AU721557B2 - Polyphenol oxidase genes from lettuce and banana - Google Patents

Description

WO 96/37617 PCTAU96/00310 -1- POLYPHENOL OXIDASE GENES FROM LETTUCE AND BANANA The present invention relates to the isolation of genes encoding polyphenol oxidase (PPO) from plants.

Browning of plant tissues often occurs following injury or damage and this generally results in spoilage of fruit and vegetables. Undesirable browning also occurs during processing of plant materials to produce food or other products.

Steps are taken during transport, storage, and processing to prevent these browning reactions. Often this involves the use of chemicals such as sulphur dioxide but the use of these substances is likely to be restricted in the future due to concerns about their safety and consumer acceptance. For example, the US Food and Drug Administration banned the use of sulphite for most fresh fruit and vegetables in 1986. The production of fruit and vegetable varieties with an inherently low susceptibility to brown would remove the need for these chemical treatments.

It will be understood that browning in plants is predominantly catalysed by the enzyme PPO. PPO is localised in the plastids of plant cells whereas the phenolic substrates of the enzyme are stored in the plant cell vacuole. This compartmentation prevents the browning reaction from occurring unless the plant cells are damaged and the enzyme and its substrates are mixed.

The prior art includes International Application PCT/AU92/00356 to the present applicant which describes the cloning of PPO genes from grapevine, broad bean leaf, apple fruit and potato tuber. This application recognises that PPO levels in plants may be manipulated by increasing or decreasing expression of PPO gene. The application also identifies two conserved copper binding sites in PPO genes, designated CuA and CuB. However, the method described in PCT/AU92/00356 which was used to clone the PPO genes from apple and potato involved the use of an oligo dT reverse primer for polymerase chain reaction (PCR). Whilst the method is acceptable, in some tissues, it does not give rise to a strong band of the predicted size or else it gives rise to many additional products making it difficult to resolve the PPO fragment.

Accordingly, it is an object of the present invention to overcome or at least alleviate one or more of the difficulties related to the prior art.

WO 96/37617 PCT/AU96/00310 -2- In a first aspect of the present invention there is provided a method for preparing nucleic acid encoding PPO, fragments and derivatives thereof, which method includes providing a source of a polypeptide having PPO activity, a first primer having a sequence corresponding to a first conserved region of PPO in sense orientation, and a second primer having a sequence corresponding to a second conserved region of PPO in antisense orientation; isolating RNA from the source of polypeptide having PPO activity; treating the RNA to construct copy DNA (cDNA) therefrom; and amplifying the cDNA so formed using the first and second primers.

Applicant has found that the method of the present invention, which involves the use of a second primer based on PPO, means that there is less likelihood that other (non-PPO) genes are amplified. Furthermore, the method of the present invention dramatically increases the amount of genuine product formed in most cases. Moreover, the added specificity provided by the second PPO-based primer makes it possible to clone PPO more readily from certain plants in which it was difficult to obtain a clone using one primer and oligo-dT.

For example, with lettuce cDNA the applicant saw only a faint smear of a range of products with GEN3/GEN8 and oligo-dT but strong bands of the predicted size with GEN3/GEN8 and REV1.

The terms "nucleic acid encoding banana/lettuce PPO" and "banana/lettuce PPO gene" as used herein should be understood to refer to a banana/lettuce PPO gene or a sequence substantially homologous therewith.

For example, these terms include sequences which differ from the specific sequences given in the Examples hereto but which, because of the degeneracy of the genetic code, encode the same protein. Applicants have found that there are families of PPO genes in most plants. Thus, there are likely to be other PPO genes in lettuce and banana, in addition to those which have been isolated.

These could be cloned using the methods of the present invention. Thus, the terms "nucleic acid encoding banana/lettuce PPO" and "banana/lettuce PPO WO 96/37617 PCT/AU96/00310 -3gene" should be understood to include banana/lettuce PPO genes other than those specific genes that have been isolated. The terms may also include presequences such as chloroplast transit sequence as well as sequences encoding mature PPO protein.

The term "derivative" as used herein includes nucleic acids that have been chemically or otherwise modified, for example mutated, or labelled, or nucleic acids incorporating a catalytic cleavage site.

The term "fragment" includes functionally active fragments of a PPO gene which encode a polypeptide or peptide having PPO activity.

The source of polypeptide having PPO activity is preferably a source of polypeptide having banana or lettuce PPO activity. The source of polypeptide having banana PPO activity may be banana fruit, preferably young banana fruit, more preferably the flesh of young banana fruit. The source of polypeptide having lettuce PPO activity may be lettuce leaves, preferably young lettuce leaves.

The RNA may be isolated by any suitable method including extraction for example with a detergent such as CTAB, use of an oligo-dT spun column as described in PCT/AU92/00356 the entire disclosure of which is incorporated herein by reference, or use of a commercially available kit such as the PolyATtract 1000 system from Promega Corporation.

The step of treating the RNA to construct cDNA according to this aspect of the present invention may include treating the RNA with reverse transcriptase and an adapter primer to form cDNA.

The adapter primer may be an oligonucleotide adapter primer including the following sequence or part thereof: I 1 I 1I 1 I II I 1 1 1-3' The first primer has a sequence corresponding to a first conserved region of PPO. Preferably the first primer has a sequence corresponding to at least a portion of or in close proximity to a first copper binding site of PPO. The second primer has a sequence corresponding to a second conserved region of PPO.

Preferably the second primer has a sequence corresponding to at least a portion WO 96/37617 PCr/AU96/00310 -4of or in close proximity to a second copper binding site of PPO. More preferably the first primer has a sequence corresponding to at least a portion of or in close proximity to one of the CuA or CuB binding sites of PPO, and the second primer has a sequence corresponding to at least a portion of or in close proximity to the other of the CuA or CuB binding sites of PPO.

The first and second primers may be degenerate. The first primer may include one of the following sequences or part thereof: 5'-GCGAATTCTT[TC][TC]TICCITT[TC]CA[TC][AC]G-3' 5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3'.

The second primer may include the following sequence or part thereof 5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3'.

The cDNA may be amplified using the polymerase chain reaction (PCR).

Those skilled in the art will appreciate that if the Cu binding sites are internal, the nucleic acid isolated will be a fragment of the PPO gene lacking 3' and 5' termini. However, it is possible to determine the complete nucleic acid sequence of the PPO gene and to prepare or isolate nucleic acid encoding such PPO or antisense to such PPO.

Accordingly, in a further aspect of the present invention there is provided a method for preparing nucleic acid encoding the 3' end of PPO, which method includes providing a source of polypeptide having PPO activity a primer in sense orientation; and an adapter primer; isolating RNA from the source of polypeptide having PPO activity; treating the RNA to construct cDNA therefrom; and amplifying the cDNA so formed using the primers.

In a further aspect of the present invention there is provided a method for preparing nucleic acid encoding the 5' end of PPO, which method includes providing a source of polypeptide having PPO activity, an anchor, WO 96/37617 PCT/AU96/00310 primers in antisense orientation; and an anchor primer; isolating RNA from the source of polypeptide having PPO activity; treating the RNA to construct cDNA therefrom; attaching the anchor to the 5' end of the cDNA so formed; and amplifying the cDNA using the primers.

The source of polypeptide having PPO activity is preferably a source of polypeptide having banana or lettuce PPO activity. The source of polypeptide having banana PPO activity may be banana fruit, preferably young banana fruit, more preferably the flesh of young banana fruit. The source of polypeptide having lettuce PPO activity may be lettuce leaves, preferably young lettuce leaves.

The RNA may be isolated by any suitable method including extraction for example with a detergent such as CTAB, use of an oligo-dT spun column as described in PCT/AU92/00356 the entire disclosure of which is incorporated herein by reference, or use of a commercially available kit such as the PolyATtract 1000 system from Promega Corporation.

The step of treating the RNA to construct cDNA according to this aspect of the present invention may include treating the RNA with reverse transcriptase and an adapter primer to form cDNA.

The adapter primer may be an oligonucleotide adapter primer including the following sequence or part thereof: I III I II I I I I I I 1 -3' The primer in sense orientation may be a lettuce PPO specific primer. The primer in sense orientation may include the following sequence or part thereof: 5'-CGCTGGGTGGGTAATTCTAGGATG-3'.

The primer in sense orientation may be a banana PPO specific primer.

The primer in sense orientation may include the following sequence or part thereof: 5'-AGTCATCCACAATGCGGCGCACATG-3'.

WO 96/37617 PC/AU96/00310 -6- The adapter primer may include the following sequence or part thereof: 5'-GACTCGAGTCGACATCG-3'.

The primers in antisense orientation may be lettuce PPO specific primers.

The primers in antisense orientation may include the following sequences or part thereof: 5'-TGCTGTTCTGTTCGAACATGGCAG-3' 5'-TATACAAGTGGCACCAGTGTCTGC-3'.

The primers in antisense orientation may be banana PPO specific primers.

The primers in antisense orientation may include the following sequences or part thereof: 5'-CCGCATTGTGGATGACTTCCATCTG-3' 5'-CCAGAATGGGATGGTGAAGGTGTCG-3'.

The anchor may be of any suitable type. The anchor may be attached by ligation for example using T4 RNA ligase. The anchor primer should be capable of hybridizing with the anchor.

The cDNA may be amplified using PCR.

Those skilled in the art will appreciate that using the methods of the present invention it is possible to determine the complete nucleic acid sequence of the PPO gene of interest and to prepare or isolate nucleic acid encoding such PPO or antisense to such PPO.

In a further aspect of the present invention, there is provided a nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Fig. 2 or Fig. 3, fragments and derivatives thereof, and substantially homologous sequences.

In a further aspect of the present invention, there is provided a nucleic acid encoding lettuce PPO or antisense to lettuce PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Fig. 1, fragments and derivatives thereof, and substantially homologous sequences.

The nucleic acid may be prepared by a method as hereinbefore described.

The nucleic acid may be modified, for example by inclusion of a catalytic cleavage site.

WO 96/37617 PCT/AU96/00310 -7- In a further aspect of the present invention there is provided a method for preparing a recombinant vector including a nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof, which method includes providing nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof; and a vector; and reacting the nucleic acid and the vector to deploy the nucleic acid within the vector.

In a further aspect of the present invention there is provided a method for preparing a recombinant vector including a nucleic acid encoding lettuce PPO or antisense to lettuce PPO, fragments and derivatives thereof, which method includes providing nucleic acid encoding lettuce PPO or antisense to lettuce PPO, fragments and derivatives thereof; and a vector; and reacting the nucleic acid and the vector to deploy the nucleic acid within the vector.

The nucleic acid may be prepared by a method as hereinbefore described.

The nucleic acid may be modified, for example by inclusion of a catalytic cleavage site.

The vector may be a plasmid expression vector. For example Bluescript SK has been found to be suitable. Alternatively, the vector may be a binary vector. The recombinant vector may contain a promoter, preferably a constitutive promoter upstream of the nucleic acid.

The cloning step may take any suitable form. A preferred form may include fractionating the cDNA, for example on a column or a gel; isolating a fragment of the expected size, for example from the column or gel; and WO 96/37617 PCr/AU96/00310 -8ligating said fragment into a suitable restriction enzyme site of the vector, for example the EcoRV site of a Bluescript SK' vector.

In order to test the clones so formed, a suitable microorganism may be transformed with the vector, the microorganism cultured and the polypeptide encoded therein expressed. The microorganism may be a strain of Escherichia coli, for example E.coli DH5 has been found to be suitable. Alternatively, appropriate vectors may be used to transform plants.

In a further aspect of the present invention there is provided a recombinant vector including a nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof, which vector is capable of being replicated, transcribed and translated in a unicellular organism or alternatively in a plant.

In a further aspect of the present invention there is provided a recombinant vector including a nucleic acid encoding lettuce PPO or antisense to lettuce PPO, fragments and derivatives thereof, which vector is capable of being replicated, transcribed and translated in a unicellular organism or alternatively in a plant.

The nucleic acid may be prepared by a method as hereinbefore described.

The nucleic acid may be modified, for example by inclusion of a catalytic cleavage site.

The vector may be a plasmid expression vector. For example Bluescript SK' has been found to be suitable. Alternatively, the vector may be a binary vector. The recombinant vector may contain a promoter, preferably a constitutive promoter upstream of the nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof.

The microorganism may be a strain of Escherichia coli, for example E.coli has been found to be suitable.

In a further aspect of the present invention there is provided a method of decreasing the level of PPO activity in a plant tissue, which method includes providing a nucleic acid encoding banana PPO, a modified nucleic acid encoding banana PPO, or a .nucleic acid antisense to banana PPO, fragments and derivatives thereof; and WO 96/37617 PCT/AU96/00310 -9a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.

In a further aspect of the present invention there is provided a method of decreasing the level of PPO activity in a plant tissue, which method includes providing a nucleic acid encoding lettuce PPO, a modified nucleic acid encoding lettuce PPO, or a nucleic acid antisense to lettuce PPO, fragments and derivatives thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.

PPO activity may be decreased by the use of sense constructs (cosuppression). Alternatively the nucleic acid may include a sequence encoding antisense mRNA to banana or lettuce PPO or a functionally active fragment thereof. Alternatively the nucleic acid may encode banana or lettuce PPO or a functionally active fragment thereof and incorporate a catalytic cleavage site (ribozyme). The nucleic acid may be included in a recombinant vector as hereinbefore described. In a preferred aspect, the nucleic acid may be included in a binary vector. In a further preferred aspect, the introduction of a binary vector into the plant may be by infection of the plant with an Agrobacterium containing the binary vector or by bombardment with nucleic acid coated microprojectiles. Methods for transforming banana with Agrobacterium are known to those skilled in the art and are described in, for example, May et al., Bio/technology (1995) 13:486-492, the entire disclosure of which is incorporated herein by reference. Methods for transforming banana by bombardment with DNA coated microprojectiles are known to those skilled in the art and are described in, for example, Sagi et al., Bio/technology (1995) 13:481-485, the entire disclosure of which is incorporated herein by reference. Methods for transforming lettuce using Agrobacterium are known to those skilled in the art and are described in, for example, Michelmore et al., Plant Cell Reports (1987) 6:439-442, and Curtis et al., Journal of Experimental Botany (1994) WO 96/37617 PCT/AU96/00310 45:1141-1149.

In a further aspect of the present invention there is provided a method of increasing the level of PPO activity in a plant tissue, which method includes providing a nucleic acid encoding banana PPO or a fragment thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.

In a further aspect of the present invention there is provided a method of increasing the level of PPO activity in a plant tissue, which method includes providing a nucleic acid encoding lettuce PPO or a fragment thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.

The nucleic acid may be included in a recombinant vector as hereinbefore described. In a preferred aspect, the nucleic acid may be included in a binary vector. In a further preferred aspect, the introduction of the binary vector into the plant may be by infection of the plant with an Aqrobacterium containing the binary vector or by bombardment with nucleic acid coated microprojectiles.

The plant may be of any suitable type. However the method is particularly applicable to banana or lettuce.

In a further aspect of the present invention there is provided a transgenic plant, which plant contains nucleic acid capable of modifying expression of the normal banana PPO gene.

The plant may be of any suitable type. Preferably, the plant is banana.

In a further aspect of the present invention there is provided a transgenic plant, which plant contains nucleic acid capable of modifying expression of the normal lettuce PPO gene.

The plant may be of any suitable type. Preferably, the plant is lettuce.

The nucleic acid may be as hereinbefore described.

P:\OPER\MRO\56803-96.CLM- 17/1/00 11 Those skilled in the art will be aware that means for inhibiting, interrupting or otherwise reducing the expression of a PPO polypeptide include means which target transcription and/or mRNA stability and/or mRNA turnover and/or accessibility of mRNA to ribosomes or polysomes. Such means include the use of antisense molecules and ribozyme molecules and the like introduced into the cell in an expressible format and expressed therein.

In the context of the present invention, an antisense molecule is an RNA molecule which is transcribed from the complementary strand of a nuclear PPO gene to that which is normally transcribed to produce a "sense" mRNA molecule capable of being translated into a PPO polypeptide. The antisense molecule is therefore complementary to the sense mRNA, or a part thereof. Although not limiting the mode of action of the antisense molecules of the present invention to any specific mechanism, the antisense RNA molecule possesses the capacity to form a double-stranded mRNA by base pairing with the PPO-encoding sense mRNA, which may prevent translation of the sense mRNA and subsequent synthesis of a PPO polypeptide product.

Ribozymes are known by those skilled in the art as RNA molecules which comprise a

U

hybridising region complementary to two regions, each of at least 5 contiguous nucleotide bases in the target sense mRNA. In addition, ribozymes possess highly specific endoribonuclease activity, which autocatalytically cleaves the target sense mRNA. A complete description of the function of ribozymes is contained in International Patent Publication No. W089/05852.

The present invention extends to a ribozyme or antisense molecule comprising a sequence of contiguous nucleotide bases which are able to form a hydrogen-bonded complex with a sense mRNA encoding a PPO polypeptide described herein, to reduce translation of said mRNA.

As will be known to those skilled in the art, preferred antisense or ribozyme molecules hybridise to at least about 10 to 20 nucleotides of the target molecule and preferably to the full-length or substantially full-length PPO mRNA.

In yet a further embodiment of the invention, expression of a PPO polypeptide may be P:\OPER\MRO\56803-96.CLM 17/1/00 -12inhibited, interrupted or otherwise reduced by introducing to the cell a sense molecule, in particular a co-suppression molecule, in an expressible format and expressing said molecule therein.

The term "sense molecule" as used herein shall be taken to refer to an isolated nucleic acid molecule which encodes or is complementary to an isolated nucleic acid molecule which encodes a banana or lettuce PPO polypeptide or a homologue, analogue or derivative thereof, wherein said nucleic acid molecule is provided in a format suitable for its expression to produce a recombinant polypeptide when said sense molecule is introduced into a host cell by transfection or transformation.

A "co-suppression molecule" is a sense molecule which is capable of producing cosuppression when introduced and optionally, expressed in a cell. Co-suppression is the reduction in expression of an endogenous gene that occurs when one or more copies of said gene, or one or more copies of a substantially similar gene are introduced into the cell. The present invention clearly extends to the use of co-suppression to inhibit the expression of a PPO gene as described herein.

S. It is understood in the art that certain modifications, including nucleotide substitutions amongst others, may be made to the co-suppression molecule, antisense, or ribozyme molecule, without destroying the efficacy of said molecules in inhibiting the expression of the PPO gene. It is therefore within the scope of the present invention to include any nucleotide *0 :4 sequence variants, homologues, analogues, or fragments of the said gene encoding same.

However, in the case of ribozymes and antisense molecules, those skilled in the art will be aware that it is necessary for such nucleotide sequence variants to be capable of hybridising .25 to the biologically active PPO gene sequence or to sense mRNA encoded therefor.

a As will be known to those skilled in the art, the efficacy of an antisense molecule or a ribozyme molecule or a co-suppression molecule is dependent upon it being introduced and preferably, expressed in the cells, tissues or organs of a banana or lettuce plant, or a 30 progenitor cell, tissue or organ thereof. Such introduction and expression may be facilitated P:\OPER\MRO\56803-96.CLM 17/1/00 13by presenting said antisense molecule or ribozyme molecule or co-suppression molecule in a genetic construct. Accordingly, the present invention clearly extends to the use of genetic constructs designed to facilitate the introduction and/or expression of an antisense molecule, ribozyme molecule, or co-suppression molecule in a plant cell.

Those skilled in the art will also be aware that expression of an antisense, ribozyme, or cosuppression molecule may require said molecule to be placed in operable connection with a promoter sequence. The choice of promoter for the present purpose may vary depending upon the level of expression required and/or the tissue, organ and species in which expression is to occur.

Those skilled in the art are aware that a "promoter" includes the transcriptional regulatory sequences of a classical eukaryotic genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Promoters may contain additional copies of one or more specific regulatory elements to further enhance expression and/or to alter the spatial expression and/or temporal expression :of a nucleic acid molecule to which it is operably connected. Placing a nucleic acid molecule 1* under the regulatory control of a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence. A promoter is usually, but not necessarily, positioned upstream or 5' of a nucleic acid molecule which it regulates.

Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of a sense, antisense, ribozyme, or co-suppression molecule, or a chimeric gene comprising same. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a •distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a fi 0-A, regulatory sequence element with respect to a heterologous gene to be placed under its control P:\OPER\MRO\56803-96.CLM 17/1/00 -14is defined by the positioning of the element in its natural setting, the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.

Examples of promoters suitable for use in genetic constructs of the present invention include promoters derived from the genes of viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants which are capable of functioning in plant cells, in particular the cells of banana or lettuce plants. Examples of promoters useful in performing this embodiment include the well-known CaMV 35S promoter, NOS promoter, octopine synthase (OCS) promoter, Arabidopsis thaliana SSU gene promoter, the meristem-specific promoter (meril),napin seed-specific promoter, and the like. In addition to the specific promoters identified herein, cellular promoters for so-called housekeeping genes are useful. Such promoters will be known to those skilled in the art.

The genetic construct may further comprise a terminator sequence and be introduced into a suitable host cell where it is capable of being expressed. The term "terminator" refers to .15 a DNA sequence at the end of a transcriptional unit which signals termination of transcription.

Terminators are 3'-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript.

Terminators active in cells derived from viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants.

n Examples of terminators known to those skilled in the art which are particularly suitable for use in the genetic constructs of the present invention include the nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator from Zea mays, the Rubisco small subunit (SSU) gene terminator sequences and subclover stunt virus (SCSV) gene sequence terminators, amongst others. Those skilled in the art will be aware of additional promoter sequences and terminator sequences which may be suitable for use in performing the invention. Such sequences may readily be used without any undue experimentation.

P:\OPER\MRO\56803-96.CLM 17/1/00 The genetic constructs of the invention may further include an origin of replication sequence which is required for replication in a specific cell type, for example a bacterial cell, when said genetic construct is required to be maintained as an episomal genetic element (eg. plasmid or cosmid molecule) in said cell. Preferred origins of replication known to those skilled in the art include, but are not limited to, thefl-ori and colEl origins of replication.

The genetic construct may further comprise a selectable marker gene or genes that are functional in a cell into which said genetic construct is introduced. As used herein, the term "selectable marker gene" includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof. Suitable selectable marker genes known to those skilled in the art and contemplated herein include the ampicillin resistance (Ampr), tetracycline resistance gene (Irc), bacterial kanamycin resistance gene (Kanr), phosphinothricin resistance gene, neomycin phosphotransferase gene (nptl), hygromycin resistance gene, P-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene and luciferase gene, amongst others.

:A sense or antisense molecule which comprises the inventive nucleotide sequences or a genetic construct comprising same, may be introduced into a plant cell using any known method for the transfection or transformation of plants. Whole plants may be regenerated from a single transformed plant cell, using any method known to those skilled in the art.

o By "transfect" is meant that the sense or antisense molecule or genetic construct comprising same is introduced into said cell without integration into the plant cell's genome.

n By "transform" is meant that the sense or antisense molecule or genetic construct comprising same or a fragment said genetic construct comprising the PPO-encoding gene sequence is stably integrated into the genome of the plant cell.

R\ Means for introducing recombinant DNA into plant tissue or cells include, but are not limited to direct DNA uptake into protoplasts (Krens et al, Nature 296, 72-74,1982; Paszkowski et P:\OPER\MRO\56803-96.CLM 17/1/00 -16al, EMBO J. 3, 2717-2722,1984), microparticle bombardment, electroporation (Fromm et al., Proc. Natl. Acad. Sci. (USA) 82,5824-5828, 1985), microinjection of DNA (Crossway et al., Mol. Gen. Genet. 202,179-185, 1986), microparticle bombardment of tissue explants or cells (Christou et al, Plant Physiol 87, 671-674, 1988), vacuum-infiltration of tissue with nucleic acid, or T-DNA-mediated transfer from Agrobacterium to the plant tissue as described essentially by An et al., EMBO J. 4, 277-284,1985; Herrera-Estrella et al., Nature 303, 209- 213, 1983; Herrera-Estrella et al. EMBO J. 2, 987-995, 1983; and Herrera-Estrella et al., In: Plant Genetic Engineering, Cambridge University Press, NY, pp 63-93, 1985.

For microparticle bombardment of cells, a microparticle is propelled into a cell to produce a transformed cell. Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp et al. Patent No. 5,122,466) and Sanford and Wolf Patent No. 4,945,050). When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.

1.5.

Examples of microparticles suitable for use in such systems include 1 to 5 jim gold spheres.

The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.

Plant tissue capable of subsequent clonal propagation, whether by organogenesis or oo embryogenesis, may be transformed with a genetic construct containing the PPO-encoding sequence of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue apical meristem, axillary buds, and root meristems), and induced meristem tissue cotyledon meristem and hypocotyl meristem).

K,4 RThe term "organogenesis", as used herein, means a process by which shoots and roots are 0 developed sequentially from meristematic centres.

P:\OPER\MRO\56803-96.CLM 18/1/00 -17- The term "embryogenesis", as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.

The regenerated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformant, and the T2 plants further propagated through classical breeding techniques.

The regenerated transformed organisms contemplated herein may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants all cells transformed contain the expression cassette); grafts of transformed and untransformed tissues in plants, a transformed root stock grafted to an untransformed scion).

t Numerous modifications and variations of the present invention are possible in light of the •above teachings and, therefore, within the scope of the appended claims, the invention may be practised otherwise than as particularly described.

o Throughout this specification and the claims which follow, unless the context requires 20: otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but oeee*[ not the exclusion of any other integer or step or group of integers or steps.

The present invention is more fully described with reference to the accompanying Figures and Examples. It should be understood that the description following is illustrative only and should not be taken in any way as limiting the generality of the invention described herein.

P:\OPER\MRO\56803-96.CLM 18/1100 -18- In the Figures: Figure 1 is a representation showing the nucleotide sequence of the LPO1 cDNA clone and derived amino acid sequence therefor, including the putative chloroplast transit sequence and mature lettuce PPO amino acid sequence.

Figure 2 is a representation showing the nucleotide sequence of the BANPPO1 cDNA clone and derived amino acid sequence therefor, including the putative chloroplast transit sequence and mature banana PPO amino acid sequence.

Figure 3 is a representation showing the nucleotide sequence of the BANPPO11 cDNA clone and derived amino acid sequence therefor, including part of the banana PPO amino acid sequence.

Figure 4 is a representation showing the nucleotide sequence of the BPPO2 cDNA clone and derived amino acid sequence therefor, including part of the banana PPO amino acid sequence.

Figure 5 is a representation showing the nucleotide sequence of the BPPO8 cDNA clone and derived amino acid sequence therefor, including part of the banana PPO amino acid sequence.

EXAMPLE 1 *Cloning Lettuce PPO Genes Messenger RNA (mRNA) was isolated directly from young leaves of lettuce using the PolyATract 1000 system from Promega Corporation. First strand cDNA was synthesised with reverse transcriptase using a Timesaver cDNA synthesis Kit (Pharmacia Biotech) utilising an oligo-dT primer adapter as described by Frohman, MA (1990) in PCR Protocols :A Guide to Methods and Applications" (MA Innis, DH Gelfrand, JJ Sninsky and TJ White, eds)Academic Press, New York pp28-38, the entire disclosure of which is incorporated herein by reference: B26: GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3').

P:\OPERNMRM\56803-96.CLM 1317/99 -19- Oligonucleotide primers were designed based on known plant PPO DNA sequences in the conserved regions of the gene which encode the copper binding sites, CuA and CuB as described in Dry, IB and Robinson, SP (1994) "Molecular cloning and characterisation of grape berry polyphenol oxidase", Plant Molecular Biology 26 495-502, the entire disclosure of which is incorporated herein by reference. Two forward primers designed around the CuA site (GEN3 and GEN8) and one reverse primer designed around the CuB site (REV1) were synthesised: GEN3 (5'-GCGAATTCTT[TC][TC]TICCITT[TC]CA[TC]AClG-3') GEN8 (5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3') REV1 (5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3') Although the primers are in the region of the Cu binding sites, one of them (GEN8) is just outside of what is traditionally accepted to be a Cu binding site of the enzyme.

15 The first strand cDNA was amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman using GEN3 and REV1 or GEN8 and REV1 primers, each at a final concentration of 1gM (Dry et al.).

Amplification involved an initial program of 2 cycles of denaturation at 94 0 C for 1 min, annealing at 37 0 C for 2 min, a slow ramp to 72 0 C over 2 min and elongation at 72 0 C for 3 min, followed by 25 cycles of denaturation at 94 0 C for 1 min, annealing at 55*C for 1 min, and elongation at 72 0 C for 3 min. A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products and the remainder was purified and concentrated using PCR Wizard Prep columns (Promega Corporation).

25 The purified DNA was cloned into Eco RV-cut Bluescript SK' vector (Stratagene) which had been T-tailed with Taq Polymerase and the ligated DNA was introduced into E.coli DH5a by electroporation. Recombinant clones which had an insert of the predicted size were selected and their DNA sequence was determined by automated sequencing. Three putative lettuce PPO clones S(LP0316, LPO812 and LP0813) were identified based on their homology to u known plant PPO genes.

P:OPER\MRO56803-%.CLM 137/99 Using this sequence information a specific forward primer (LET3P) and two reverse primers (LET5P1 and LET5P2) were synthesised: LET3P (5'-CGCTGGGTGGGTAATTCTAGGATG'3-) LET5P1 (5'-TGCTGTTCTGTTCGAACATGGCAG-3') LET5P2 (5'-TATACAAGTGGCACCAGTGTCTGC-3') To obtain the 3'-end of the lettuce PPO gene, the first strand cDNA described above was amplified by the same PCR procedure using 1M LET3P primer and 100 nM adapter primer: (5'-GACTCGAGTCGACATCG-3').

The amplified cDNA was purified as described above and run on a 2% Nusieve GTG (FMC Bioproducts) agarose gel. A 1000bp fragment was excised from the gel and the DNA was cloned into T-tailed, Eco RV-cut Bluescript SK to yield the end clones LPO9 and LPO10, which were sequenced.

The 5'-end of the lettuce PPO gene was cloned by a modification of the 15 RACE procedure originally described by Frohman using a 5'-AmpliFINDER RACE kit (Clontech Laboratories). First strand cDNA was synthesised from mRNA with reverse transcriptase using the LET5P2 primer and an AmpliFINDER anchor was ligated onto the 5'-end of the cDNA. The cDNA was amplified by PCR with LET5P1 primer and the AmpliFINDER anchor primer. The amplified cDNA was 20 purified as described above and run on a 2% -eve GTG (FMC Bioproducts) o agarose gel. An 850bp fragment was excised from the gel and the DNA was cloned into T-tailed Eco RV-cut Bluescript SK* to give the 5'-end clones LPO4, LP05, LPO6, and LPO7, which were sequenced.

The and 3'-clones were found to have the predicted overlapping 25 sequences with the original clone and the complete sequence of lettuce PPO (LPO1) was derived by combining the sequences from the various clones (Figure P:\OPER\MRM\56803-%.CLM 1317199 -21 EXAMPLE 2 Cloning Banana PPO Genes Total RNA was isolated from young banana fruit. Fruit tissue (3g) was frozen and ground to a fine powder in liquid nitrogen with a coffee grinder then added to 20 ml of extraction buffer hexadecyltrimethylammonium bromide (CTAB), 2% polyvinyl pyrolidone, 100 mM Tris-HCI, pH 8.0, 25 mM EDTA, 2 M NaCI, 0.05% spermidine, 2% p-mercaptoethanol) at 65 0 C. The extract was mixed with 20 ml of chloroform IAA then centrifuged for 20 minutes at 5,000 RPM and the aqueous phase was re-extracted with chloroform IAA. The aqueous phase was filtered through Miracloth and 0.25 volumes of 10 M LiCI were added then the sample was incubated overnight at 4 0 C before centrifuging for 20 minutes at 8,000 RPM. The supematant was removed and the pellet was resuspended in ml of 1 M NaCI, 0.5% SDS, 10 mM Tris, pH 8.0, 1 mM EDTA. The RNA was extracted once with an equal volume of chloroform IAA and 2 volumes of ethanol was added. After incubation for 40 mins at -70 0 C the solution was centrifuged for 15 minutes at 10,000 RPM The supematant was removed and the pellet was rinsed with 80% ethanol, drained, and dried. The pellet was resuspended in 50 ill of sterile water.

First strand cDNA was synthesised from 10 I.g total RNA with reverse transcriptase as described in Dry, I.B. and Robinson, S.P. (1994) "Molecular cloning and characterisation of grape berry polyphenol oxidase", Plant Molecular Biology 26 495-502, the entire disclosure of which is incorporated herein by reference, utilising an oligo-dT primer adapter (Frohman, M.A. (1990) in "PCR Protocols A Guide to Methods and Applications" Innis, D.H. Gelfrand, J.J.

25 Sninsky and T.J. White, eds.) Academic Press, New York pp 28-38, the entire disclosure of which is incorporated herein by reference): B26 (5'-GACTCGAGTCGACATCGAI II I I I I II I l I1-3') Oligonucleotide primers were designed based on known plant PPO DNA sequences in the conserved regions of the gene which encode the copper binding RA sites, CuA and CuB (Dry et A forward primer designed around the CuA site (GEN3) and a reverse primer designed around the CuB site (REV1) were synthesised P:\OPER\MRO\568039%.CLM 13/7/99 -22- GEN3: (5'-GCGAATTCTC][TC]TICCITT[TC]CA[TC][ACIG-3') REV1 (5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3') The first strand reaction was amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman using GEN3 and REV1 primers, each at a final concentration of 1 pM (Dry et Amplification involved an initial program of 2 cycles of denaturation at 940 C for 1 min, annealing at 370 C for 2 min, a slow ramp to 720 C over 2 min and elongation at 720 C for 3 min, followed by 25 cycles of denaturation at 940 C for 1 min, annealing at 550 C for 1 min, and elongation at 720 C for 3 min. A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products and the remainder was purified and concentrated using

PCR

Wizard Prep columns (Promega Corporation).

The purified DNA was cloned into Eco RV-cut Bluescript SK' vector (Stratagene) which had been T-tailed with Taq Polymerase and the ligated

DNA

15 was introduced into E. coli DH5a by electroporation. Recombinant clones which had an insert of the predicted size were selected and their DNA sequence was determined by automated sequencing. A putative banana PPO clone (BPO3) was identified based on its homology to known plant PPO genes.

Using this sequence information a specific forward primer (BAN1) and two 20 specific reverse primers (BAN2R and BAN3R) were synthesised: BAN 1: (5'-AGTCATCCACAATGCGGCGCACATG-3') BAN2R BAN2R: (5'-CCGCATTGTGGATGACTTCCATCTG-3') BAN3R: (5'-CCAGAATGGGATGGTGAAGGTGTCG-3') To obtain the 3'-end of this banana PPO gene, the first strand cDNA 25 described above was amplified by the same PCR procedure using 1 M BAN1 primer and 100nM adapter primer: (5'-GACTCGAGTCGACATCG- 3 The DNA was amplified using 25 cycles of denaturation at 94 0 C for 1 min, RA annealing at 55 0 C for 1 min, and elongation at 72 0 C for 3 min. The amplified A DNA was purified using a QlAquick Spin PCR Purification Kit (QIAGEN) and run on a 2% Nusieve GTG (FMC Bioproducts) agarose gel. A 1000bp fragment was S excised from the gel and the DNA was cloned into T-tailed Eco RV-cut Bluescript P:\OPER\MRO\6803-%.CLM -13/7/99 -23- SK to yield the 3'-end clone BPO17, which was sequenced and shown to encode the 3'-end of BPO3.

The 5'-end of BPO3 was cloned by a modification of the 5'-RACE procedure originally described by Frohmann. First strand cDNA was synthesised from banana fruit RNA as described above but utilising the banana PPO specific primer BAN2R. The DNA was tailed with Terminal transferase as described in Frohmann and amplified by PCR with BAN3R and B26 primers, each at a final concentration of 1lM. The DNA was amplified using 30 cycles of denaturation at 94 0 C for 1 min, annealing at 55°C for 1 min, and elongation at 72 0 C for 3 min.

The amplified DNA was run on a 1.8% Nusieve GTG (FMC Bioproducts) agarose gel and a 700bp fragment was excised from the gel. The DNA was extracted with a QIAquick Gel Extraction Kit and cloned into T-tailed Eco RV-cut Bluescript SK* to yield the 5'-end clone BP026 which was sequenced and shown to encode the 5'-end of BPO3.

The overlapping clones BPO3, BPO17 and BP026 were fully sequenced in both directions and the sequence of this banana PPO gene (BANPPO1) was derived by combining the sequences (Figure 2).

In the course of these experiments a number of clones were obtained from the banana fruit cDNA by PCR amplification using the B25 primer with one of the degenerate primers based on conserved sequences in other plant PPO genes: ."GEN7 (5'-GCGAATTCAA[TC]GTIGA[TC][AC]GIATTGTG-3') using the methods described above. Most of thes clones were identical to BANPPOI but one clone, designated BANPPO11, was found to be distinctly different and its sequence is shown in Figure 3.

25 EXAMPLE 3 Cloning Banana Peel PPO genes Total RNA was isolated from the peel of young banana fruit. Fruit tissue (3g) was frozen and ground to a fine white powder in liquid nitrogen with a coffee grinder then added to 20 ml of extraction buffer (2% T RA hexadeycitrimethylammonium bromide (CTAB), 2% polyvinyl pyrolidone, 100 mM Tris-CHI, pH 8.0, 25 mM EDTA, 2 M NaCI, 0.05% spermidine, 2% Pmercaptoethanol) at 65C. The extract was mixed with 20 ml of chloroformllAA P:\OPER\MRO\56803-96CLM 1317199 -24then centrifuged for 20 minutes at 5,000 RPM and the aqueous phase was reextracted with chloroform/IAA. The aqueous phase was filtered through Miracloth and 0.25 volumes of 10 M LiCI were added then the sample was incubated overnight at 4C before centrifuging for 20 minutes at 8,000 RPM. The supematant was removed and the pellet was resuspended in 0.5 ml of 1 M NaCI, SDS, 10 mM Tris, pH 8.0, 1 mM EDTA. The RNA was extracted once with an equal volume of chloroform/IAA and 2 volumes of ethanol was added. After incubation for 40 mins at -70 0 C the solution was centrifuged for 15 minutes at 10,000 RPM. The supematant was removed and the pellet was rinsed with ethanol, drained, and dried. The pellet was resuspended in 50 pl of sterile water.

First strand cDNA was synthesised from 10 jig total RNA with reverse transcriptase as described in Ref 2, utilising an oligo-dT primer adapter (Ref 1): B26 (5'-GACTCGAGTCGACATCGA I I I I I I I I I I I I I I I Oligonucleotide primers were designed based on known plant PPO DNA sequences. Comparison of a number of PPO sequences from a range of different plants allowed identification of the conserved regions of the gene, which r p are mostly in or near the regions which encode the two copper binding sites, CuA and CuB Forward primers designed around the CuA site (GEN8, GEN9 and GEN10) are reverse primers designed around the CuB site (REV1 and REV2) were synthesised: GEN8: (5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3') GEN9: (5'-GCGAATTCTICA[TC]TG[TC]GCITA[TC]TG-3') GEN10: (5'-GCGAATTCTTICCIT[TA][TCITGGAA[TC]TGGG-3') REV1: (5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AGTT-3') REV2: (5'-GCCTGCAGTTTC]TC[AG]TC[AGITAGAA-3') The first strand reaction of amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman using GEN and REV primers, each at a final concentration of 1 IM Amplification involved an initial 1- program of 2 cycles of denaturation at 94°C for 1 min, annealing at 37 0 C for 2 min, a slow ramp to 72 0 C over 2 min and elongation at 72 0 C for 3 min, followed Sby 33 cycles of denaturation at 94 0 C for 1 min, annealing at 55°C for 1 min, and P:\OPER\MRO\5603-96.CLM 130/99 elongation at 72 0 C for 3 min. A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products. The remainder was run on a low point agarose gel and the bands of interest was excised. DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen).

The purification DNA was cloned into Eco RV-cut Bluescript SK vector (Stratagene) which had been T-tailed with Taq Polymerase and the ligated DNA was introduced into E. coli DH5a by electroporation. Recombinant clones which had an insert of the predicted size were selected and their DNA sequence was determined by automated sequencing. Two putative banana PPO clones (BPPO2 and BPPO8) were identified based on their homology to known plant PPO genes.

REFERENCES

15 1. Frohman, MA (1990) in "PCR Protocols: A Guide to Methods and Applications" (MA Innis, DH Gelfrand, JJ Sninsky and TJ White, eds) Academic Press, New York pp 28-38.

2. Dry, IB and Robinson, SP (1994) "Molecular cloning and characterisation of grape berry polyphenol oxidase". Plant Molecular Biology 26:495-502.

Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

Claims (22)

1. A method of preparing nucleic acid encoding PPO of banana or lettuce, or a fragment or derivatives thereof, which method includes: providing: a source of a polypeptide having lettuce or banana PPO activity; a first primer having a sequence corresponding to at least a portion of or in close proximity to a first copper (Cu) binding site of PPO in sense orientation; a second primer having a sequence corresponding at least a portion of or in close proximity to a second Cu binding site of PPO in antisense orientation; and an adaptor primer; (ii) isolating RNA from said source; (iii) treating said RNA to produce cDNA therefrom; and (iv) amplifying the cDNA so formed using the first and second primers.

2. The method according to claim 1 wherein the first primer includes a nucleotide sequence selected from the group consisting of (i)GEN3: 5'-GCGAATTCTTTC[TC][TC]TICCITT[TC] [CA] [TC] [AC]G-3'; (ii)GEN7: 5'-GCGAATTCAA[TC]GTIGA[TC][AC]GIATGTGG-3'; and (iii)GEN8: 5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3'; or a fragment of said primer that is capable of hybridizing to mRNA or cDNA encoding PPO, and wherein I is inosine or any nucleotide residue.

3. The method according to claim 1 or claim 2 wherein the second primer includes the following sequence or part thereof: 5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3' and wherein I is inosine or any nucleotide residue. The method according to any one of claims 1 to 3, wherein the step of treating the P:\OPER\MRO\56803-96.CLM 17/1/00 -27- RNA to construct cDNA includes treating the RNA with reverse transcriptase and an adaptor primer to form cDNA, and wherein said adaptor primer includes the nucleotide sequence: 5'-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3' or a fragment thereof that is capable of hybridising to mRNA encoding PPO. The method according to any one of claims 1 to 4, further including providing a primer in the antisense orientation and an anchor primer for attaching to the 5' end of the cDNA formed and amplifying the cDNA using said primers to prepare nucleic acid encoding the N-terminal portion of PPO.

6. The method according to any one of claims 1 to 5, further including providing a primer in sense orientation and an adapter primer and amplifying the cDNA using said i* primers to prepare nucleic acid encoding the C-terminal portion of PPO.

7. The method according to claim 5 wherein the primer in the antisense orientation includes a nucleotide sequence selected from the group consisting of: S. 5'TGCTGTTCTGTTCGAACATGGCAG-3'; and (ii) 5'-TATACAAGTGGCACCAGTGTCTGC-3' or a fragment thereof that is capable of hybridising to mRNA or cDNA encoding PPO.

8. The method according to claim 6 wherein the primer in the sense orientation includes ;the following sequence or a fragment thereof that is capable of hybridising to the complement of mRNA or cDNA encoding PPO: 5'CGCTGGGTGGGTAATTCTAGGATG-3'.

9. The method according to claim 5 wherein the primer in the antisense orientation includes a nucleotide sequence selected from the group consisting of: 5'-CCGCATTGTGGATGACTTCCATCTG-3'; and (ii) 5'-CCAGAATGGGATGGTGAAGGTGTCG-3' or a fragment thereof that is capable of hybridising to mRNA or cDNA encoding PPO. P:\OPER\MRO\56803-96.CLM 17/1/00 -28- The method according to claim 6 wherein the primer in the sense orientation includes the following sequence or a fragment thereof that is capable of hybridising to the complement of mRNA or cDNA encoding PPO: 5'-AGTCATCCACAATGCGGCGCACATG-3'.

11. The method according to any one of claims 6, 8 or 10, wherein the adaptor primer includes the following sequence or a fragment thereof that is capable of hybridising to mRNA or cDNA encoding PPO: 5'-GACTCGAGTCGACATCG-3'.

12. An isolated nucleic acid molecule that comprises a nucleotide sequence which encodes or is complementary to a nucleotide sequence which encodes a PPO polypeptide of lettuce or banana having an amino acid sequence set forth in any one of Figures 1 to 3 or comprising the copper-binding site of any one of said amino acid sequences.

13. An isolated nucleic acid molecule that encodes a PPO polypeptide of lettuce or banana, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a nucleotide sequence set forth in any one of Figures 1 to 3; (ii) a fragment of comprising a nucleotide sequence that encodes the copper- binding site of a PPO polypeptide; (iii) a degenerate nucleotide sequence of or and (iv) a nucleotide sequence that is complementary to or (ii) or (iii). a

14. The isolated nucleotide sequence according to claims 12 or 13 wherein the copper- binding site is the CuA binding site of said banana or lettuce PPO polypeptide. The isolated nucleotide sequence according to claims 12 or 13 wherein the copper- binding site is the CuB binding site of said banana or lettuce PPO polypeptide.

16. The isolated nucleic acid molecule according to any one of claims 12 to 15, wherein i the PPO polypeptide of banana is at least expressed in banana fruit. P:\OPER\MRO\56803-96.CLM -17/1/00 -29-

17. The isolated nucleic acid molecule according to any one of claims 12 to 15, wherein the PPO polypeptide of lettuce is at least expressed in lettuce leaves.

18. A recombinant vector comprising the isolated nucleic acid molecule according to any one of claims 12 to 17 inserted within a vector molecule.

19. The recombinant vector according to claim 18 wherein the vector is a plasmid expression vector. The recombinant vector according to claim 19 wherein the plasmid expression vector is Bluescript SK+.

21. The recombinant vector according to claim 18 wherein the vector is a binary vector suitable for introducing into a plant cell, tissue or organ. 9

22. The recombinant vector according to any one of claims 18 to 21 wherein the vector is capable of being replicated and the PPO-encoding nucleotide sequence is capable of being transcribed and translated in a unicellular organism or in a plant. *999

23. A method of increasing the level of banana or lettuce PPO activity in a plant or a cell, tissue or organ thereof, said method comprising: introducing a nucleotide sequence to said plant or a cell, tissue or organ thereof which sequence encodes a PPO polypeptide of lettuce or banana having an amino acid sequence set forth in any one of Figures 1 to 3 or an enzymatically-active PPO polypeptide comprising the copper-binding site of any one of said amino acid sequences; and (ii) expressing said nucleotide sequence to produce an enzymatically-active PPO polypeptide. f IRA 24. A method of increasing the level of banana or lettuce PPO activity in a plant or a cell, Stissue or organ thereof, said method comprising: P:\OPER\MRO\56803-96.CLM 17/1/00 C. C. C C C introducing a nucleic acid molecule to said plant or a cell, tissue or organ thereof which nucleic acid molecule comprises the nucleotide sequence set forth in any one of Figures 1 to 3 or a degenerate nucleotide sequence thereof; and (ii) expressing said nucleic acid molecule to produce an enzymatically-active PPO polypeptide. A method of decreasing the level of PPO activity in a plant or a cell, tissue or organ thereof, said method comprising introducing a nucleic acid molecule to said plant or a cell, tissue or organ thereof which comprises a nucleotide sequence selected from the group consisting of: a nucleotide sequence which encodes a PPO polypeptide of banana or lettuce having an amino acid sequence set forth in any one of Figures 1 to 3 or the copper- binding site of any one of said amino acid sequences; (ii) a nucleotide sequence set forth in any one of Figures 1 to 3; (iii) a fragment of (ii) comprising a nucleotide sequence that encodes the copper- binding site of a PPO polypeptide; and (iv) a nucleotide sequence that is complementary to or (ii) or (iii).

26. The method according to claim 25 further comprising expressing the introduced nucleic acid molecule to produce sense or antisense RNA therefrom.

27. The method according to claims 25 or 26 wherein the PPO activity is decreased by co- suppression of the endogenous PPO-encoding genes that would otherwise be expressed in the plant or a cell, tissue or organ thereof.

28. The method according to claims 25 or 26, wherein the PPO activity is decreased by the expression of antisense RNA that is complementary to RNA encoded by an endogenous PPO-encoding gene that would otherwise be expressed in the plant or a cell, tissue or organ thereof.

129. The method according to any one of claims 23 to 28 wherein the nucleic acid molecule P:\OPER\MRO\56803-96.CLM 17/1/00 -31 is introduced into the plant or a cell, tissue or organ thereof by means of Agrobacterium- mediated transformation. The method according to any one of claims 23 to 28 wherein the nucleic acid molecule is introduced into the plant or a cell, tissue or organ thereof by means of microparticle bombardment using a nucleic acid-coated microprojectile. 31. A transformed plant comprising the isolated nucleic acid molecule according to any one of claims 12 to 17 or a plant part, progeny or propagule thereof that also comprises said nucleic acid molecule. 32. A transformed plant comprising the recombinant vector according to any one of claims 18, 19, 21 or 22 or a plant part, progeny or propagule thereof that also comprises said nucleic acid molecule. o 33. The transformed plant, plant part, progeny or propagule according to claim 31 or claim 32 being a banana plant or a part, progeny or propagule thereof. 34. The transformed plant, plant part, progeny or propagule according to claim 31 or claim 32 being a lettuce plant or a part, progeny or propagule thereof. o 35. The method according to any one of claims 1 to 11 substantially as hereinbefore described with reference to the Figures and/or Examples other than Example 3 or Figures 4 or 36. The isolated nucleic acid molecule according to any one of claims 12 to 17 substantially as hereinbefore described with reference to the Figures and/or Examples other than Example 3 or Figures 4 or 37. The recombinant vector according to any one of claims 18 to 22 substantially as hereinbefore described with reference to the Figures and/or Examples other than Example 3 P:\OPER\MRO\56803-96.CLM 18/1/00 -32- or Figures 4 or 38. The method according to any one of claims 23 to 30 substantially as hereinbefore described with reference to the Figures and/or Examples other than Example 3 or Figures 4 or 39. The transformed plant according to any one of claims 31 to 34 substantially as hereinbefore described with reference to the Figures and/or Examples other than Example 3 or Figures 4 or DATED this EIGHTEENTH day of JANUARY, 2000. Commonwealth Scientific and Industrial Research Organisation by DAVIES COLLISON CAVE Patent Attorneys for the Applicant .e*