%, or 100% sequence identity to SEQ ID NO:152; PPO2 polynucleotides include polynucleotide sequences having 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% sequence identity to SEQ ID NO:152; PPO3 polynucleotides include polynucleotide sequences having 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% sequence identity to SEQ ID NO:153. or 100% sequence identity to SEQ ID NO: 154; the PPO4 polynucleotides include polynucleotide sequences having 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% sequence identity to SEQ ID NO: 154; PPO5 polynucleotides include polynucleotide sequences having 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 155; PPO5 polynucleotides include polynucleotide sequences having 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% sequence identity to SEQ ID NO: 155;The PPO6 polynucleotide comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO:156; A PPO7 polynucleotide comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 157; The PO8 polynucleotide comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO:158; or The PPO9 polynucleotides include a polynucleotide sequence having 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% sequence identity to SEQ ID NO:159. In certain embodiments of the DNA sequences, DNA constructs, vectors, plant cells transformed with vectors, banana plant cells transformed with vectors, and methods of expressing polyphenol oxidase in plant cells, the PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase polynucleotide is selected from the group consisting of SEQ ID NO:151 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; SEQ ID NO:152 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; SEQ ID NO:158 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:158;SEQ ID NO: 159 (PPO9), or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159; and SEQ ID NO: 154 (PPO4), or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 154;

[0121] The present invention further provides a synthetic banana polyphenol oxidase guide RNA comprising a variable region selected from the group consisting of SEQ ID NOs: 32-39 and 57-97. The present invention further provides a synthetic banana polyphenol oxidase guide RNA comprising a variable region selected from the group consisting of SEQ ID NOs: 180-196, 199-202, and 207-225. In some embodiments, the synthetic banana polyphenol oxidase guide RNA comprises a variable region selected from the group consisting of SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 62; SEQ ID NO: 76; and SEQ ID NO: 77.

[0122] The present invention further provides a recombinant DNA construct comprising a promoter operably linked to a nucleotide sequence encoding at least one banana polyphenol oxidase guide RNA, the guide RNA can form a complex with a CRISPR-associated endonuclease or modified CRISPR-associated endonuclease, and the complex can bind to at least one endogenous banana polyphenol oxidase gene and generate a double-stranded or single-stranded break. In some of these embodiments, the at least one endogenous polyphenol oxidase gene is selected from the group consisting of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO1 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO2 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO3 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO4 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO5 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO6 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO7 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO8 gene. In some embodiments, the at least one endogenous polyphenol oxidase gene is a PPO9 gene. In some embodiments, the endogenous polyphenol oxidase gene is two or more of the PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, and PPO9 genes. In some embodiments, the endogenous polyphenol oxidase genes are the PPO1 and PPO2 genes. In certain embodiments, the PPO1 gene isThe present invention relates to a coding sequence having 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% sequence identity to SEQ ID NO:5. In certain embodiments, the PPO2 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:6. In certain embodiments, the PPO3 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:7. In certain embodiments, the PPO4 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:8. In certain embodiments, the PPO5 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:9. In certain embodiments, the PPO6 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO: 10. In certain embodiments, the PPO7 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO: 11.In certain embodiments, the PPO8 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:12. In certain embodiments, the PPO9 gene comprises a coding sequence having 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% sequence identity to SEQ ID NO:13. In certain embodiments, the PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene comprises a coding sequence selected from the group consisting of SEQ ID NO:5 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; SEQ ID NO:6 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; SEQ ID NO:12 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; SEQ ID NO:13 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:13; and SEQ ID NO:8 (PPO4) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8. In certain embodiments, the PPO1 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:40; the PPO2 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:41.the PPO3 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:42; the PPO4 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:43; the PPO5 gene encodes a polyphenol oxidase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, the PPO6 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:45; the PPO7 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:46; the PPO8 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:47;or the PPO9 gene encodes a polyphenol oxidase having 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% sequence identity to SEQ ID NO:48. In certain embodiments, the PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene encodes a polyphenol oxidase selected from the group consisting of SEQ ID NO:40 (PPO1) or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; SEQ ID NO:41 (PPO2) or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; SEQ ID NO:47 (PPO8) or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; SEQ ID NO:48 (PPO9) or a polypeptide having at least 75% sequence identity to SEQ ID NO:48; and SEQ ID NO:43 (PPO4) or a polypeptide having at least 75% sequence identity to SEQ ID NO:43. In certain embodiments, the PPO1 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO:151. In certain embodiments, the PPO2 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 152. In certain embodiments, the PPO3 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO: 153.In certain embodiments, the PPO4 gene comprises a polynucleotide sequence having 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the PPO4 gene. No. 154. In certain embodiments, the PPO5 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO:155. In certain embodiments, the PPO6 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO:156. In certain embodiments, the PPO7 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO:157. In certain embodiments, the PPO8 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO:158. In certain embodiments, the PPO9 gene comprises a polynucleotide sequence having 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% sequence identity to SEQ ID NO:159.In certain embodiments, the polyphenol oxidase gene of PPO1, PPO2, PPO8, PPO9, or PPO4 comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:151 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; SEQ ID NO:152 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; SEQ ID NO:158 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:158; SEQ ID NO:159 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:159; and SEQ ID NO:154 (PPO4) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:154.

[0123] In some of these embodiments, the one or more banana polyphenol oxidase guide RNAs comprise a variable region having a sequence selected from the group consisting of SEQ ID NOs: 32-39 and 57-97; and any combination of the foregoing. In some embodiments, the one or more banana polyphenol oxidase guide RNAs comprise a variable region having a sequence selected from the group consisting of SEQ ID NOs: 180-196, 199-202, and 207-225; and any combination of the foregoing. In some embodiments, the synthetic banana polyphenol oxidase guide RNAs comprise a variable region selected from the group consisting of SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 62; SEQ ID NO: 76; and SEQ ID NO: 77; and any combination of the foregoing. In some embodiments of the invention, the one or more guide RNAs are a pair of guide RNAs comprising variable regions selected from the group consisting of SEQ ID NOs: 32 and 33; SEQ ID NOs: 34 and 35; SEQ ID NOs: 32 and 34; SEQ ID NOs: 32 and 35; SEQ ID NOs: 33 and 34; SEQ ID NOs: 33 and 35; SEQ ID NOs: 57 and 58; SEQ ID NOs: 38 and 39; SEQ ID NOs: 62 and 33; and SEQ ID NOs: 76 and 77. In some of these embodiments, the CRISPR-associated endonuclease is a Cas9 endonuclease.

[0124] Additional embodiments of the methods and compositions of the present invention are provided herein. Such embodiments include:

[0125] 1. A method for reducing the level or activity of at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase encoded by a PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene in a banana plant or banana plant cell.

[0126] 2. The polyphenol oxidase gene of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 is (A) (a) SEQ ID NO:5 (PPO1), or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (b) SEQ ID NO:6 (PPO2), or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (c) SEQ ID NO:7 (PPO3), or a polynucleotide having at least 75% sequence identity to SEQ ID NO:7; (d) SEQ ID NO:8 (PPO4), or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8; (e) SEQ ID NO:9 (PPO5), or a polynucleotide having at least 75% sequence identity to SEQ ID NO:9; (f) SEQ ID NO:10 (PPO6), or a polynucleotide having at least 75% sequence identity to SEQ ID NO:10; (g) SEQ ID NO:11 (PPO7), or a polynucleotide having at least 75% sequence identity to SEQ ID NO:11; (h) SEQ ID NO:12 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; and (i) SEQ ID NO: 13 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13 comprising a coding sequence selected from the group consisting of: (B) (a) SEQ ID NO:40 (PPO1), or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; (b) SEQ ID NO:41 (PPO2), or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; (c) SEQ ID NO:42 (PPO3), or a polypeptide having at least 75% sequence identity to SEQ ID NO:42; (d) SEQ ID NO: 43 (PPO4), or a polypeptide having at least 75% sequence identity to SEQ ID NO: 43; (e) SEQ ID NO:44 (PPO5), or a polypeptide having at least 75% sequence identity to SEQ ID NO:44; (f) SEQ ID NO:45 (PPO6), or a polypeptide having at least 75% sequence identity to SEQ ID NO:45; (g) SEQ ID NO:46 (PPO7), or a polypeptide having at least 75% sequence identity to SEQ ID NO:46; (h) SEQ ID NO: 47 (PPO8) or a polypeptide having at least 75% sequence identity to SEQ ID NO: 47; and (i) SEQ ID NO: 48 (PPO9) or a polypeptide having at least 75% sequence identity to SEQ ID NO: 48 or (C) (a) SEQ ID NO:151 (PPO1), or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; (b) SEQ ID NO: 152 (PPO2), or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 152; (c) SEQ ID NO: 153 (PPO3), or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 153; (d) SEQ ID NO: 154 (PPO4), or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 154; (e) SEQ ID NO: 155 (PPO5), or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 155; (f) SEQ ID NO: 156 (PPO6), or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 156; (g) SEQ ID NO: 157 (PPO7), or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 157; (h) SEQ ID NO: 158 (PPO8), or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; and (i) SEQ ID NO: 159 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159 2. The method of embodiment 1, comprising a polynucleotide sequence selected from the group consisting of:

[0127] 3. (a) reducing the level of at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase in the banana plant or banana plant cell; (b) reducing the function of at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase in the banana plant or banana plant cell; or (c) the method of any one of the preceding claims, wherein the function of at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase in the banana plant or banana plant cell is abolished.

[0128] 4. (a) delaying browning of the flesh and / or peel of a banana plant compared to the flesh and / or peel of a banana plant that does not have a reduced level or activity of the at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase; and / or (b) reducing browning of the pulp and / or peel of a banana plant compared to the pulp and / or peel of a banana plant not having a reduced level or activity of the at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase.

[0129] 5. (a) providing to the banana plant cell or part of the banana plant a silencing RNA targeting a transcript of the at least one PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene, optionally wherein the silencing RNA is provided by introducing into the banana plant cell or part of the banana plant an endonuclease, the endonuclease activating the at least one PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene. a gene encoding an endogenous non-coding RNA can be modified to encode the silencing RNA that targets a transcript of a polyphenol oxidase gene of PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9, and optionally, the endonuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a homing endonuclease, a CRISPR-associated endonuclease, and a modified CRISPR-associated endonuclease; or (b) introducing a modification into a PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene encoding the at least one PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase; optionally, the modification is provided to the banana plant cell and is capable of targeting the at least one PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene. and wherein the endonuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a homing endonuclease, a CRISPR-associated endonuclease, and a modified CRISPR-associated endonuclease; and wherein the CRISPR-associated endonuclease is a Cas9 endonuclease. The method according to any one of embodiments 1 to 4, comprising:

[0130] 6. The method of any one of embodiments 1-4, comprising providing the banana plant cell with a CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and one or more guide RNAs specific for the at least one PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene, wherein the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and the one or more guide RNAs form a complex that allows the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease to introduce a double-stranded or single-stranded break into the at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene; optionally, the CRISPR-associated endonuclease is a Cas9 endonuclease.

[0131] 7. The method further comprises identifying at least one banana plant cell comprising a modification of at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene, wherein the modification is (a) at least one nucleotide insertion; (b) at least one nucleotide deletion; (c) insertion-deletion (indel); (d) inversion; (e) at least one nucleotide substitution; and (f) Any combination of (a) to (e) 7. The method of embodiment 5 or 6, selected from the group consisting of:

[0132] 8. The method of embodiment 6 or 7, wherein the one or more guide RNAs are provided to the banana plant cell within one or more recombinant DNA constructs encoding the one or more guide RNAs operably linked to one or more promoters.

[0133] 9. The method of embodiment 6 or 7, wherein the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and / or the one or more guide RNAs are provided to the banana plant cell in the form of RNA.

[0134] 10. The method according to embodiment 6 or 7, wherein the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease is provided to the banana plant cell in the form of a protein, and the one or more guide RNAs are provided to the banana plant cell in the form of an RNA; optionally, 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.

[0135] 11. The endonuclease, the one or more guide RNAs, and / or the one or more recombinant DNA constructs (a) Microprojectile gun; (b) Agrobacterium transformation; (c) protoplast transfection; (d) electroporation; and (e) Nanoparticle-mediated transfection The method according to any one of embodiments 5 to 10, wherein the banana plant cell is provided using a method selected from the group consisting of:

[0136] 12. The method of any one of embodiments 5 to 11, wherein the endonuclease is provided to the banana plant cell as a polynucleotide encoding an endonuclease polypeptide; optionally, the endonuclease is a Cas9 endonuclease and the polynucleotide is a Cas9 polynucleotide encoding a Cas9 polypeptide.

[0137] 13. The method of embodiment 6 or 7, wherein the one or more guide RNAs and the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease are provided to the banana plant cell via Agrobacterium transformation of one or more plasmids encoding the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease, at least one selectable marker gene, and the one or more guide RNAs.

[0138] 14. The method according to any one of embodiments 6 to 13, wherein the one or more guide RNAs comprise a variable region having a sequence selected from the group consisting of SEQ ID NOs: 32-39 and 57-97; and any combination thereof, or the one or more guide RNAs comprise a variable region having a sequence selected from the group consisting of SEQ ID NOs: 180-196, 199-202, and 207-225; and any combination thereof.

[0139] 15. The one or more guide RNAs (a) SEQ ID NOs: 32 and 33; (b) SEQ ID NOs:34 and 35; (c) SEQ ID NOs: 32 and 34; (d) SEQ ID NOs:32 and 35; (e) SEQ ID NOs:33 and 34; (f) SEQ ID NOs:33 and 35; (g) SEQ ID NOs: 57 and 58; (h) SEQ ID NOs:38 and 39; (i) SEQ ID NOs: 62 and 33; and (j) SEQ ID NOs: 76 and 77 The method according to any one of embodiments 6 to 13, wherein the pair of guide RNAs comprises a variable region selected from the group consisting of:

[0140] 16. The method according to any one of the preceding embodiments, wherein the banana plant cells are embryogenic cells and / or are contained in an embryogenic cell suspension.

[0141] 17. A banana plant cell obtainable by the method according to any one of embodiments 1 to 16.

[0142] 18. Further comprising regenerating a banana plant from said banana plant cell; 17. The method of any one of embodiments 1-16, optionally further comprising harvesting fruit from the banana plant.

[0143] 19. A banana plant or plant part obtainable by the method according to embodiment 18; optionally comprising: (a) set forth in SEQ ID NO: 179; (b) expressing a truncated PPO1 protein set forth in SEQ ID NO: 177; or (c) having a coding sequence set forth in SEQ ID NO: 178 Contains a mutated PPO1 gene; Further optionally, the banana plant or plant part, wherein the mutation is present in only one allele of the PPO1 gene.

[0144] 20. Fruit harvested from a banana plant obtainable by the method according to embodiment 18, wherein the pulp and / or peel of the fruit is characterized by a delayed and / or reduced browning phenotype compared to the pulp and / or peel of a banana plant not having a reduced level or activity of the at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase.

[0145] 21. A method for producing a banana plant characterized by a phenotype of delayed and / or reduced browning of the flesh and / or skin compared to a wild-type banana plant, comprising: (a) providing to a banana plant cell a CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and one or more guide RNAs, wherein the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and the one or more guide RNAs are (A) (i) SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (ii) SEQ ID NO:6 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (iii) SEQ ID NO:7, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:7; (iv) SEQ ID NO:8, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8; (v) SEQ ID NO:9, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:9; (vi) SEQ ID NO:10, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:10; (vii) SEQ ID NO:11, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:11; (viii) SEQ ID NO:12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; and (ix) SEQ ID NO: 13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13 comprising a coding sequence selected from the group consisting of: (B) (i) SEQ ID NO:40, or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; (ii) SEQ ID NO:41, or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; (iii) SEQ ID NO:42, or a polypeptide having at least 75% sequence identity to SEQ ID NO:42; (iv) SEQ ID NO:43, or a polypeptide having at least 75% sequence identity to SEQ ID NO:43; (v) SEQ ID NO:44, or a polypeptide having at least 75% sequence identity to SEQ ID NO:44; (vi) SEQ ID NO:45, or a polypeptide having at least 75% sequence identity to SEQ ID NO:45; (vii) SEQ ID NO:46, or a polypeptide having at least 75% sequence identity to SEQ ID NO:46; (viii) SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; and (ix) SEQ ID NO: 48 or a polypeptide having at least 75% sequence identity to SEQ ID NO: 48 or (C) (i) SEQ ID NO:151, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; (ii) SEQ ID NO:152, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; (iii) SEQ ID NO: 153, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 153; (iv) SEQ ID NO:154, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:154; (v) SEQ ID NO:155, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:155; (vi) SEQ ID NO:156, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:156; (vii) SEQ ID NO:157, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:157; (viii) SEQ ID NO: 158, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; and (ix) SEQ ID NO: 159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159 The polynucleotide sequence is selected from the group consisting of forming a complex capable of introducing a single-strand break or a double-strand break into at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene; (b) identifying at least one banana plant cell comprising a modification of the at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene, the modification comprising: (i) at least one nucleotide insertion; (ii) at least one nucleotide deletion; (iii) at least one nucleotide substitution; or (iv) Any combination of (b)(i) to (b)(iii). being selected from the group consisting of: (c) regenerating a banana plant from the banana plant cell, the banana plant being characterized by a phenotype of delayed and / or reduced browning of the flesh and / or skin compared to a wild-type banana plant. The method includes:

[0146] 22. The method of embodiment 21, further comprising harvesting fruit from the banana plant, wherein the fruit is characterized by a delayed and / or reduced browning phenotype compared to fruit from a banana plant that does not have a reduced or lost level or activity of the at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase.

[0147] 23. A banana plant or plant part comprising at least one modified endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene in its genome, wherein the modification results in a reduction or decreased function of at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase encoded by the modified endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene, and the modification is (A) (a) SEQ ID NO:5, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (b) SEQ ID NO:6, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (c) SEQ ID NO:7, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:7; (d) SEQ ID NO:8, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8; (e) SEQ ID NO:9, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:9; (f) SEQ ID NO:10, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:10; (g) SEQ ID NO:11, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:11; (h) SEQ ID NO:12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; and (i) SEQ ID NO: 13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13 comprising a coding sequence selected from the group consisting of: (B) (a) SEQ ID NO:40, or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; (b) SEQ ID NO:41, or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; (c) SEQ ID NO:42, or a polypeptide having at least 75% sequence identity to SEQ ID NO:42; (d) SEQ ID NO:43, or a polypeptide having at least 75% sequence identity to SEQ ID NO:43; (e) SEQ ID NO:44, or a polypeptide having at least 75% sequence identity to SEQ ID NO:44; (f) SEQ ID NO:45, or a polypeptide having at least 75% sequence identity to SEQ ID NO:45; (g) SEQ ID NO:46, or a polypeptide having at least 75% sequence identity to SEQ ID NO:46; (h) SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; and (i) SEQ ID NO: 48 or a polypeptide having at least 75% sequence identity to SEQ ID NO: 48 or (C) (a) SEQ ID NO:151, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; (b) SEQ ID NO:152, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; (c) SEQ ID NO:153, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:153; (d) SEQ ID NO:154, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:154; (e) SEQ ID NO:155, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:155; (f) SEQ ID NO:156, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:156; (g) SEQ ID NO:157, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:157; (h) SEQ ID NO: 158, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; and (i) SEQ ID NO: 159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159 The polynucleotide sequence is selected from the group consisting of 1. A banana plant or plant part, comprising at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene.

[0148] 24. A banana plant or plant part according to embodiment 19 or 23, which is non-transgenic.

[0149] 25. Banana fruit harvested from a banana plant according to embodiment 23 or 24, wherein the fruit is characterized by a delayed and / or reduced browning phenotype compared to fruits of banana plants not having reduced levels or activity of the at least one endogenous PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase.

[0150] 26. A method for obtaining a banana fruit food product, comprising processing a banana fruit according to embodiment 25.

[0151] 27. (A) (a) SEQ ID NO:5, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (b) SEQ ID NO:6, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (c) SEQ ID NO:7, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:7; (d) SEQ ID NO:8, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8; (e) SEQ ID NO:9, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:9; (f) SEQ ID NO:10, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:10; (g) SEQ ID NO:11, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:11; (h) SEQ ID NO:12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; and (i) SEQ ID NO: 13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13 comprising a coding sequence selected from the group consisting of: (B) (a) SEQ ID NO:40, or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; (b) SEQ ID NO:41, or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; (c) SEQ ID NO:42, or a polypeptide having at least 75% sequence identity to SEQ ID NO:42; (d) SEQ ID NO:43, or a polypeptide having at least 75% sequence identity to SEQ ID NO:43; (e) SEQ ID NO:44, or a polypeptide having at least 75% sequence identity to SEQ ID NO:44; (f) SEQ ID NO:45, or a polypeptide having at least 75% sequence identity to SEQ ID NO:45; (g) SEQ ID NO:46, or a polypeptide having at least 75% sequence identity to SEQ ID NO:46; (h) SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; and (i) SEQ ID NO: 48 or a polypeptide having at least 75% sequence identity to SEQ ID NO: 48 or (C) (a) SEQ ID NO:151, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; (b) SEQ ID NO:152, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; (c) SEQ ID NO:153, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:153; (d) SEQ ID NO:154, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:154; (e) SEQ ID NO:155, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:155; (f) SEQ ID NO:156, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:156; (g) SEQ ID NO:157, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:157; (h) SEQ ID NO: 158, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; and (i) SEQ ID NO: 159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159 The polynucleotide sequence is selected from the group consisting of A DNA sequence comprising a banana PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase polynucleotide.

[0152] 28. (A) (a) SEQ ID NO:5, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (b) SEQ ID NO:6, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (c) SEQ ID NO:7, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:7; (d) SEQ ID NO:8, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8; (e) SEQ ID NO:9, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:9; (f) SEQ ID NO:10, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:10; (g) SEQ ID NO:11, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:11; (h) SEQ ID NO:12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; and (i) SEQ ID NO:13, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:13; comprising a coding sequence selected from the group consisting of: (B) (a) SEQ ID NO:40, or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; (b) SEQ ID NO:41, or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; (c) SEQ ID NO:42, or a polypeptide having at least 75% sequence identity to SEQ ID NO:42; (d) SEQ ID NO:43, or a polypeptide having at least 75% sequence identity to SEQ ID NO:43; (e) SEQ ID NO:44, or a polypeptide having at least 75% sequence identity to SEQ ID NO:44; (f) SEQ ID NO:45, or a polypeptide having at least 75% sequence identity to SEQ ID NO:45; (g) SEQ ID NO:46, or a polypeptide having at least 75% sequence identity to SEQ ID NO:46; (h) SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; and (i) SEQ ID NO: 48 or a polypeptide having at least 75% sequence identity to SEQ ID NO: 48 or (C) (a) SEQ ID NO:151, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; (b) SEQ ID NO:152, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; (c) SEQ ID NO:153, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:153; (d) SEQ ID NO:154, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:154; (e) SEQ ID NO:155, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:155; (f) SEQ ID NO:156, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:156; (g) SEQ ID NO:157, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:157; (h) SEQ ID NO: 158, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; and (i) SEQ ID NO: 159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159 The polynucleotide sequence is selected from the group consisting of A DNA construct or vector comprising a banana PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase polynucleotide.

[0153] 29. A plant cell transformed with a vector or embodiment 28, optionally being a banana plant cell.

[0154] 30. (a) encoded by any one of SEQ ID NOs: 5 to 13 or encoded by a polynucleotide having at least 75% sequence identity to any one of SEQ ID NOs: 5 to 13; (b) comprising any one of SEQ ID NOs: 40 to 48 or comprising a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 40 to 48; or (c) A polyphenol oxidase protein encoded by any one of SEQ ID NOs: 151 to 159 or encoded by a polynucleotide having at least 75% sequence identity to any one of SEQ ID NOs: 151 to 159.

[0155] 31. A method for expressing polyphenol oxidase in a plant cell, comprising: (A) (a) SEQ ID NO:5, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (b) SEQ ID NO:6, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (c) SEQ ID NO:7, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:7; (d) SEQ ID NO:8, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8; (e) SEQ ID NO:9, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:9; (f) SEQ ID NO:10, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:10; (g) SEQ ID NO:11, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:11; (h) SEQ ID NO:12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; and (i) SEQ ID NO: 13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13 comprising a coding sequence selected from the group consisting of: (B) (a) SEQ ID NO:40, or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; (b) SEQ ID NO:41, or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; (c) SEQ ID NO:42, or a polypeptide having at least 75% sequence identity to SEQ ID NO:42; (d) SEQ ID NO:43, or a polypeptide having at least 75% sequence identity to SEQ ID NO:43; (e) SEQ ID NO:44, or a polypeptide having at least 75% sequence identity to SEQ ID NO:44; (f) SEQ ID NO:45, or a polypeptide having at least 75% sequence identity to SEQ ID NO:45; (g) SEQ ID NO:46, or a polypeptide having at least 75% sequence identity to SEQ ID NO:46; (h) SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; and (i) SEQ ID NO: 48 or a polypeptide having at least 75% sequence identity to SEQ ID NO: 48 or (C) (a) SEQ ID NO:151, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; (b) SEQ ID NO:152, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; (c) SEQ ID NO:153, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:153; (d) SEQ ID NO:154, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:154; (e) SEQ ID NO:155, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:155; (f) SEQ ID NO:156, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:156; (g) SEQ ID NO:157, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:157; (h) SEQ ID NO: 158, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; and (i) SEQ ID NO: 159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159 The polynucleotide sequence is selected from the group consisting of A method comprising introducing a banana polyphenol oxidase polynucleotide.

[0156] 32. A synthetic banana polyphenol oxidase guide RNA comprising a variable region selected from the group consisting of SEQ ID NOs: 32-39 and 57-97, or selected from the group consisting of SEQ ID NOs: 180-196, 199-202, and 207-225.

[0157] 33. A recombinant DNA construct comprising a promoter operably linked to a nucleotide sequence expressing at least one banana PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase guide RNA, wherein the guide RNA is capable of forming a complex with a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease, and wherein the complex is capable of expressing a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease in the banana genome. (A) (a) SEQ ID NO:5, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (b) SEQ ID NO:6, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (c) SEQ ID NO:7, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:7; (d) SEQ ID NO:8, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:8; (e) SEQ ID NO:9, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:9; (f) SEQ ID NO:10, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:10; (g) SEQ ID NO:11, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:11; (h) SEQ ID NO:12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:12; and (i) SEQ ID NO: 13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13 comprising a coding sequence selected from the group consisting of: (B) (a) SEQ ID NO:40, or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; (b) SEQ ID NO:41, or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; (c) SEQ ID NO:42, or a polypeptide having at least 75% sequence identity to SEQ ID NO:42; (d) SEQ ID NO:43, or a polypeptide having at least 75% sequence identity to SEQ ID NO:43; (e) SEQ ID NO:44, or a polypeptide having at least 75% sequence identity to SEQ ID NO:44; (f) SEQ ID NO:45, or a polypeptide having at least 75% sequence identity to SEQ ID NO:45; (g) SEQ ID NO:46, or a polypeptide having at least 75% sequence identity to SEQ ID NO:46; (h) SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; and (i) SEQ ID NO: 48 or a polypeptide having at least 75% sequence identity to SEQ ID NO: 48 or (C) (a) SEQ ID NO:151, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:151; (b) SEQ ID NO:152, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:152; (c) SEQ ID NO:153, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:153; (d) SEQ ID NO:154, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:154; (e) SEQ ID NO:155, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:155; (f) SEQ ID NO:156, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:156; (g) SEQ ID NO:157, or a polynucleotide having at least 75% sequence identity to SEQ ID NO:157; (h) SEQ ID NO: 158, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; and (i) SEQ ID NO: 159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159 The polynucleotide sequence is selected from the group consisting of 1. A recombinant DNA construct capable of binding to at least one endogenous banana PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, or PPO9 polyphenol oxidase gene and generating a double-stranded or single-stranded break.

[0158] 34. The recombinant DNA construct of embodiment 33, wherein one or more banana polyphenol oxidase guide RNAs comprise a variable region having a sequence selected from the group consisting of SEQ ID NOs: 32-39 and 57-97; and any combination of the foregoing; or one or more banana polyphenol oxidase guide RNAs comprise a variable region having a sequence selected from the group consisting of SEQ ID NOs: 180-196, 199-202, and 207-225; and any combination of the foregoing.

[0159] 35. The recombinant DNA construct of embodiment 33 or 34, wherein the CRISPR-associated endonuclease is Cas9 endonuclease.

[0160] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present 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.

[0161] The terms "comprises," "comprising," "includes," "including," "having," and their cognates mean "including, but not limited to." The term "consisting of" means "including and limited to." The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or parts so long as the additional ingredients, steps, and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0162] As used herein, the singular forms "a," "an," and "the" include plural referents 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%. EXAMPLES

[0163] The following examples are for illustrative purposes and are not to be construed as limiting the scope of the invention.

[0164] The nomenclature and laboratory procedures used herein include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are explained in the literature. See, e.g., "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, 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. New York (1998); the methodologies described 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, JE, ed. (1994); “Culture of Animal Cells-A Manual of Basic Technique” by Freshney, Wiley-Liss, NY (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, WHFreeman and Co., New York (1980); available immunoassays are described extensively in the patent and scientific literature, see, e.g., U.S. Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521; "Oligonucleotide Synthesis" Gait,MJ,ed.(1984);“Nucleic Acid Hybridization” Hames,BD,and Higgins SJ,eds.(1985);“Transcription and Translation” Hames,BD,and Higgins SJ,eds.(1984);“Animal Cell Culture” Freshney,RI,ed.(1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “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); Marshak et al., "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); 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 information contained therein is incorporated herein by reference.

[0165] Example 1 - Measurement of browning in bananas Browning can be measured based on the color of the peel, where a browning index is constructed to correlate the visual assessment of the peel color with the number of days it takes for the banana fruit to reach such a color after flower emergence (see “Dole Retail Banana Ripening Guide” https: / / www.dolenz.co.nz / uploads / media / 59236082048a9 / banana-trade-section-web.pdf and Gooding et al., “Molecular cloning and characterisation of banana fruit polyphenol oxidase”, Planta, Sep 2001;123(5):748-57). This is based on the fact that the fruit matures approximately 60-90 days after flower emergence. To construct the index, banana fruits were harvested as early as 40 days (stage 1) until they turned dark brown (stage 10), where each color correlates with the number of days after flower emergence. The index predicts that stage 10 will be reached approximately 90 days after flower emergence. Fruit color is visually assessed on a 10-point scale based on color descriptions defined, for example, in the "Dole Retail Banana Ripening Guide." Stage 1 is when the skin of all the fingers of the bunch turns green (40 days after flower appearance), stage 2 is when the color first changes to light green, usually seen on the shoulder (45 days after flower appearance), stage 3 is when the banana has more green than yellow parts (50 days after flower appearance), stage 4 is when the banana has more yellow than green parts (60 days after flower appearance), and stage 5 is when the banana has turned yellow with a green tip. Stage 1 is when the banana has turned yellow (65 days after flower appearance), stage 2 is when the banana has turned yellow (70 days after flower appearance), stage 3 is when the banana has turned yellow (65 days after flower appearance), stage 4 is when the banana has turned yellow (70 days after flower appearance), stage 5 is when the banana has turned yellow (70 days after flower appearance), stage 6 is when the whole banana has turned yellow (70 days after flower appearance), stage 7 is when all the fingers of the bunch have yellow spots and the skin has turned brown (75 days after flower appearance), stage 8 is when browning has progressed (80 days after flower appearance), stage 9 is when browning has progressed further (85 days after flower appearance), and stage 10 is when all the fingers of the bunch have dark brown skin (90 days after flower appearance).Colorimetric coordinates are taken with a Minolta Chroma Meter CR 400 or Minolta CR-300 Chroma Meter equipped with a DP-301 data processor to standardize the color scale. The CR-300 measurement head uses diffuse illumination / 0° viewing geometry (with specular components) to provide measurements of a wide variety of surfaces that correlate well with color when viewed under diffuse lighting conditions, as described in Bruno Bonnet, C., Hubert, O., Mbeguie-A-Mbeguie, D. et al., Effect of physiological harvest stages on the composition of bioactive compounds in Cavendish bananas. J. Zhejiang Univ. Sci. B14, 270-278 (2013). This allows the measurement of reflected color at each fruit development stage (1-10), and these data are used to correlate skin color with fruit ripening and browning.

[0166] Browning can also be measured based on the Banana Browning Guide, which utilizes visual assessment of sliced ​​and strained banana flesh over time (0-180 hours). Three fingers are taken from banana bunches representing stages 3 to 10 (see above) and the peel is gently washed with 0.2% sodium hypochlorite for 5 minutes (to avoid mechanical damage that may cause browning). A banana puree is then prepared from each individual banana finger after peeling, cutting into small pieces, and homogenizing in an electric mixer or food processor. The resulting puree is poured into a Petri dish and images are taken after 0, 15, 30, 60, and 120 minutes, and after 24, 48, and 72 hours. Additionally, bananas are sliced ​​and placed in Petri dishes and images are taken after 0, 12, 24, 36, 48, and 72 hours for banana bunches at color stages 3 and 4. For banana digits at stages 5 to 10, images of the banana slices are taken every 8 h from 0 h to 180 h (approximately 22 time points). This measurement method is based on Chi et al. (2014) (Chi, M., Bhagwat, B., Lane, WD et al. 14, 62 (2014). https: / / doi.org / 10.1186 / 1471-2229-14-62) and Escalante-Minakata, P., Ibarra-Junquera, V., Ornelas-Paz, Jdet al., Comparative study of the banana pulp browning process of 'Giant Dwarf' and FHIA-23 during fruit ripening based on image analysis and the Based on the results of polyphenol oxidase and peroxidase biochemical properties.3 Biotech 8,30 (2018).The images are processed to correlate color to browning, which is expected to vary anywhere from the color of freshly cut banana slices or banana puree (yellow / off-white) to brown bananas (dark brown).

[0167] Browning can also be measured based on assessment of flesh firmness and peel firmness. Flesh firmness and peel firmness are measured using a TA-XT2 penetrometer as described in Bruno Bonnet, C., Hubert, O., Mbeguie-A-Mbeguie, D. et al., Effect of physiological harvest stages on the composition of bioactive compounds in Cavendish bananas. J. Zhejiang Univ. Sci. B14, 270-278 (2013). Three banana fingers are harvested from bunches representing stages 3 to 10 (proven based on skin color; see above). A 4.9 mm cylindrical metal perforator is used to penetrate clean, fresh, unpeeled fruit to a depth of 10 mm at a constant speed (2 mm / s). The maximum force applied to break the peel represents the peel firmness, and the slope of the force / time curve represents the fruit firmness.

[0168] Browning can also be measured based on the correlation of skin color (and firmness) with flesh color / texture, utilizing a catalog of color (visual and colorimetric), skin firmness, and flesh firmness in wild-type plants versus banana ripeness stage and skin and flesh browning over time, providing a benchmark against which reduction of browning in banana skin and flesh can be assessed.

[0169] Example 2 - Identification of PPO in bananas To identify banana PPO genes, bioinformatics analysis was performed. The PPO peptide sequence from Arctic apple provided in US Pat. No. 9,580,723 (SEQ ID NOs: 1-4) was used as a query sequence to search for homologs in the banana genome and in the genomes of other plant species known to have PPO genes (apricot, sweet potato, pokeweed, tobacco, tomato, potato, and grapevine). The "TBlastN" tool was used to align the query protein sequence with the translated nucleotide sequences of selected genomes. As a result, 72 sequences homologous to the query sequence were found from various species, including 9 sequences from banana (Malayan wild cucumber).

[0170] The obtained sequences were subjected to multiple sequence alignment (MSA), after which a phylogenetic tree was constructed. The genes were organized into clusters. A confidence score was assigned to each cluster by combining the Maximum Likeliness and Neighbour Joining algorithms. The rationale behind it is that banana genes that cluster with known PPO genes with high confidence are more likely to have conserved PPO activity as well. The analysis showed that all nine banana genes identified by "TBlastN" cluster with PPO genes from other species with high confidence. Thus, all nine genes were predicted to be PPO genes with conserved functions. This analysis and the retrieved PPOs were further confirmed by performing phylogenetic analysis. These PPOs have 35%-97% homology with each other as shown in Figure 1 and about 39%-97% homology to the query sequence as shown in Figure 2. The identified banana PPOs were further confirmed by aligning the DWL domains to the DWL domain of the query sequence as can be seen in Figure 3. The nine PPO banana peptide homologs are listed in Table 1 with their corresponding accession numbers obtained from the Banana Genome Hub and set forth in SEQ ID NOs: 40-48 (the corresponding gene sequences are listed in SEQ ID NOs: 5-13).

[0171] [Table 1]

[0172] Example 3 - Selection of PPO candidates for targeting To identify banana tissues expressing the PPOs in Table 1, mRNA was produced from various banana tissues, specifically embryonic cell suspension (ECS), embryo differentiated from ECS after incubation in embryo development medium (EDM), curled leaf, top leaf, old leaf, brown peel, yellow peel, green peel, brown fruit, yellow fruit, and green fruit. RNA extraction was performed by flash freezing banana tissues in liquid nitrogen, lyophilizing, and homogenizing. Samples were then placed in tubes containing extraction buffer and thawed with mixing. Samples were centrifuged and the supernatant transferred to a new tube. Phenol-chloroform extraction was then used, followed by centrifugation to obtain an RNA pellet. mRNA extraction was then completed using the Plant / Fungi Total RNA Purification Kit (Norgen Biotek Corp).

[0173] Expression of mRNAs encoding the nine identified PPOs was examined in all the above tissues using semi-quantitative PCR with three to six biological replicates for each tissue / PPO combination. DNA was removed from RNA samples using Turbo DNA-free kit (Invitrogen). DNAse-treated RNA was used to synthesize cDNA using Superscript III (Thermofisher) with a mixture of oligo-dT and random hexamers to ensure complete coverage of the transcript. For each PCR, 12 ng of cDNA was used as template and standard PCR reactions were performed with GoTaq G2 Master Mix (Promega) for 30 cycles. The oligonucleotides used in the PCR reactions are shown in Table 2 (and SEQ ID NOs: 14 to 31):

[0174] [Table 2]

[0175] The expression profile is presented in Figure 4, which represents the average expression level of each PPO in each tissue tested. The units are arbitrary, derived by visually quantitating, expressing the quantification as a number, and linearly converting the number onto a scale from - to +, ++, +++.

[0176] Additional expression analysis was performed in some tissues by comparing total RNA sequencing data from various tissues and measuring expression by TMM normalization (trimmed mean of M-values). Briefly, samples of roots, top leaves, curled leaves, and banana flesh and peel (both green-yellow and yellow ripe stages) were collected from greenhouse Grand Nine banana plants and commercial banana fruits, respectively. Roots, top leaves, and curled leaves were collected from plants grown for 9 months to 1.5 years under greenhouse conditions. Samples were flash frozen and freeze-dried for 2 days before sampling treatment. Freeze-dried samples were ground using a mortar and pestle. RNA-seq sample preparation from these samples consisted of total RNA extraction, mRNA enrichment using polyadenylated tail binding, reverse transcription to generate cDNA, and preparation of sequencing libraries using adapter ligation. Libraries were sequenced to a raw read depth of at least 44 million per sample, and after QC analysis of the raw data (including adapter trimming), sequencing reads were aligned to the banana genome. The number of reads aligned to each gene was quantified and normalized between samples using the "trimmed mean M value" (TMM) method (Robinson, MD, Oshlack, AA scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11, R25 (2010)). As can be seen from Figure 5, high expression of PPO1, PPO2, PPO8, and PPO9 was observed in the flesh and / or peel at green-yellow and / or yellow ripening stages. Semi-quantitative PCR data also suggested similar expression of PPO1, PPO2, PPO8, and PPO9.

[0177] The annotations of the identified PPO genes were further validated in the banana genome hub (https: / / banana-genome-hub.southgreen.fr / ) by loading additional annotation tracks from publicly available datasets into the genome to show expression patterns. Annotated exonic mRNA sequences in the genome were assessed to confirm their expression. This validation confirmed, for example, the annotations of PPO1 and PPO2. This validation further confirmed that PPO3 was not expressed in any tissue where RNA sequencing data were compared to the annotations. PPO1 and PPO2 were observed to be expressed in the flesh and skin, suggesting that they are involved in fruit browning, but not in the embryo / ECS.

[0178] Additional RNA-seq expression analysis was performed on Grand Nine bananas obtained directly from a commercial distributor over the course of natural ripening without the application of exogenous ethylene. Peel and flesh samples were taken from five ripening stages: all green (immature), green-yellow (first turning point), all yellow (mature), yellow-brown (second turning point), and all brown (overripe). High-quality RNA was obtained from all flesh samples and from the peel at the all-green stage. Tissue samples were also taken from leaves and roots of in vitro Grand Nine plants, as well as from in vitro cultures of embryos and embryogenic cells. Relative mRNA abundance was quantified as above using TMM normalization. As can be seen in Figure 8, PPO1, PPO4, and PPO9 account for >90% of PPO expression in Grand Nine banana peel at the immature green stage, with PPO1 being the dominant PPO gene expressed in the flesh of Grand Nine bananas, with the exception of the overripe brown stage where PPO8 is more highly expressed.

[0179] Example 4 - Confirmation of PPO activity To confirm that the identified PPOs, such as PPO1 and PPO2, are indeed PPO genes, the proteins are synthesized and the PPO activity is tested using a colorimetric assay. Briefly, for each synthetic PPO, 0.2 mL of synthetic enzyme solution and 2.8 mL of 10 mM 4-methylcatechol (0.2 M phosphate buffer, pH 6.3) are mixed, and the PPO activity is measured as a function of the change in absorbance at 420 nm over time (an increase in absorbance indicates PPO activity). The linear portion of the data plot is used to calculate the relative enzyme activity.

[0180] Example 5 - Creation of genetically modified plants edited in PPO1, PPO2, PPO8, or PPO9 Genetically modified banana plants are created in which either PPO1, PPO2, PPO8, or PPO9 has been edited to measure the effect of reducing the levels or activity of PPO1, PPO2, PPO8, or PPO9. Plants in which PPO3 has been edited are used as controls. DNA encoding CAS9 and sgRNA targeting PPO1, PPO2, PPO3, PPO8, or PPO9 are introduced into the genome of banana plants using Agrobacterium. Embryogenic cell suspensions (ECS) are transformed with Agrobacterium strains carrying plasmids encoding the Cas9 machinery and expressing sgRNA targeting PPO and a kanamycin resistance gene (nptII). Agrobacterium transformation is performed according to Ganapathi et al., Plant Cell Reports (2001) 20:157-162 and Kanna et al., Molecular Breeding October 2004, Volume 14, Issue 3, pp 239-252. Embryogenic cells are co-cultured with Agrobacterium for 1-3 days and then transferred to regeneration medium containing G418 as the selection agent until shoots develop.

[0181] CRISPR vector constructs are used that contain pairs of sgRNAs as shown in Tables 3 and 3A below, respectively, targeting each PPO gene separately. Tables 3 and 3A show the variable sequences of the sgRNAs used with the scaffold listed as SEQ ID NO:98, respectively. To maximize the probability of generating double mutant plants, constructs were made that contain combinations of sgRNA1 and sgRNA2, as shown in Table 3. An example of editing PPO2 is shown in FIG. 6. As a control, genetically modified plants are generated that target PPO3. The following combinations of sgRNA variable regions were used: SEQ ID NO:32 and 33, SEQ ID NO:62 and 33, SEQ ID NO:34 and 35, SEQ ID NO:36 and 37, SEQ ID NO:38 and 39, SEQ ID NO:32 and 34, SEQ ID NO:32 and 35, SEQ ID NO:33 and 34, SEQ ID NO:33 and 35; SEQ ID NO:38 and 39; and SEQ ID NO:57 and 58.

[0182] [Table 3]

[0183] [Table 3A]

[0184] Once the genetically modified plants are regenerated, they are grown in the field until fruit set. Evaluation of the PPO editing phenotype in mature plants / fruit is performed during field trials by comparing PPO expression and fruit browning between edited and wild-type plants. Approximately 20 plants from each edited and control line are divided into 4 groups of 5 plants each and randomly spaced in the field. Plants are allowed approximately 12 weeks to flower, and fruit development is evaluated approximately 14-17 weeks after flowering. For fruit, 3 groups are harvested 24 weeks after planting, and each bunch is split and either kept without ethylene ripening (representative of the yellow stage) or ripened with ethylene to test browning / PPO expression both with and without ethylene-induced ripening.

[0185] Browning can be measured as described in Example 1 above.

[0186] Control plants in which a PPO gene expressed in the leaves has been edited (e.g., PPO9 has been edited) are compared to wild-type plants for expression levels and / or activity of PPO at earlier developmental stages (since expression can be tested in leaves first). Once it has been identified which genetically modified plants show reduced browning and / or reduced expression of PPO in fruit, non-genetically modified banana plants (without CRISPR / CAS9 machinery integrated into the genome) that have been transiently edited for the selected PPO gene are generated.

[0187] Example 6 - Generation of banana plants with mutations in the PPO gene using transient CAS9 expression To generate banana plants with a mutated PPO gene, a vector encoding the CAS9 machinery and expressing an sgRNA targeting the PPO gene is introduced into banana embryogenic cell suspensions (ECS). To generate ECS, embryogenic callus is first generated from early explants such as immature male flowers or shoot tips as described in Ma, Proceedings of Symposium on Tissue culture of horticultural crops, Taipei, Taiwan, 8-9 March 1988, pp. 181-188 and Schoofs, H. (1997)-"The origin of embryogenic cells in Musa", PhD thesis, KULeuven, Belgium. Then, culture of embryogenic cell suspensions (ECS) is initiated from the newly generated highly embryogenic callus in liquid medium. Then, 80% of the medium is replaced every 12-14 days until the cell suspension is fully established (6-9 months).

[0188] The embryogenic cell suspension is then bombarded with a plasmid encoding the CAS9 machinery and expressing an sgRNA targeting PPO. Bombardment of cells can be performed according to any method known in the art, for example, the method described in Hamada et al., Sci Rep. 2018; 8: 14422. All plasmids used for bombardment contain four transcription units. The first transcription unit contains a CaMV-35S promoter and a tobacco mosaic virus (TMV) terminator driving the expression of Streptococcus Cas9 (human codon optimized). The next transcription unit consists of another CaMV-35S promoter and a tNOS (nopaline synthase) terminator driving the expression of the mCherry fluorescent marker. The third and fourth transcription units each contain a wheat U6 promoter expressing an sgRNA for a selected target gene (each vector contains two sgRNAs). The sgRNA contained in the plasmid used for bombardment is designed to target the PPO gene. sgRNAs were designed, for example, to target: - a region found in exon 1 of the PPO gene PPO1 Ma06_g31080 (SEQ ID NO: 5); - a region found in exon 1 of the PPO gene PPO2 Ma07_g03540 (SEQ ID NO: 6); - a region found in exon 2 of the PPO gene PPO3 Ma07_g03650 (SEQ ID NO: 7); - a region found in exon 1 of the PPO gene PPO8 Ma08_34740 (SEQ ID NO: 12); and - A region found in exon 2 of the PPO gene PPO9 Ma10_g20510 (SEQ ID NO: 13).

[0189] The sgRNAs used and the target genes for their design are summarized in Table 4 (all sgRNA sequences are listed in the 5' to 3' direction. All sgRNAs were cloned without the sequence of the PAM motif (marked in bold in Table 4).

[0190] [Table 4]

[0191] [Table 4A]

[0192] Three days after bombardment with the vectors containing the sgRNAs shown above, the cells are transferred to growth medium, followed by embryo development medium (EDM) and then maturation medium (relevant media can be found, for example, in Strosse H., R. Domergue, B. Panis, JV Escalant and F. Cote, 2003, Banana and plantain embryogenic cell suspensions (A. Vezina and C. Picq, eds). INIBAP Technical Guidelines 8, The International Network for the Improvement of Banana and Plantain, Montpellier, France). Mature embryos are germinated on germination medium (Strosse H., R. Domergue, B. Panis, JV Escalant and F. Cote. 2003. Banana and plantain embryogenic cell suspensions (A. Vezina and C. Picq, eds). INIBAP Technical Guidelines 8. The International Network for the Improvement of Banana and Plantain, Montpellier, France) and young shoots are transferred to shoot maturation medium until they reach a height of approximately 1 cm. Shoots are transferred to rooting medium for generating plantlets.

[0193] Next, leaf samples are taken from each plantlet to extract genomic DNA, and the DNA samples are subjected to genotyping using next-generation sequencing. The goal of genotyping is to identify whether a Cas9-driven gene editing event has occurred in any of the PPO genes. Plantlets are sampled individually. Small pieces of leaves are cut and placed in sample tubes (approximately 25 mg). Genomic DNA is extracted from these samples using the Sbeadex kit (Biosearch Technologies) in an OktoPure system (LGC). The resulting genomic DNA is diluted to 5 ng / μL and then mixed to form a pool of 12 plantlets. PCR amplification of the target genes is performed using primers flanking the predicted editing sites to form 1 Kbp amplicons using 10 ng of template DNA. The PCR amplicons of each pooled sample are mixed and sent for AmpSeq sequencing. Here, a library is constructed using transposase-mediated fragmentation, and then miseq sequencing is performed to generate sequencing reads for each amplicon. The amplicon sequences are then analyzed using "Geneious", "pindel", "varscan" and "freebase" software to identify potential indels. If an editing event is found, it is further verified by PCR reactions using the primers listed in Table 5. If editing is found, it indicates that the target gene was cut by the transiently expressed Cas9 in the ECS from which the plantlet was derived. To confirm that Cas9 was indeed transiently expressed, the absence of Cas9 and the carrier vector backbone is confirmed by PCR and qRT-PCR using the standard primer sets listed in Table 6.

[0194] An alternative method uses Sanger sequencing for genotyping: genomic DNA is used to amplify the target region using primers flanking the predicted editing site (Table 5), and the 1 kbp amplicons are analyzed by Sanger sequencing to identify edits in the target gene.

[0195] [Table 5]

[0196] [Table 6]

[0197] Once the edited plantlets are identified, the plantlets are micropropagated to produce additional identical plantlets.Micropropagation methods are known in the art and are described, for example, in Munir Iqbal et al. (2013), International Journal of Agriculture Innovations and Research Volume 2, Issue 1, ISSN (Online) 2319-1473.The PPO level and browning of the edited plants are then confirmed in the field as described above.

[0198] Browning can be measured as described in Example 1 above.

[0199] Example 7 - Generation of banana plants with mutations in the PPO gene using transient Cas9 expression in Agrobacterium tumefaciens transformed embryogenic cells An alternative method to that described in Example 6 for generating banana plants with a mutated PPO gene utilizes Agrobacterium-mediated transformation of embryogenic banana cells. Embryogenic cell suspensions (ECS) are prepared as described in Example 6 and transformed using Agrobacterium tumefaciens following procedures such as those described in Khanna et al., Mol. Breed. 2004;14:239 and Tripathi et al., In Vitro Cell Dev. Biol.-Plant 2012;48:216. All plasmids used for Agrobacterium-mediated transformation contain four transcription units. The first transcription unit drives expression of a resistance gene that confers resistance to a selection agent. The next transcription unit drives expression of a human codon-optimized Streptococcus pyogenes Cas9. The third and fourth transcription units each drive expression of an sgRNA against a selected target gene (each vector contains two sgRNAs). The sgRNA contained in the plasmid used for Agrobacterium-mediated transformation is designed to target the PPO gene. The sgRNAs used and the target genes for which they were designed are summarized in Table 4 (all sgRNA sequences are listed in the 5' to 3' direction, and sgRNAs without the sequence of the PAM motif were cloned and are marked in bold in Table 4).

[0200] After co-cultivation of the banana embryogenic cells with Agrobacterium tumefaciens cells carrying the above plasmids, the banana cells are resuspended in liquid growth medium containing the selection agent in a 250 mL Erlenmeyer flask and cultured for 5 days with gentle shaking. This selection treatment allows enrichment of banana cells that have been successfully transformed and thereby expressed the resistance gene, while selecting for non-transformed banana cells and Agrobacterium tumefaciens cells. The banana cells are then washed 4 times with liquid growth medium to remove the selection agent, and then cultured in growth medium, followed by embryogenesis medium, maturation medium, and germination medium (the relevant media can be found, for example, in Strosse H., R. Domergue, B. Panis, JV Escalant and F. Cote, 2003, Banana and plantain embryogenic cell suspensions (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 shoot maturation medium until they reach a height of approximately 1 cm, and then transferred to rooting medium to generate plantlets. Targeted gene editing in regenerated plants is identified by extracting genomic DNA from leaf samples and analyzing the target site by PCR and sequencing using gene-specific primers listed in Table 5, as described in Example 6. The absence of plasmid sequences in edited plant lines is confirmed by qRT-PCR using primers listed in Table 6. Finally, edited plants are micropropagated to generate clones, as discussed in Example 6, and the level of enzymatic browning in plants is verified using the method described in Example 1. Using the above procedure, banana plants containing targeted edits in the PPO1 gene and no foreign plasmid sequences integrated into the genome were successfully obtained.Banana embryogenic cells were transformed with plasmid pMOL_0019 (see Table 4), which contains sgRNAs sg857 (SEQ ID NO: 176) and sg858 (SEQ ID NO: 50), both of which target the first exon of PPO1. Cells transformed with pMOL_0019 were transiently selected and regenerated into shoots, which were screened by Sanger sequencing and qRT-PCR as described above.

[0201] The PPO1-edited plants have a single base pair deletion in the first exon of one of the three alleles of PPO1, resulting in the production of a truncated PPO1 protein from the edited allele. The resulting protein, coding, and gene sequences are provided in SEQ ID NOs: 177, 178, and 179, respectively. The target region of the PPO1 gene is shown in FIG. 7A, highlighting the deleted nucleotide (cytosine-bp371), sgRNA, and genotyping primers. The sequences of the edited and unedited PPO1 proteins are shown in FIG. 7B. As shown in Table 7, the plasmid-specific primers were unable to amplify the target sequence from genomic DNA extracted from PPO1-edited banana plants. This was also the case for DNA from the negative control wild-type plants, but these primers did amplify the plasmid sequence from genomic DNA extracted from the positive control transgenic plants. As an internal control, the endogenous banana genomic region was amplified in all samples. Thus, these analyses confirmed the absence of plasmid sequences in the genome of PPO1-edited banana plants.

[0202] [Table 7]

[0203] Example 8 - Further sgRNA design Additional PPO sgRNAs were designed to maximize targeting specificity (minimal number of potential off-target edits), editing efficiency (taking into account potential RNA secondary structures and SNPs that affect targeting), and mutation predictability (likelihood to induce frameshift mutations based on sequence microhomology around DSBs). As listed in Table 8, multiple sgRNAs were designed for all PPO genes for both Cas9 and base editor targeting strategies, respectively, to generate either indels or programmable base substitutions that result in premature stop codons in the coding sequence of the target gene. sgRNAs designed for Cas9 editing are predicted to be most effective for editing with Cas9, but may also be useful for editing with base editors. Similarly, sgRNAs designed for base editing are predicted to be most effective for editing with base editors, but may also be useful for editing with Cas9. sgRNAs against PPO3 and PPO7 were also designed, primarily for use as controls. The sequences in Table 8 do not include PAM sites. PAM sites can be identified by aligning the guide sequence to the target sequence.

[0204] [Table 8-1]

[0205] [Table 8-2]

[0206] While the present 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.

[0207] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety 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

1. A method for reducing the level or activity of at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase encoded by a PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene in a banana plant or banana plant cell.

2. The polyphenol oxidase gene of PPO1, PPO2, PPO8, PPO9, or PPO4 is (A) (a) SEQ ID NO:5 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (b) SEQ ID NO:6 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (c) SEQ ID NO: 12 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 12; (d) SEQ ID NO: 13 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13; and (e) SEQ ID NO: 8 (PPO4) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 8 comprising a coding sequence selected from the group consisting of: (B) (a) SEQ ID NO: 40 (PPO1) or a polypeptide having at least 75% sequence identity to SEQ ID NO: 40; (b) SEQ ID NO: 41 (PPO2) or a polypeptide having at least 75% sequence identity to SEQ ID NO: 41; (c) SEQ ID NO: 47 (PPO8) or a polypeptide having at least 75% sequence identity to SEQ ID NO: 47; (d) SEQ ID NO: 48 (PPO9) or a polypeptide having at least 75% sequence identity to SEQ ID NO: 48; and (e) SEQ ID NO: 43 (PPO4) or a polypeptide having at least 75% sequence identity to SEQ ID NO: 43 a polyphenol oxidase selected from the group consisting of: (C) (a) SEQ ID NO: 151 (PPO1) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 151; (b) SEQ ID NO: 152 (PPO2) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 152; (c) SEQ ID NO: 158 (PPO8) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; (d) SEQ ID NO: 159 (PPO9) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159; and (e) SEQ ID NO: 154 (PPO4) or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 154 or comprising a polynucleotide sequence selected from the group consisting of: (D) (a) Accession number Ma06_31080 (PPO1); (b) Accession number Ma07_03540 (PPO2); (c) Accession number Ma08_34740 (PPO8); (d) Accession No. Ma10_20510 (PPO9); and (e) Accession number Ma08_09150 (PPO4) The method of claim 1 , comprising a sequence characterized by:

3. (a) reducing the level of at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase in the banana plant or banana plant cell; (b) reducing the function of at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase in the banana plant or banana plant cell; or 3. The method of claim 1 or 2, wherein (c) the function of at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase in the banana plant or banana plant cell is abolished.

4. (a) delaying browning of the pulp and / or peel of a banana plant compared to the pulp and / or peel of a banana plant that does not have a reduced level or activity of the at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase; and / or 3. The method of claim 1 or 2, wherein (b) the browning of the pulp and / or peel of the banana plant is reduced compared to the pulp and / or peel of a banana plant that does not have a reduced level or activity of the at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase.

5. (a) providing to said banana plant cell or part of said banana plant a silencing RNA that targets a transcript of said at least one PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene, optionally wherein said silencing RNA is provided by introducing into said banana plant cell or part of said banana plant an endonuclease, wherein said endonuclease is capable of targeting a transcript of said at least one PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene; a gene encoding an endogenous non-coding RNA can be modified to encode the silencing RNA targeting a transcript of the polyphenol oxidase gene of 4, and optionally, the endonuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a homing endonuclease, a CRISPR-associated endonuclease, and a modified CRISPR-associated endonuclease; or (b) introducing a modification into a PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene encoding said at least one PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase; optionally, said modification is provided to said banana plant cell and is introduced into said PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene by an endonuclease capable of targeting said at least one PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene. 2, PPO8, PPO9, or PPO4 polyphenol oxidase gene; further optionally, the endonuclease is selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), homing endonucleases, CRISPR-associated endonucleases, and modified CRISPR-associated endonucleases; further optionally, the CRISPR-associated endonuclease is a Cas9 endonuclease; and / or (c) providing the banana plant cell with a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease and one or more guide RNAs specific for the at least one PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene, wherein the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and the one or more guide RNAs form a complex that enables the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease to introduce a double-stranded or single-stranded break in the at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene; and optionally, the CRISPR-associated endonuclease is a Cas9 endonuclease. The method according to claim 1 or 2.

6. and identifying at least one banana plant cell comprising a modification of the at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene, wherein the modification (a) at least one nucleotide insertion; (b) at least one nucleotide deletion; (c) insertion-deletion (indel); (d) inversion; (e) at least one nucleotide substitution; and (f) Any combination of (a) to (e) 6. The method of claim 5, wherein the compound is selected from the group consisting of:

7. (a) providing the one or more guide RNAs to a banana plant cell within one or more recombinant DNA constructs encoding the one or more guide RNAs operably linked to one or more promoters; (b) the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and / or the one or more guide RNAs are provided to a banana plant cell in the form of RNA; (c) the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease is provided to the banana plant cell in the form of a protein, and the one or more guide RNAs are provided to the banana plant cell in the form of RNA; optionally, 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; or 6. The method of claim 5, wherein (d) the one or more guide RNAs and the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease are provided to the banana plant cell via Agrobacterium transformation of one or more plasmids encoding the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease, at least one selectable marker gene, and the one or more guide RNAs.

8. the endonuclease, the one or more guide RNAs, and / or the one or more recombinant DNA constructs (a) Microprojectile bombardment; (b) Agrobacterium transformation; (c) protoplast transfection; (d) electroporation; and (e) Nanoparticle-mediated transfection The method of claim 5, wherein the banana plant cell is provided with a method selected from the group consisting of:

9. 6. The method of claim 5, wherein the endonuclease is provided to the banana plant cell as a polynucleotide encoding an endonuclease polypeptide; optionally, the endonuclease is a Cas9 endonuclease and the polynucleotide is a Cas9 polynucleotide encoding a Cas9 polypeptide.

10. (A) the one or more guide RNAs (a) SEQ ID NO: 32; (b) SEQ ID NO: 33; (c) SEQ ID NO: 34; (d) SEQ ID NO: 35; (e) SEQ ID NO: 57; (f) SEQ ID NO: 58; (g) SEQ ID NO: 38; (h) SEQ ID NO: 39; (i) SEQ ID NO: 62; (j) SEQ ID NO: 76; (k) SEQ ID NO: 77; and (l) Any combination of (a) to (k) or comprising a variable region having a sequence selected from the group consisting of: (B) the one or more guide RNAs (a) SEQ ID NOs: 32 and 33; (b) SEQ ID NOs: 34 and 35; (c) SEQ ID NOs: 32 and 34; (d) SEQ ID NOs: 32 and 35; (e) SEQ ID NOs: 33 and 34; (f) SEQ ID NOs: 33 and 35; (g) SEQ ID NOs: 57 and 58; (h) SEQ ID NOs: 38 and 39; (i) SEQ ID NOs: 62 and 33; and (j) SEQ ID NOs: 76 and 77 The method of claim 5, wherein the pair of guide RNAs comprises a variable region selected from the group consisting of:

11. 3. The method of claim 1 or 2, wherein the banana plant cells are embryogenic cells and / or are contained in an embryogenic cell suspension.

12. 3. Banana plant cells obtainable by the method according to claim 1 or 2.

13. further comprising regenerating a banana plant from said banana plant cell; 3. The method of claim 1 or 2, optionally further comprising harvesting fruit from the banana plant.

14. A banana plant or plant part obtainable by the method according to claim 13; optionally comprising: (a) set forth in SEQ ID NO: 179; (b) expressing a truncated PPO1 protein set forth in SEQ ID NO: 177; or (c) comprising a mutated PPO1 gene having a coding sequence set forth in SEQ ID NO: 178; Further optionally, the mutation is present in only one allele of the PPO1 gene, and further optionally, the banana plant or plant part is non-transgenic.

15. 14. Fruit harvested from a banana plant obtainable by the method of claim 13, wherein the pulp and / or peel of the fruit is characterized by a phenotype of delayed and / or reduced browning compared to the pulp and / or peel of a banana plant that does not have a reduced level or activity of the at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase.

16. 1. A banana plant or plant part comprising at least one modified endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene in its genome, wherein said modification results in a reduction or decreased function of at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase encoded by said modified endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene, and wherein said modification is (A) (a) SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (b) SEQ ID NO:6 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (c) SEQ ID NO: 12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 12; (d) SEQ ID NO: 13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13; and (e) SEQ ID NO: 8 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 8 comprising a coding sequence selected from the group consisting of: (B) (a) SEQ ID NO:40 or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; (b) SEQ ID NO:41 or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; (c) SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; (d) SEQ ID NO:48 or a polypeptide having at least 75% sequence identity to SEQ ID NO:48; and (e) SEQ ID NO: 43 or a polypeptide having at least 75% sequence identity to SEQ ID NO: 43 a polyphenol oxidase selected from the group consisting of: (C) (a) SEQ ID NO: 151, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 151; (b) SEQ ID NO: 152 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 152; (c) SEQ ID NO: 158 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; (d) SEQ ID NO: 159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159; and (e) SEQ ID NO: 154 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 154 or comprising a polynucleotide sequence selected from the group consisting of: (D) (a) Accession number Ma06_31080 (PPO1); (b) Accession number Ma07_03540 (PPO2); (c) Accession number Ma08_34740 (PPO8); (d) Accession No. Ma10_20510 (PPO9); and (e) Accession number Ma08_09150 (PPO4) and a sequence characterized by: Optionally, non-transgenic A banana plant or plant part located in at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene.

17. 17. Banana fruit harvested from the banana plant of claim 16, wherein the fruit is characterized by a phenotype of delayed and / or reduced browning compared to fruit from a banana plant that does not have a reduced level or activity of the at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase.

18. 20. A method for obtaining a banana fruit food product, comprising processing the banana fruit of claim 17.

19. (A) (a) SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (b) SEQ ID NO:6 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (c) SEQ ID NO: 12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 12; (d) SEQ ID NO: 13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13; and (e) SEQ ID NO: 8 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 8 comprising a coding sequence selected from the group consisting of: (B) (a) SEQ ID NO:40 or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; (b) SEQ ID NO:41 or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; (c) SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; (d) SEQ ID NO:48 or a polypeptide having at least 75% sequence identity to SEQ ID NO:48; and (e) SEQ ID NO: 43 or a polypeptide having at least 75% sequence identity to SEQ ID NO: 43 a polyphenol oxidase selected from the group consisting of: (C) (a) SEQ ID NO: 151, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 151; (b) SEQ ID NO: 152 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 152; (c) SEQ ID NO: 158 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; (d) SEQ ID NO: 159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159; and (e) SEQ ID NO: 154 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 154 or comprising a polynucleotide sequence selected from the group consisting of: (D) (a) Accession number Ma06_31080 (PPO1); (b) Accession number Ma07_03540 (PPO2); (c) Accession number Ma08_34740 (PPO8); (d) Accession No. Ma10_20510 (PPO9); and (e) Accession number Ma08_09150 (PPO4) comprising a sequence characterized by: A DNA sequence comprising a banana PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase polynucleotide.

20. (A) (a) SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (b) SEQ ID NO:6 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (c) SEQ ID NO: 12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 12; (d) SEQ ID NO: 13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13; and (e) SEQ ID NO: 8 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 8 comprising a coding sequence selected from the group consisting of: (B) (a) SEQ ID NO:40 or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; (b) SEQ ID NO:41 or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; (c) SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; (d) SEQ ID NO:48 or a polypeptide having at least 75% sequence identity to SEQ ID NO:48; and (e) SEQ ID NO: 43 or a polypeptide having at least 75% sequence identity to SEQ ID NO: 43 a polyphenol oxidase selected from the group consisting of: (C) (a) SEQ ID NO: 151, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 151; (b) SEQ ID NO: 152 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 152; (c) SEQ ID NO: 158 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; (d) SEQ ID NO: 159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159; and (e) SEQ ID NO: 154 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 154 or comprising a polynucleotide sequence selected from the group consisting of: (D) (a) Accession number Ma06_31080 (PPO1); (b) Accession number Ma07_03540 (PPO2); (c) Accession number Ma08_34740 (PPO8); (d) Accession No. Ma10_20510 (PPO9); and (e) Accession number Ma08_09150 (PPO4) Contains sequences characterized by A DNA construct or vector comprising a banana PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase polynucleotide.

21. A plant cell transformed with the vector of claim 20, optionally a banana plant cell.

22. (a) encoded by SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:8, or encoded by a polynucleotide having at least 75% sequence identity to SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:8; (b) comprising SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:43, or comprising a sequence having at least 75% sequence identity to SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:43; (c) encoded by SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:158, SEQ ID NO:159, or SEQ ID NO:154, or encoded by a polynucleotide having at least 75% sequence identity to SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:158, SEQ ID NO:159, or SEQ ID NO:154; or (d) encoded by a sequence characterized by accession number Ma06_31080 (PPO1), accession number Ma07_03540 (PPO2), accession number Ma08_34740 (PPO8), accession number Ma10_20510 (PPO9), or accession number Ma08_09150 (PPO4); Polyphenol oxidase protein.

23. A method for expressing polyphenol oxidase in a plant cell, comprising: (A) (a) SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (b) SEQ ID NO:6 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (c) SEQ ID NO: 12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 12; (d) SEQ ID NO: 13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13; and (e) SEQ ID NO: 8 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 8 comprising a coding sequence selected from the group consisting of: (B) (a) SEQ ID NO:40 or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; (b) SEQ ID NO:41 or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; (c) SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; (d) SEQ ID NO:48 or a polypeptide having at least 75% sequence identity to SEQ ID NO:48; and (e) SEQ ID NO: 43 or a polypeptide having at least 75% sequence identity to SEQ ID NO: 43 a polyphenol oxidase selected from the group consisting of: (C) (a) SEQ ID NO: 151, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 151; (b) SEQ ID NO: 152 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 152; (c) SEQ ID NO: 158 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; (d) SEQ ID NO: 159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159; and (e) SEQ ID NO: 154 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 154 or comprising a polynucleotide sequence selected from the group consisting of: (D) (a) Accession number Ma06_31080 (PPO1); (b) Accession number Ma07_03540 (PPO2); (c) Accession number Ma08_34740 (PPO8); (d) Accession No. Ma10_20510 (PPO9); and (e) Accession number Ma08_09150 (PPO4) Contains sequences characterized by A method comprising introducing a banana polyphenol oxidase polynucleotide.

24. (a) SEQ ID NO: 32; (b) SEQ ID NO: 33; (c) SEQ ID NO: 34; (d) SEQ ID NO: 35; (e) SEQ ID NO: 57; (f) SEQ ID NO: 58; (g) SEQ ID NO: 38; (h) SEQ ID NO: 39; (i) SEQ ID NO: 62; (j) SEQ ID NO: 76; and (k) SEQ ID NO: 77 A synthetic banana polyphenol oxidase guide RNA comprising a variable region selected from the group consisting of:

25. 1. A recombinant DNA construct comprising a promoter operably linked to a nucleotide sequence that expresses at least one banana PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase guide RNA, wherein the guide RNA is capable of forming a complex with a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease, and wherein the complex is capable of expressing a CRISPR-associated endonuclease in the banana genome. (A) (a) SEQ ID NO:5 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:5; (b) SEQ ID NO:6 or a polynucleotide having at least 75% sequence identity to SEQ ID NO:6; (c) SEQ ID NO: 12 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 12; (d) SEQ ID NO: 13 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 13; and (e) SEQ ID NO: 8 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 8 comprising a coding sequence selected from the group consisting of: (B) (a) SEQ ID NO:40 or a polypeptide having at least 75% sequence identity to SEQ ID NO:40; (b) SEQ ID NO:41 or a polypeptide having at least 75% sequence identity to SEQ ID NO:41; (c) SEQ ID NO:47 or a polypeptide having at least 75% sequence identity to SEQ ID NO:47; (d) SEQ ID NO:48 or a polypeptide having at least 75% sequence identity to SEQ ID NO:48; and (e) SEQ ID NO: 43 or a polypeptide having at least 75% sequence identity to SEQ ID NO: 43 a polyphenol oxidase selected from the group consisting of: (C) (a) SEQ ID NO: 151, or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 151; (b) SEQ ID NO: 152 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 152; (c) SEQ ID NO: 158 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 158; (d) SEQ ID NO: 159 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 159; and (e) SEQ ID NO: 154 or a polynucleotide having at least 75% sequence identity to SEQ ID NO: 154 or comprising a polynucleotide sequence selected from the group consisting of: (D) (a) Accession number Ma06_31080 (PPO1); (b) Accession number Ma07_03540 (PPO2); (c) Accession number Ma08_34740 (PPO8); (d) Accession No. Ma10_20510 (PPO9); and (e) Accession number Ma08_09150 (PPO4) and a sequence characterized by: Optionally, the one or more banana polyphenol oxidase guide RNAs comprise: (a) SEQ ID NO: 32; (b) SEQ ID NO: 33; (c) SEQ ID NO: 34; (d) SEQ ID NO: 35; (e) SEQ ID NO: 57; (f) SEQ ID NO: 58; (g) SEQ ID NO: 38; (h) SEQ ID NO: 39; (i) SEQ ID NO: 62; (j) SEQ ID NO: 76; (k) SEQ ID NO: 77; and (l) Any combination of (a) to (k) and / or comprising a variable region having a sequence selected from the group consisting of The CRISPR-associated endonuclease is a Cas9 endonuclease. A recombinant DNA construct capable of binding to at least one endogenous banana PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene and generating a double-strand break or a single-strand break.

Images