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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D489/00—Heterocyclic compounds containing 4aH-8, 9 c- Iminoethano-phenanthro [4, 5-b, c, d] furan ring systems, e.g. derivatives of [4, 5-epoxy]-morphinan of the formula:
  • C07D489/02—Heterocyclic compounds containing 4aH-8, 9 c- Iminoethano-phenanthro [4, 5-b, c, d] furan ring systems, e.g. derivatives of [4, 5-epoxy]-morphinan of the formula: with oxygen atoms attached in positions 3 and 6, e.g. morphine, morphinone
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01247—Codeinone reductase (NADPH) (1.1.1.247)
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  • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
  • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
  • C12Y114/11031—Thebaine 6-O-demethylase (1.14.11.31)
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  • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
  • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
  • C12Y114/11032—Codeine 3-O-demethylase (1.14.11.32)

Definitions

• the present invention relates to genetically modified plants of the species Papaver bracteatum wherein the type or amount of one or more alkaloids produced by the plant has been modified.
• Opium poppies (Papaver somniferum) are commercially cultivated in a number of countries under regulatory control.
• the latex obtained by the incision of unripe capsules is known as opium and is the source of several pharmacologically important alkaloids.
• Morphine, codeine, thebaine, narcotine and papaverine are the most important alkaloids produced by the plant, and are exploited by the pharmaceutical industry as analgesics, antitussives and antispasmodics.
• poppy straw and straw concentrate are the most commonly used raw materials for the production of morphine and other poppy-derived alkaloids.
• more than 50% of the world's morphine is now manufactured from poppy straw or poppy straw concentrate.
• Papaver bracteatum (herein referred to as P. bracteatum) is one perennial species of poppy which produces significant quantities of the alkaloid thebaine.
• Thebaine itself is not used therapeutically, however, this alkaloid is used as a feedstock for the production of other alkaloids, including the production of oxycodone.
• a further significant problem affecting the cultivation of P. bracteatum in at least some poppy growing regions is that many varieties do not produce seed capsules and significant quantities of alkaloid in the first growing season after seed germination.
• the present invention provides a genetically modified poppy plant of the species Papaver bracteatum, or a hybrid thereof, wherein the expression of one or more of thebaine 6-O-demethylase, codeine O-demethylase and/or codeinone reductase is increased relative to wild type P. bracteatum and wherein said genetically modified poppy plant produces an increased quantity of an alkaloid selected from codeine, oripavine and/or morphine relative to a wild type P. bracteatum.
• At least the expression of thebaine 6-O-demethylase is increased relative to wild type P. bracteatum.
• the poppy plant comprises one or more transgenes which encode thebaine 6-O-demethylase, codeine O-demethylase and/or codeinone reductase.
• the one or more transgenes comprise the nucleotide sequence set forth in any one or more of SEQ ID NOs: 1 to 8, 12 and 13.
• the thebaine 6-O-demethylase comprises the amino acid sequence set forth in one of SEQ ID NOs: 9 to 1 1.
• the poppy plant produces seed capsules and an alkaloid selected from codeine, oripavine and/or morphine in the first growing season after germination in a temperate poppy growing region.
• the poppy plant produces seed capsules and an alkaloid selected from codeine, oripavine and/or morphine in the first growing season after germination at a latitude of between 40° and 44°.
• the poppy plant produces seed capsules and an alkaloid selected from codeine, oripavine and/or morphine in the first growing season after germination in Georgia, Australia.
• the poppy plant comprises an indehiscent seed capsule or a dehiscent seed capsule.
• the present invention provides a progeny plant having a poppy plant of a first aspect of the invention as a parent, wherein the progeny plant comprises increased expression of one or more of thebaine 6-O-demethylase, codeine O-demethylase and/or codeinone reductase relative to wild type P. bracteatum and wherein said progeny plant produces an increased quantity of an alkaloid selected from codeine, oripavine and/or morphine relative to a wild type P. bracteatum.
• the present invention provides a mutant or derivative plant of the plant of a first or second aspect of the invention wherein the mutant or derivative comprises increased expression of one or more of thebaine 6-O-demethylase, codeine O-demethylase and/or codeinone reductase relative to wild type P. bracteatum and wherein said mutant or derivative produces an increased quantity of an alkaloid selected from codeine, oripavine and/or morphine relative to a wild type P. bracteatum.
• the present invention provides reproductive material derived from the plant of any one of the first, second or third aspects of the invention.
• the reproductive material comprises a seed.
• the present invention provides straw produced from the plant of any one of the first, second or third aspects of the invention.
• the present invention provides a straw concentrate produced from the plant of any one of the first, second or third aspects of the invention.
• the present invention provides latex derived from the plant of any one of the first, second or third aspects of the invention.
• the present invention provides a stand of stably reproducing poppy plants of any one of the first, second or third aspects of the invention.
• the present invention provides an isolated cell derived from the plant of any one of the first, second or third aspects of the invention.
• the present invention provides an in-vitro culture comprising one or more of the cells of ninth aspect of the invention.
• said culture produces an alkaloid selected from codeine, oripavine and/or morphine.
• the present invention provides a method of producing an alkaloid selected from codeine, oripavine and/or morphine, the method comprising growing a plant of any one of the first, second or third aspects of the invention such that the plant produces an alkaloid selected from codeine, oripavine and/or morphine; and extracting the codeine, oripavine and/or morphine from the poppy plant or a part thereof.
• the present invention provides an alkaloid selected from codeine, oripavine and/or morphine produced according to the method of the eleventh aspect of the invention.
• the present invention provides an expression construct comprising one or more transgenes which encode thebaine 6-O-demethylase, codeine O-demethylase and/or codeinone reductase, wherein said one or more transgenes are operably connected to a transcriptional control sequence which is active in P. bracteatum.
• the one or more transgenes comprise the nucleotide sequence set forth in any one or more of SEQ ID NOs: 1 to 8, 12 and 13.
• the thebaine 6-O- demethylase comprises the amino acid sequence set forth in one of SEQ ID NOs: 9 to 1 1.
• the present invention provides an isolated nucleic acid molecule comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8, 12 and 13.
• the present invention provides an isolated nucleic acid molecule selected from the group consisting of: (i) a nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8, 12 and 13; and
• nucleic acid molecule which hybridizes to a nucleic acid molecule comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8, 12 and 13 under stringent conditions.
• the present invention provides an isolated polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 9 to 1 1.
• the present invention provides an isolated polypeptide comprising an amino acid sequence which is at least 50% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 9 to 11.
• Figure 1 is a schematic diagram showing the biosynthesis of morphine from thebaine in Papaver somniferum. T60DM - thebaine 6-O-demethylase; CODM - codeine O-demethylase; COR - codeinone reductase.
• Figure 2 is a photograph of an electrophoresis gel showing the products of PCR amplification of DNA isolated from P. somniferum and P. bracteatum using primers to the T60DM and CODM genes.
• the primer combinations corresponding to each lane in the gel are provided in Table 2.
• P. somniferum genomic DNA as a template (top panel);
• P. bracteatum genomic DNA as a template (bottom panel).
• Figure 3 is a schematic showing the location of the primers in the CODM and T60DM genes used to PCR amplify the products shown in Figure 2.
• Figure 4 is a nucleotide sequence alignment of T60DM variants (clones 21 , 22, 23 and 1 1 ) with a reference cDNA sequence (GQ500139) for T60DM (referred to as "cDNA" in the alignment). Coding sequences are boxed in black and intronic sequences are boxed in grey. Nucleotide sequence differences are boxed in white.
• Figure 5 is an amino acid sequence alignment of T60DM variants encoded by the nucleotide sequences of Figure 4.
• the amino acid sequence of variant clones 21 , 22 and 23 is shown compared to the amino acid sequence encoded by the reference cDNA sequence (GQ500139) for T60DM (referred to as "cDNA" in the alignment).
• Figure 6 is a nucleotide sequence alignment of a CODM variant (clone 21 ) with a reference cDNA sequence (GQ500141 ) for CODM (referred to as "cDNA" in the alignment). Coding sequences are boxed in black and intronic sequences are boxed in grey. The nucleotide sequence difference is boxed in white.
• Figure 7 is a schematic showing a vector map of the T60DM construct generated in Example 4.
• Figure 8 is a schematic showing a vector map of the bar (Basta) construct generated in Example 4.
• Figure 9 is a schematic showing a vector map of the CODM construct generated in Example 4.
• Figure 10 is a schematic showing a vector map of the COR1.1 construct generated in Example 4.
• Figure 1 1 provides photos evidencing callus induction and plant regeneration of P. bracteatum var PB-1.
• A Callus from seed after 8 weeks:
• B shoot regeneration after 20 weeks;
• C plantlets with roots after 24 weeks;
• D, E and F new callus growth under selection after agrobacterium mediated transformation with 35S::Basta, 35S::CODM, and 35S::T60DM, respectively.
• SEQ ID NO: Nucleotide and amino acid sequences are referred to herein by a sequence identifier number (SEQ ID NO:). A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided at the end of the specification. TABLE 1
• SEQ ID NO: 34 als gene PCR amplification primer-1 (Example 4) 400 ⁇ 34>
• SEQ ID NO: 35 als gene PCR amplification primer-2 (Example 4) 400 ⁇ 35>
• the present invention provides a genetically modified poppy plant of the species P. bracteatum, or a hybrid thereof, wherein the expression of one or more of thebaine 6-O-demethylase, codeine O-demethylase and/or codeinone reductase is increased relative to wild type P. bracteatum and wherein said genetically modified poppy plant produces an increased quantity of an alkaloid selected from codeine, oripavine and/or morphine relative to a wild type P. bracteatum.
• Reference herein to a "poppy plant” may refer to a whole poppy plant, but may also refer to a part of a poppy plant, including, for example, reproductive material (such as seeds) derived from a poppy plant; a cell, tissue or organ derived from a poppy plant; and the like.
• reproductive material such as seeds
• the present invention also provides poppy plant cells, tissues, organs, reproductive material and the like.
• the poppy plants of the present invention are of the perennial poppy species, Papaver bracteatum.
• Typical P. bracteatum, or Persian Poppy grows to a height of up to 1.5 metres. It has 6 petals which are blood red in colour with a dark basal blotch.
• the capsules are typically about 40 mm diameter with a concave cap and persistent bracts.
• the above morphological description may not apply to all members of the species and thus the morphological description should not be considered to limit the scope of the term P. bracteatum as used herein.
• a poppy plant of the present invention should be understood to include P. bracteatum species as well as hybrid plants wherein at least one of the parents is a plant of the species P. bracteatum.
• hybrids may include, for example, an intrageneric hybrid plant wherein one of the parents is a plant of the first aspect of the invention and the other parent is a plant within the genus Papaver (e.g. Papaver somniferum or Papaver orientale).
• the hybrid may be an intergeneric hybrid plant wherein one of the parents is a plant of the first aspect of the invention and the other parent is a plant of a genus other than Papaver.
• the present invention contemplates genetically modified poppy plants in which the expression of one or more of thebaine 6-O-demethylase, codeine O- demethylase and/or codeinone reductase is increased relative to wild type P. bracteatum.
• Reference herein to a "wild type P. bracteatum" should be understood to be native or non-genetically modified or non-hybridised plants of the species P. bracteatum.
• the genetically modified poppy plants of the present invention comprise increased expression of one or more of thebaine 6-O-demethylase (T60DM), codeine O-demethylase (CODM) and/or codeinone reductase (COR) relative to wild type Papaver bracteatum.
• T60DM baine 6-O-demethylase
• CODM codeine O-demethylase
• COR codeinone reductase
• increased expression is intended, for example to refer to a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or greater increase in the enzymatic activity of T60DM, CODM and/or COR in the genetically modified poppy plants of the present invention relative to wild type P. bracteatum.
• increased expression may comprise the introduction of T60DM, CODM and/or COR activity into a poppy plant of the present invention where such activity is absent in wild type P. bracteatum.
• T60DM catalyses the demethylation of thebaine to neopinone and also catalyses the demethylation of oripavine to morphinone.
• CODM catalyses the demethylation of thebaine to oripavine and also catalyses the demethylation of codeine to morphine.
• COR catalyses the reduction of codeinone to codeine and also catalyses the reduction of morphinone to morphine.
• wild-type P. bracteatum accumulates significant amounts of thebaine, while later the alkaloids codeine, oripavine and morphine are either absent, or at very low levels, in wild type P. bracteatum.
• the inventors have determined that one or more of the biosynthetic enzymes responsible for the conversion of thebaine through to morphine (via codeine and/or oripavine) are either absent or disabled in wild type P. bracteatum.
• Example 2 As shown in Example 2, the present inventors have data to indicate that the COR gene is present in P. bracteatum. Furthermore, Brochmann-Hanssen & Wunderly (J Pharm Sci 67(1 ): 103-106, 1978) have reported that when codeinone was administered to living P. bracteatum, it was converted to codeine rapidly and efficiently. From this report, the present inventors have determined that in at least some P. bracteatum cultivars, COR may also be active. However, even in such cultivars where COR is active, it may be desirable to increase the activity of COR in order to increase the yield of morphine and/or codeine in the plant.
• the present inventors have determined that in order to obtain production of an alkaloid selected from codeine, oripavine and/or morphine in P. bracteatum, the expression of one or more of T60DM, CODM and/or COR activity may be increased in P. bracteatum.
• At least the expression of T60DM is increased in the poppy plants of the present invention relative to wild type P. bracteatum.
• genetically modified poppy plant should be understood to refer to a poppy plant that has had a genetic modification made to it.
• a “genetic modification” may include any genetic modification that effects an increase in the expression of an enzymatic activity of interest (eg. T60DM, CODM and/or COR) in a genetically modified plant relative to a non-genetically modified form of the plant.
• Exemplary types of genetic modification include: random mutagenesis such as transposon, chemical, UV and phage mutagenesis together with selection of mutants which overexpress an enzymatic activity of interest; transient or stable introduction of one or more nucleic acid molecules into a cell which direct the expression and/or overexpression of an enzyme of interest in the cell; and the like.
• the present invention contemplates increasing the expression of T60DM, CODM and/or COR in P. bracteatum, by increasing the expression of a T60DM, CODM and/or COR encoding nucleic acid in one or more cells of the plant and/or increasing the copy number of a T60DM, CODM and/or COR encoding nucleic acid in one or more cells of the plant.
• a T60DM, CODM and/or COR encoding nucleic acid By “increasing the expression of a T60DM, CODM and/or COR encoding nucleic acid” is intended, for example a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or greater increase in the transcription and/or translation of a T60DM, CODM and/or COR encoding nucleic acid relative to wild type P. bracteatum.
• Increasing the expression of a T60DM, CODM and/or COR encoding nucleic acid should also be understood to comprise introducing the expression of a T60DM, CODM and/or COR encoding nucleic acid into a poppy plant of the present invention where such activity is absent in wild type P. bracteatum.
• the genetically modified poppy plant of the present invention comprises one or more transgenes which encode T60DM, CODM and/or COR.
• the genetically modified poppy plant at least comprises one or more transgenes which encode thebaine 6-O-demethylase.
• a "transgene” as referred to herein should be understood to include any nucleotide sequence which is introduced into and/or is non-native to the wild type P. bracteatum genome. As such, a transgene may be an additional or replacement copy of a nucleotide sequence already in the P. bracteatum genome or may be a nucleotide sequence which is exogenous to the wild type P. bracteatum genome.
• Reference herein to "thebaine 6-O-demethylase” or "T60DM” should be understood to include any enzyme which can catalyse the O-demethylation of thebaine to neopinone and/or the O-demethylation of oripavine to morphinone.
• nucleotide sequence which encodes thebaine 6-O-demethylase should be understood to include any nucleotide sequence which encodes a thebaine 6-O- demethylase enzyme.
• An example of a nucleotide sequence which encodes thebaine 6- O-demethylase includes the nucleotide sequence set forth in GenBank accession number GQ500139 or a functional homolog or variant thereof.
• codeine O-demethylase or “CODM” should be understood to include any enzyme which can catalyse the O-demethylation of thebaine to oripavine and/or the O-demethylation of codeine to morphine.
• reference herein to a nucleotide sequence which encodes codeine O-demethylase should be understood to include any nucleotide sequence which encodes a codeine O-demethylase enzyme.
• An example of a nucleotide sequence which encodes codeine O-demethylase includes the nucleotide sequence set forth in GenBank accession number GQ500141 or a functional homolog or variant thereof.
• codeinone reductase or “COR” should be understood to include any enzyme which can catalyse the reduction of codeinone to codeine and/or the reduction of morphinone to morphine.
• reference herein to a nucleotide sequence which encodes codeinone reductase should be understood to include any nucleotide sequence which encodes a codeinone reductase enzyme.
• An example of a nucleotide sequence which encodes codeinone reductase includes the nucleotide sequence set forth in GenBank accession number FJ596162 or a functional homolog or variant thereof.
• Reference herein to a "functional homolog or variant" of a particular reference sequence may be a nucleic acid which has one or more nucleotide insertions, deletions or substitutions relative to the reference sequence; a mutant form or allelic variant of the reference sequence; an ortholog of the reference sequence; analogs that contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons, and the like.
• “Modified” bases include, for example, tritylated and unusual bases such as inosine.
• the functional homolog or variant retains the ability to encode an enzyme having equivalent activity to the reference sequence.
• a functional homolog or variant comprises at least 50% sequence identity to the reference nucleotide sequence, including at least 55% nucleotide sequence identity, at least 60% nucleotide sequence identity, at least 65% nucleotide sequence identity, at least 70% nucleotide sequence identity, at least 75% nucleotide sequence identity, at least 80% nucleotide sequence identity, at least 85% nucleotide sequence identity, at least 90% nucleotide sequence identity or at least 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to the reference nucleotide sequence.
• the inventors have identified variants of the reference sequences for T60DM and CODM.
• T60DM four variants have been identified which respectively comprise the cDNA nucleotide sequences set forth in SEQ ID NOs: 1 to 4. These nucleotide sequences are derived from genomic DNA clones comprising the nucleotide sequences set forth in SEQ ID NOs: 5 to 8, respectively.
• the identified T60DM nucleotide sequence variants collectively encode T60DM enzyme variants comprising the amino acid sequences set forth in SEQ ID NOs: 9 to 1 1.
• CODM a single nucleotide sequence variant which comprises the cDNA nucleotide sequence set forth in SEQ ID NO: 12.
• This nucleotide sequence is derived from a genomic DNA clone comprising the nucleotide sequence set forth in SEQ ID NO: 13.
• the identified CODM variant does not encode a variant CODM polypeptide.
• the compared sequences should be compared over a comparison window of at least 100 nucleotide residues, at least 200 nucleotide residues, at least 300 nucleotide residues, at least 400 nucleotide residues, at least 500 nucleotide residues, at least 600 nucleotide residues, at least 800 nucleotide residues, at least 1000 nucleotide residues, or over the full length of the reference sequence.
• the comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such as the BLAST family of programs as, for example, disclosed by Altschul et al. 1997 (Nucl. Acids Res. 25: 3389- 3402).
• Global alignment programs may also be used to align similar sequences of roughly equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk/Tools/psa/emboss_needle/) which is part of the EMBOSS package (Rice P et al, 2000, Trends Genet, 16: 276-277), and the GGSEARCH program (available at fasta.bioch. Virginia.
• the transgene may be operably connected to one or more transcriptional control sequences and/or promoters.
• a T60DM, CODM and/or COR encoding transgene may be operably connected to a transcriptional control sequence such as a native T60DM, CODM and/or COR promoter or a heterologous promoter.
• a transcriptional control sequence is regarded as "operably connected" to a given gene or other nucleotide sequence when the transcriptional control sequence is able to promote, inhibit or otherwise modulate the transcription of the gene or other nucleotide sequence.
• a promoter may regulate the expression of an operably connected nucleotide sequence constitutively, or differentially, with respect to the cell, tissue, organ or developmental stage at which expression occurs, in response to external stimuli such as physiological stresses, pathogens, or metal ions, amongst others, or in response to one or more transcriptional activators.
• the promoter used in accordance with the methods of the present invention may include, for example, a constitutive promoter, an inducible promoter, a tissue-specific promoter or an activatable promoter.
• Plant constitutive promoters typically direct expression in nearly all tissues of a plant and are largely independent of environmental and developmental factors.
• Examples of constitutive promoters that may be used in accordance with the present invention include plant viral derived promoters such as the Cauliflower Mosaic Virus 35S and 19S (CaMV 35S and CaMV 19S) promoters; bacterial plant pathogen derived promoters such as opine promoters derived from Agrobacterium spp., e.g.
• rbcS rubisco small subunit gene
• Pubi plant ubiquitin promoter
• Pact rice actin promoter
• a CaMV 35S promoter may be used that comprises dual enhancer motifs (see Example 4).
• Such a promoter is represented by the sequence set forth in SEQ ID NO: 14.
• “Inducible” promoters include, but are not limited to, chemically inducible promoters and physically inducible promoters.
• Chemically inducible promoters include promoters which have activity that is regulated by chemical compounds such as alcohols, antibiotics, steroids, metal ions or other compounds. Examples of chemically inducible promoters include: alcohol regulated promoters (eg. see European Patent 637 339); tetracycline regulated promoters (eg. see US Patent 5,851 ,796 and US Patent 5,464,758); steroid responsive promoters such as glucocorticoid receptor promoters (eg. see US Patent 5,512,483), estrogen receptor promoters (eg.
• the inducible promoter may also be a physically regulated promoter which is regulated by non-chemical environmental factors such as temperature (both heat and cold), light and the like.
• physically regulated promoters include heat shock promoters (eg. see US Patent 5,447858, Australian Patent 732872, Canadian Patent Application 1324097); cold inducible promoters (eg. see US Patent 6,479,260, US Patent 6,184,443 and US Patent 5,847,102); light inducible promoters (eg. see US Patent 5,750,385 and Canadian Patent 132 1563); light repressible promoters (eg. see New Zealand Patent 508103 and US Patent 5,639,952).
• heat shock promoters eg. see US Patent 5,447858, Australian Patent 732872, Canadian Patent Application 1324097
• cold inducible promoters eg. see US Patent 6,479,260, US Patent 6,184,443 and US Patent 5,847,102
• light inducible promoters eg. see US Patent
• tissue specific promoters include promoters which are preferentially or specifically expressed in one or more specific cells, tissues or organs in an organism and/or one or more developmental stages of the organism. It should be understood that a tissue specific promoter may also be inducible.
• plant tissue specific promoters include: root specific promoters such as those described in US Patent Application 2001047525; fruit specific promoters including ovary specific and receptacle tissue specific promoters such as those described in European Patent 316 441 , US Patent 5,753,475 and European Patent Application 973 922; and seed specific promoters such as those described in Australian Patent 612326 and European Patent application 0 781 849 and Australian Patent 746032.
• the promoter may also be a promoter that is activatable by one or more transcriptional activators, referred to herein as an "activatable promoter".
• the activatable promoter may comprise a minimal promoter operably connected to an Upstream Activating Sequence (UAS), which comprises, inter alia, a DNA binding site for one or more transcriptional activators.
• UAS Upstream Activating Sequence
• the term "minimal promoter” should be understood to include any promoter that incorporates at least an RNA polymerase binding site and, optionally a TATA box and transcription initiation site and/or one or more CAAT boxes.
• the minimal promoter may be derived from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter.
• the CaMV 35S derived minimal promoter may comprise, for example, a sequence that substantially corresponds to positions -90 to +1 (the transcription initiation site) of the CaMV 35S promoter (also referred to as a -90 CaMV 35S minimal promoter), -60 to +1 of the CaMV 35S promoter (also referred to as a -60 CaMV 35S minimal promoter) or -45 to +1 of the CaMV 35S promoter (also referred to as a -45 CaMV 35S minimal promoter).
• the activatable promoter may comprise a minimal promoter fused to an Upstream Activating Sequence (UAS).
• UAS Upstream Activating Sequence
• the UAS may be any sequence that can bind a transcriptional activator to activate the minimal promoter.
• Exemplary transcriptional activators include, for example: yeast derived transcription activators such as Gal4, Pdr1 , Gcn4 and Ace1 ; the viral derived transcription activator, VP16; Hap1 (Hach et ai, J Biol Chem 278: 248-254, 2000); Gaf1 (Hoe et ai, Gene 215(2): 319-328, 1998); E2F (Albani et al., J Biol Chem 275: 19258-19267, 2000); HAND2 (Dai and Cserjesi, J Biol Chem 277: 12604-12612, 2002); NRF-1 and EWG (Herzig et al., J Cell Sci 113: 4263-4273,
• the UAS comprises a nucleotide sequence that is able to bind to at least the DNA-binding domain of the GAL4 transcriptional activator.
• UAS sequences which can bind transcriptional activators that comprise at least the GAL4 DNA binding domain, are referred to herein as UAS G -
• the UAS sequence in the activatable promoter may comprise a plurality of tandem repeats of a DNA binding domain target sequence.
• UASG comprises four tandem repeats of the DNA binding domain target sequence.
• the term "plurality" as used herein with regard to the number of tandem repeats of a DNA binding domain target sequence should be understood to include, for example, at least 2 tandem repeats, at least 3 tandem repeats or at least 4 tandem repeats.
• the transcriptional control sequence may also include a terminator.
• the term "terminator” refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences generally containing a polyadenylation signal, which facilitate the addition of polyadenylate sequences to the 3'-end of a primary transcript. As with promoter sequences, the terminator may be any terminator sequence which is operable in the cells, tissues or organs in which it is intended to be used.
• Suitable terminator sequences which may be useful in plant cells include: the nopaline synthase (nos) terminator, the CaMV 35S terminator, the octopine synthase (ocs) terminator, potato proteinase inhibitor gene (pin) terminators, such as the pinll and pinlll terminators and the like.
• the transcriptional control sequence to which a transgene is connected may be introduced into a cell with the transgene, or alternatively, the transgene may be inserted into the genome of the plant cell such that it becomes operably connected to an endogenous transcriptional control sequence.
• the insertion of the transgene in the genome such that it is under the control of an endogenous transcriptional control sequence may be the result of either non-site directed or random DNA insertion (eg. T-DNA or transposon mediated insertion) or the result of site-directed insertion (for example as described in Terada et al., Nat. Biotechnol. 20: 1030-1034, 2002).
• the one or more transgenes may be contained within a vector or construct for transformation into P. bracteatum.
• the vector or construct comprises the one or more transgenes operably connected to transcriptional control sequences which are active in P. bracteatum
• the vector or construct may be referred to as an expression vector or construct.
• the vector or construct may further comprise, for example, one or more of: an origin of replication for one or more hosts (including bacteria such as E. coli or Agrobacterium spp.); a selectable marker gene which is active in one or more hosts; and/or one or more additional transcriptional control sequences.
• selectable marker gene includes any gene that 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.
• Selectable marker genes include any nucleotide sequences which, when expressed by a cell, confer a phenotype on the cell that facilitates the identification and/or selection of these transformed cells.
• a range of nucleotide sequences encoding suitable selectable markers are known in the art.
• Exemplary nucleotide sequences that encode selectable markers include: antibiotic resistance genes such as ampicillin- resistance genes, tetracycline-resistance genes, kanamycin-resistance genes, the AURI- C gene which confers resistance to the antibiotic aureobasidin A, neomycin phosphotransferase genes (eg.
• nptl and nptll nptl and nptll
• hygromycin phosphotransferase genes eg. hpt
• herbicide resistance genes including glufosinate, phosphinothricin or bialaphos resistance genes such as phosphinothricin acetyl transferase encoding genes (eg. bar), glyphosate resistance genes including 3-enoyl pyruvyl shikimate 5-phosphate synthase encoding genes (eg. aroA), bromyxnil resistance genes including bromyxnil nitrilase encoding genes, sulfonamide resistance genes including dihydropterate synthase encoding genes (eg.
• sul) and sulfonylurea resistance genes including acetolactate synthase encoding genes; enzyme-encoding reporter genes such as GUS and chloramphenicolacetyltransferase (CAT) encoding genes; fluorescent reporter genes such as the green fluorescent protein-encoding gene; and luminescence-based reporter genes such as the luciferase gene, amongst others.
• enzyme-encoding reporter genes such as GUS and chloramphenicolacetyltransferase (CAT) encoding genes
• fluorescent reporter genes such as the green fluorescent protein-encoding gene
• luminescence-based reporter genes such as the luciferase gene, amongst others.
• the vector or construct is adapted to be at least partially transferred into a plant cell via Agrobacterium-mediaied transformation. Accordingly, in some embodiments, the construct may comprise left and/or right T-DNA border sequences.
• T-DNA border sequences would be readily ascertained by one of skill in the art.
• T-DNA border sequences should be understood to include, for example, any substantially homologous and substantially directly repeated nucleotide sequences that delimit a nucleic acid molecule that is transferred from an Agrobacterium sp. cell into a plant cell susceptible to Agrobacterium-mediaied transformation.
• the vector or construct is adapted to be transferred into a plant via Agrobacterium-mediaied transformation
• the present invention also contemplates any suitable modifications to the genetic construct that facilitate bacterial mediated insertion into a plant cell via bacteria other than Agrobacterium sp., for example as described in Broothaerts et al. ⁇ Nature 433: 629-633, 2005).
• the present invention provides an expression construct comprising one or more one or more transgenes which encode thebaine 6-0- demthylase, codeine O-demethylase and/or codeinone reductase wherein said one or more transgenes are operably connected to a transcriptional control sequence which is active in P. bracteatum.
• a transgene or a construct including a transgene must be introduced into P. bracteatum via a suitable transformation method.
• Methods for the transformation of plant cells include, for example: Agrobacterium- mediated transformation, microprojectile bombardment based transformation methods and direct DNA uptake based methods.
• Roa-Rodriguez et al. Agrobacterium-mec /atec transformation of plants, 3 rd Ed. CAMBIA Intellectual Property Resource, Canberra, Australia, 2003
• Bacterial mediated transformation using bacteria other than Agrobacterium sp. may also be used, for example as described in Broothaerts et al. ⁇ Nature 433: 629-633, 2005).
• Microprojectile bombardment may also be used to transform plant tissue and methods for the transformation of plants, including cereal plants, and such methods are reviewed by Casas et al. (Plant Breeding Rev. 13: 235-264, 1995).
• Direct DNA uptake transformation protocols such as protoplast transformation and electroporation are described in detail in Galbraith et al. (eds.), Methods in Cell Biology Vol. 50, Academic Press, San Diego, 1995).
• a range of other transformation protocols may also be used. These include infiltration, electroporation of cells and tissues, electroporation of embryos, microinjection, pollen-tube pathway, silicon carbide- and liposome mediated transformation.
• the method of Solouki et al. ( Trakia Journal of Sciences 7(2): 1 -7, 2009) is an example of a method known to be suitable for the transformation of P. bracteatum.
• P. bracteatum may be transformed according to methods set out in the Examples (see later).
• the genetically modified poppy plants of the present invention produce an increased quantity of an alkaloid selected from codeine, oripavine and/or morphine relative to a wild type P. bracteatum.
• the "increased quantity of codeine, oripavine and/or morphine” may occur in one or more tissues of the genetically modified poppy plant, or in the latex of the genetically modified poppy plant, relative to wild-type P. bracteatum.
• the increased quantity of codeine, oripavine and/or morphine in the genetically modified poppy plants of the present invention may occur in the latex and/or one or more tissues selected from root tissue, leaf tissue and/or capsule.
• increased quantity is intended for example, to refer to a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or greater increase in the quantity of codeine, oripavine and/or morphine in the poppy plants of the present invention relative to wild type P. bracteatum.
• the poppy plants of the present invention produce an extractable quantity of codeine, oripavine and/or morphine.
• An "extractable quantity" of codeine, oripavine and/or morphine should be understood to include an amount of codeine, oripavine and/or morphine which is extractable and detectable from one or more tissues of the plant. The one or more tissues may be selected from those referred to above.
• an "extractable quantity" of codeine, oripavine and/or morphine should be understood to include wherein any of codeine, oripavine and/or morphine, or a combination of codeine, oripavine and/or morphine, represents at least 1 %, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% by weight of the total extractable alkaloid from any one or more of the latex, capsules, roots or leaves of the poppy plants of the present invention.
• the genetically modified poppy plants contemplated by the present invention are typically perennial.
• a "perennial" poppy plant encompasses poppy plants that produce seed capsules and an increased and/or extractable quantity of an alkaloid (such as codeine, oripavine and/or morphine) for more than one growing season without the need to replant.
• the poppy plants of the present invention produce seed capsules and an increased and/or extractable quantity of codeine, oripavine and/or morphine in the first growing season after germination in a temperate poppy growing region.
• Production of seed capsules and an increased and/or extractable quantity of codeine, oripavine and/or morphine "in the first growing season after germination" should be understood as the plants producing seed capsules and an increased and/or extractable quantity of codeine, oripavine and/or morphine within one year of seed germination.
• a temperate poppy growing region should be understood as a region in which poppy plants may be cultivated which is located at a latitude that lies between the tropics and the polar circles.
• the north temperate zone extends from the Tropic of Cancer at about 23.5 degrees north latitude to the Arctic Circle at about 66.5 degrees north latitude.
• the south temperate zone extends from the Tropic of Capricorn at about 23.5 degrees south latitude to the Antarctic Circle at about 66.5 degrees south Latitude.
• the temperate poppy growing region may include a region where the poppy plants of the present invention will grow which is at one or more North or South latitudes of about 34°, about 35°, about 36°, about 37°, about 38°, about 39°, about 40°, about 41 °, about 42°, about 43°, about 44°, about 45°, about 46°, about 47°, about 48°, about 49°, and/or about 50°.
• the poppy plants of the present invention produce seed capsules and an increased and/or extractable quantity of codeine, oripavine and/or morphine in the first growing season after germination in at least a temperate poppy growing region in the Southern hemisphere.
• the poppy plants of the present invention may produce seed capsules and an increased and/or extractable quantity of codeine, oripavine and/or morphine in the first growing season after germination at one or more latitudes of about 34° S, about 35° S, about 36° S, about 37° S, about 38° S, about 39° S, about 40° S, about 41 ° S, about 42° S, about 43° S, about 44° S, about 45° S, about 46° S, about 47° S, about 48° S, about 49° S, and/or about 50° S.
• the poppy plants of the present invention produce seed capsules and an increased and/or extractable quantity of codeine, oripavine and/or morphine in the first growing season after germination at a latitude of between about 40° S and about 44° S.
• the poppy plants of the present invention produce seed capsules and an increased and/or extractable quantity of codeine, oripavine and/or morphine in the first growing season after germination in Georgia, Australia or a region having substantially equivalent climate.
• Reference herein to a "region having substantially equivalent climate” refers to a region having equivalent day length, rainfall and/or seasonal temperature minima and maxima such that conditions under which poppies may be grown are equivalent to those in poppy growing regions of Georgia, Australia.
• the poppy plants of the present invention may have either a dehiscent (open) seed capsule or an indehiscent (closed) seed capsule at seed maturity.
• Wild type P. bracteatum, and most other Papaver species form pores of dehiscence in the seed capsule at seed maturity, which allows the mature seed in the seed capsule to be released from the seed capsule.
• the poppy cultivar may have seed capsules that have reduced or no pores of dehiscence at the time the seed in the seed capsule has matured.
• the trait of an indehiscent or closed seed capsule at maturity leads to the retention of mature seed in the seed capsule in cultivars having the trait.
• the present invention also provides a progeny plant, wherein the progeny plant has a plant of the first aspect of the invention as a parent, wherein the progeny plant comprises increased expression of one or more of thebaine 6-O-demethylase, codeine O-demethylase and/or codeinone reductase relative to wild type P. bracteatum, and wherein said progeny plant produces an increased and/or extractable quantity of an alkaloid selected from codeine, oripavine and/or morphine relative to a wild type P. bracteatum.
• a "progeny" plant may be any plant for which a plant of the first aspect of the invention is at least one of the parents.
• the progeny plant may be a plant resulting from self-fertilisation of a plant of the first aspect of the invention; a plant resulting from a non-self fertilisation wherein a plant of the first aspect of the invention is the male or female parent; an intrageneric hybrid plant wherein one of the parents is a plant of the first aspect of the invention and the other parent is a plant within the genus Papaver (e.g.
• Papaver somniferum or Papaver orientate an intergeneric hybrid plant wherein one of the parents is a plant of the first aspect of the invention and the other parent is a plant of a genus other than Papaver, an asexually produced progeny of a plant of the first aspect of the invention, such as vegetatively reproduced progeny or progeny produced by apomixis.
• the present invention also provides a mutant or derivative plant of the poppy plant of the first aspect of the invention wherein the mutant or derivative comprises increased expression of one or more of thebaine 6-O-demethylase, codeine O-demethylase and/or codeinone reductase relative to wild type P. bracteatum, and wherein said mutant or derivative produces an increased and/or extractable quantity of an alkaloid selected from codeine, oripavine and/or morphine relative to a wild type P. bracteatum.
• a "mutant or derivative" of the subject poppy plants should be understood to encompass, for example, any spontaneous or induced mutant, breeding progeny or further genetically modified forms of the poppy plants of the first aspect of the invention.
• Mutagenisation techniques that may be used to generate derivatives or mutants of the poppy plants of the present invention include, for example, chemical mutagenesis (eg. EMS mutagenesis), ionising radiation-induced mutagenesis (eg. X-ray mutagenesis, ⁇ -ray mutagenesis and UV mutagenesis), genetic insertion mutagenesis methods (eg. transposon mutagenesis or T-DNA mutagenesis) and the like.
• Techniques for the production of mutagenized seed are well known in the art. For example, methods of seed mutagenesis as well as chemical mutagens suitable for use in these methods are described in, for example, The Manual on Mutation Breeding, 2nd ed.
• Example mutagens include ethyl methanesulfonate (EMS), diepoxybutane (DEB) ethyl-2-chloroethyl sulphide, 2-chloroethyl-dimethylamine, ethylene oxide, ethyleneimine, dimethyl sulphonate, diethyl sulphonate, propane sulphone, beta-propiolactone, diazomethane, N-methyl-N-nitrosourethane, acridine orange and sodium azide.
• EMS ethyl methanesulfonate
• DEB diepoxybutane
• 2-chloroethyl-dimethylamine 2-chloroethyl-dimethylamine
• ethylene oxide ethyleneimine
• dimethyl sulphonate diethyl sulphonate
• propane sulphone beta-propiolactone
• diazomethane N-methyl-N-nitrosourethane
• the progeny, mutant or derivative plants of the present invention produce seed capsules and an increased and/or extractable quantity of codeine, oripavine and/or morphine in the first growing season after germination in one or more of the geographical locations hereinbefore described.
• the present invention also provides reproductive material derived from the plants described herein.
• reproductive material should be understood as any material from which a plant may be reproduced.
• "reproductive material” may include seeds, flowers, cuttings, ovaries, ovules, embryo sacs, egg cells, anthers, pollen, regenerable de-differentiated plant tissue such as callus, embryogenic callus or suspension culture, isolated plant embryos and the like.
• the reproductive material comprises a seed.
• a plant “seed” should be understood to refer to a mature or immature plant seed.
• the term “seed” includes, for example, immature seed carried by a maternal plant or seed released from the maternal plant.
• seed should also be understood to include any seed plant sporophyte between the developmental stages of fertilisation and germination.
• the term seed refers to a mature plant seed.
• the present invention also provides straw produced from a plant of the first aspect of the invention, or a progeny, mutant or derivative thereof.
• the "straw" of a poppy plant includes fresh or dried tissue of a poppy plant.
• This tissue may include all or part of the plant, such as root tissue, shoot tissue, floral tissue or a seed capsule.
• poppy "straw” includes fresh or dried poppy plant tissue which includes a mature seed capsule.
• the contemplated seed capsule may include seeds, or may be a capsule in which the seeds have been removed.
• a solvent for example, water or a super critical fluid, such as supercritical C0 2
• a super critical fluid such as supercritical C0 2
• the present invention also provides a poppy straw concentrate produced from a plant of the first aspect of the invention or a progeny, mutant or derivative thereof.
• poppy straw concentrate should be understood to include any material arising when poppy straw has entered into a process for the concentration of its alkaloids.
• poppy straw concentrates should also be understood to include any crude or purified extracts of poppy straw in either liquid, solid or powder form which contain one or more phenanthrene alkaloids of an opium poppy.
• poppy straw concentrates When in liquid form, poppy straw concentrates may be further concentrated from a crude extract. Such concentrates may be either liquid concentrates, wherein a portion of the solvent has been removed, or powder form concentrates which result from removing substantially all of the solvent used for extraction of the poppy straw.
• a poppy straw concentrate may include all of the alkaloids that may be extracted from a poppy straw, or may include a subset of the extractable alkaloids.
• a poppy straw concentrate of the present invention at least includes morphine and/or codeine.
• the present invention also provides latex derived from the plant of the first aspect of the invention or a progeny, mutant or derivative thereof.
• latex from a poppy plant, including P. bracteatum
• Methods for obtaining latex from a poppy plant, including P. bracteatum are well known in the art.
• latex may be obtained by incising an immature seed capsule of the plant, from which latex is exuded.
• the present invention also provides a stand of stably reproducing poppy plants, the stand comprising one or more plants according to the first aspect of the invention, or a progeny, mutant or derivative thereof.
• the present invention provides an isolated cell, tissue or organ derived from the plant of the first aspect of the invention or a progeny plant thereof.
• the present invention also provides an in-vitro culture including one or more of the cells described above.
• Exemplary "in-vitro cultures” contemplated herein include, for example, callus cultures, embryogenic callus cultures, embryo cultures, plantlet cultures and suspension cultures which include one or more of the cells of the invention. Techniques for the establishment and maintenance of plant cell or tissue cultures are well known in the art. In this regard, reference is made to 'Plant Tissue Culture: An Alternative for Production of Useful Metabolites' (FAO Agricultural Services Bulletin No. 108, Food and Agriculture Organization of the United Nations Rome, 1994).
• an in-vitro culture of P. bracteatum may be established using the method described in US Patent 4,1 14,314, with the modification that cells derived from the plants of the present invention are used instead of those disclosed in US Patent 4,114,314.
• the in-vitro cultures of the present invention may be used to produce codeine, oripavine and/or morphine.
• the production of codeine, oripavine and/or morphine by the culture may be de novo production, or may be via the conversion of a complex substrate, such as an intermediate in the morphine or codeine biosynthetic pathway.
• the present invention also provides a method of producing an alkaloid selected from codeine, oripavine and/or morphine, the method comprising growing a poppy plant of the first aspect of the invention, or a progeny, mutant or derivative thereof, such that the plant produces an alkaloid selected from codeine, oripavine and/or morphine; and extracting the codeine, oripavine and/or morphine from the poppy plant or a part thereof, or a progeny, mutant or derivative thereof.
• the method of the present invention comprises a method for producing codeine, oripavine and/or morphine in one or more of the geographical locations hereinbefore described.
• the present invention also provides codeine, oripavine and/or morphine produced according to the method above.
• the inventors have identified nucleotide sequence variants of the T60DM and CODM enzymes. Accordingly, in a further aspect, the present invention provides an isolated nucleic acid molecule comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8, 12 and 13.
• the present invention also provides T60DM and CODM nucleotide sequence variants which display at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95% at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8, 12 and 13,
• the T60DM and CODM nucleotide sequence variants provided by the aspect of the present invention above do not consist of the cDNA sequences for T60DM and CODM set forth in GenBank accessions GQ500139 and GQ500141 , respectively.
• the compared sequences should be compared over a comparison window of at least 100 nucleotide residues, at least 200 nucleotide residues, at least 400 nucleotide residues, at least 600 nucleotide residues or over the full length of the relevant one of SEQ ID NOs: 1 to 8, 12 and 13.
• the comparison window may comprise additions or deletions (ie. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such as the BLAST family of programs as, for example, disclosed by Altschul et al. ⁇ Nucl. Acids Res. 25: 3389-3402, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology” John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).
• the present invention provides a nucleotide sequence which hybridises to a nucleic acid comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8, 12 and 13 under stringent conditions.
• stringent hybridisation conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least 30°C. Stringent conditions may also be achieved with the addition of destabilising agents such as formamide. In some embodiments, stringent hybridisation conditions may be low stringency conditions, medium stringency conditions or high stringency conditions.
• Exemplary moderate stringency conditions include hybridisation in 40 to 45% formamide, 1.0 M NaCI, 1 % SDS at 37°C, and a wash in 0.5x to 1xSSC at 55 to 60°C.
• Exemplary high stringency conditions include hybridisation in 50% formamide, 1 M NaCI, 1 % SDS at 37°C, and a wash in O. lxSSC at 60 to 65°C.
• wash buffers may comprise about 0.1 % to about 1 % SDS. Duration of hybridisation is generally less than about 24 hours, usually about 4 to about 12 hours.
• T m 81.5°C +16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridisation solution, and L is the length of the hybrid in base pairs.
• the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe.
• T m is reduced by about 1 °C for each 1 % of mismatching; thus, T m , hybridisation, and/or wash conditions can be adjusted to hybridise to sequences of different degrees of complementarity. For example, sequences with >90% identity can be hybridised by decreasing the T m by about 10°C. Generally, stringent conditions are selected to be about 5°C lower than the T m for the specific sequence and its complement at a defined ionic strength and pH.
• high stringency conditions can utilise a hybridisation and/or wash at, for example, 1 , 2, 3, or 4°C lower than the T m ; medium stringency conditions can utilise a hybridisation and/or wash at, for example, 6, 7, 8, 9, or 10°C lower than the T m ; low stringency conditions can utilise a hybridisation and/or wash at, for example, 1 1 , 12, 13, 14, 15, or 20°C lower than the T m .
• the T60DM nucleotide sequence variants identified by the present inventors encode a T60DM enzymes with variant amino acid sequences. Accordingly in a further aspect, the present invention provides an isolated polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9 to 11.
• the present invention provides T60DM amino acid sequence variants which display at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95% at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 11.
• the T60DM amino acid sequence variants provided in accordance with this aspect of the invention do not consist of the amino acid sequence encoded by the reference sequence for T60DM, as represented by GenBank accession number ADD85329.1.
• the compared sequences should be compared over a comparison window of at least 50 amino acid residues, at least 100 amino acid residues, at least 150 amino acid residues, or over the full length of the relevant one of SEQ ID NOs: 9 to 1 1.
• the comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such as the BLAST family of programs as, for example, disclosed by Altschul et al. (Nucl. Acids Res. 25: 3389-3402, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).
• the nucleotide sequence set forth in GenBank accession number GQ500139 (which encodes T60DM) is cloned into a suitable expression vector, such as pBI121 (Clontech) under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S) which directs constitutive expression in P. bracteatum.
• a suitable expression vector such as pBI121 (Clontech) under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S) which directs constitutive expression in P. bracteatum.
• the transgene is introduced into P. bracteatum via Agrobacterium-mediaied transformation using the method described in Solouki et al. (2009, supra). Transformants are selected using paromomycin and regenerated according to the method described in Solouki et al. (2009, supra). Transformants are then screened for alkaloid content.
• CODM activity may also be increased in P. bracteatum in order to increase the conversion of codeine to morphine and also to increase the conversion of thebaine to orpiavine.
• Increasing the activity of CODM in the transformants may be achieved by transformation of P. bracteatum with the nucleotide sequence set forth in GQ500141 (which encodes CODM) using a similar method to that outlined above for transformation of P. bracteatum with T60DM.
• T60DM and CODM may be contransformed into P. bracteatum on a single construct or, alternatively, the two transgenes may be sequentially transformed into P. bracteatum. Where sequential transformations are performed it may become necessary to introduce an additional selectable marker gene with each sequential transformation in order to allow selection of transformants transformed with both transgenes.
• any residual CODM activity in the plants may be reduced.
• Reduction of CODM activity in P. bracteatum may be achieved using suitable genetic modification of P. bracteatum.
• suitable genetic modifications to reduce CODM expression may include, for example: random mutagenesis such as transposon, chemical, UV and phage mutagenesis together with selection of mutants which underexpress CODM;
• PTGS post-transcriptional gene silencing
• RNAi of a CODM encoding nucleic acid
• the expression of COR may be increased in the transformed Papaver bracteatum by transformation with a COR-encoding nucleotide sequence, as hereinbefore described.
• the COR-encoding nucleotide sequence may be transformed into P. bracteatum using the method of Solouki et al. (2009, supra) and may be contransformed with T60DM and/or CODM or may be sequentially transformed. Again, where sequential transformations are performed it may become necessary to introduce an additional selectable marker gene with each sequential transformation.
• a series of primers were utilised to amplify PCR products of different sizes at different locations in the T60DM and CODM genes in P. somniferum. The same primers were also used to detect whether the T60DM and CODM genes, or similar sequences, were present within the P. bracteatum genome.
• the PCR amplification conditions used were as follows: initial denaturation at 95°C for 120 seconds; 30 cycles of denaturation at 95°C for 30 seconds, primer annealing at 59°C for 30 seconds and extension at 72°C for 150 seconds; and a final elongation step of 72°C for 5 minutes. Reactions were performed with the GoTaq Green Polymerase Mix (Promega) as per the manufacturer's instructions.
• Table 2 below provides a summary of the primer combinations used to amplify various regions of the T60DM and CODM genes. The results of the PCR amplifications are shown in Figure 2 and the location of the primers in the CODM and T60DM genes is shown in Figure 3.
• T60DM and CODM genes were amplified from the genome of Papaver somniferum using the following primer sets.
• Genomic DNA was isolated from Papaver somniferum by grinding tissue samples in liquid nitrogen and mixing with three times the sample volume of extraction buffer (1 % sarcosyl, 100 mM Tris-CI, 100 mM NaCI, 10 mM EDTA, pH 8.5) followed by phenol chloroform extraction, precipitation with 0.1 volumes 3M sodium acetate (pH 4.8) and 1 volume 100% isopropanol, washing with 1 ml. 70% ethanol and resuspension in an appropriate volume of sterile distilled water with 40 ⁇ g/mL RNAse A.
• PCR conditions were as follows: initial denaturation at 98°C for 30 seconds; 25 cycles of denaturation at 98°C for 10 seconds, primer annealing at 59°C for 15 seconds and extension at 72°C for 120 seconds; and a final elongation step of 72°C for 10 minutes. Reactions were performed with the Phusion High-Fidelity DNA Polymerase (Thermo Scientific) as per the manufacturer's instructions using 200 ng of genomic DNA as template. PCR products were gel extracted (Qiagen), cloned into pGEM-T Easy (Promega) and transformed into JM109 competent cells (Promega), all according to the manufacturer's instructions.
• the cloned products were sequenced and this revealed a 2288 bp T60DM gene (relative to the 1095 bp T60DM mRNA) and a 1663 bp CODM gene (relative to the 1083 bp CODM mRNA).
• Figures 4 and 5 show nucleotide and amino acid sequence alignments, respectively, of the T60DM variants identified in comparison to the reference sequence for T60DM (i.e. GQ500139 and the encoded polypeptide represented by GenBank accession number ADD85329.1 ).
• a single nucleotide sequence variant of the CODM gene was identified which comprises the cDNA nucleotide sequence set forth in SEQ ID NO: 12.
• This cDNA nucleotide sequence variant was derived from a genomic DNA clone comprising the nucleotide sequences set forth in SEQ ID NOs: 13.
• this nucleotide sequence variant encodes a CODM enzyme with the same amino acid sequence as that encoded by the CODM reference, as represented by GenBank accession number ADD85331.1.
• Figure 6 shows a nucleotide sequence alignment of the CODM variant identified in comparison to the reference sequence for CODM (i.e. GQ500141 ).
• the cauliflower mosaic virus 35S promoter with dual enhancer motifs was amplified from the pMDC32 plasmid constructed by Curtis and Grossniklaus (Plant Phys. 133: 462-469, 2003) and inserted upstream of the cDNA sequences to drive expression.
• This 35S promoter was obtained by PCR using the 5 -CAAACGCGTCAGGTCAACATGGTGGAGCAC-3' (SEQ ID NO: 30) and 5 -CAAAGATCTGATCCTCTAGAGTCGAGGTCCTCTC-3' (SEQ ID NO: 31 ) primers, which contain the Mlu ⁇ and BglW restriction enzyme recognition sites, respectively, to amplify the promoter from 5 ng of the pMDC32 plasmid.
• the PCR amplification conditions used were as follows: initial denaturation at 98°C for 30 seconds; 30 cycles of denaturation at 98°C for 10 seconds, primer annealing at 72°C for 15 seconds and extension at 72°C for 60 seconds; and a final elongation step of 72°C for 10 minutes. Reactions were performed with the Phusion High-Fidelity DNA Polymerase (Thermo Scientific) as per the manufacturer's instructions. PCR products were gel extracted (Qiagen), cloned into pGEM-T Easy (Promega) and transformed into JM109 competent cells (Promega), all according to the manufacturer's instructions. The sequence of the amplified promoter is set forth in SEQ ID NO: 14. The promoter was excised from pGEM-T Easy by restriction digest and inserted upstream of the cDNA sequences by ligating the fragment into the Mlu ⁇ and BglW restriction enzyme recognition sites in the synthetic vectors from Mr Gene.
• the 35S::cDNA::nos terminator DNA cassette was excised by restriction enzyme digestion and inserted into the multiple cloning site of the pCAMBIA 1200 plant transformation vector.
• the 35S::T60DM::nos terminator DNA cassette was inserted into the native pCAMBIA 1200 vector containing the selectable marker gene hptll that encodes hygromycin phosphotransferase.
• a vector map for the T60DM construct is shown in Figure 7.
• the 35S::CODM::nos terminator and the 35S::COR1.1 ::nos terminator DNA cassettes were transferred into modified pCAMBIA 1200 vectors that had been modified to confer resistance to glufosinate and bispyribac sodium (BPS).
• BPS bispyribac sodium
• the Basta-resistance gene (pat) was isolated by PCR from the pTOOL2 vector (Invitrogen) and a BPS- resistance gene allele (acetolactate synthase - a/s), originally described by Oldach et al. (Annals of Botany 101 : 997-1005, 2008), was isolated from seeds of a BPS-resistant Medicago variety ("Angel").
• Both resistance genes were isolated by PCR with the Phusion High-Fidelity DNA Polymerase (Thermo Scientific) as per the manufacturer's instructions. PCR products were gel extracted (Qiagen), cloned into pGEM-T Easy (Promega) and transformed into JM109 competent cells (Promega), all according to the manufacturer's instructions. The genes were excised from pGEM-T Easy by restriction digest with Xho ⁇ and inserted into the pCAMBIA 1200 vector backbone, from which the hygromycin resistance gene had been previously excised by digestion with Xho ⁇ .
• the pat gene was amplified from 5 ng of pTOOL2 DNA with the 5'-GATCTCGAGCTTTCGATGAGCCCAGAACGACGC-3' (SEQ ID NO: 32) and 5 -AAACTCGAGCTTGTCGATCGACATCAGATTTCGGTGACGGG -3' (SEQ ID NO: 33) primers and the following amplification conditions: initial denaturation at 98°C for 30 seconds; 25 cycles of denaturation at 98°C for 10 seconds, primer annealing at 60°C for 15 seconds and extension at 72°C for 60 seconds; and a final elongation step of 72°C for 10 minutes.
• the a/s gene was amplified from 100ng of "Angle" genomic DNA with the 5'-GTTCTCGAGCTTTCGATGGCAGCCACCACCACCACC-3' (SEQ ID NO: 34) and 5'-TTACTCGAGCTTGTCGATCGACATCAATAACTCCTTCTTCCATCACC-3' (SEQ ID NO: 35) primers and the following amplification conditions: initial denaturation at 98°C for 30 seconds; 25 cycles of denaturation at 98°C for 10 seconds, primer annealing at 62°C for 15 seconds and extension at 72°C for 120 seconds; and a final elongation step of 72°C for 10 minutes.
• Vector maps showing the bar, CODM and COR1.1 constructs are shown in Figures 8 to 10, respectively.
• Agrobacterium tumefaciens strain AGL0 was used for transformations. Bacterial cells were maintained in 40% glycerol stock at -80°C. Freeze-thaw method was used for transforming AGL0 competent cells with various binary vectors (Table 3). TABLE 3
• Tubes of frozen AGLO competent cells were allowed to thaw at room temperature. 5 ⁇ of the plasmid DNA was added to 100 ⁇ of AGLO and mixed by gently flicking the tubes. The bacterial cells were frozen in liquid nitrogen for 1 min and then subjected to heat shock in a water bath at 37°C for 5 mins. 500 ⁇ of LB (Luria-Bertani) broth was added and the tubes were incubated for 2 hrs at 28°C with shaking (150 rpm). 150 ⁇ of each transformation culture was plated on to duplicate LB agar medium supplemented with antibiotics, Rifampicin (50 ⁇ g ml "1 ) and Chloramphenicol (100 ⁇ g ml " 1 ). The plates were incubated at 28°C for 2-4 d. Colony PCR was carried out for amplifying genes of interest and positive colonies were selected for plant transformation.
• PCR selected agrobacterium colonies were inoculated in 10 ml LB broth supplemented with antibiotics, Rifampicin (50 ⁇ g ml "1 ) and Chloramphenicol (100 ⁇ g ml "1 ) and incubated at 28°C for 4 d. The bacterial culture was centrifuged at 4°C, 4000 rpm for 10 mins and cells were harvested.
• Agrobacterium infection solution was prepared by adding small volumes of harvested cells to filter sterilized (Millipore ExpressTM PLUS 0.22 ⁇ ) pre-treatment medium (MS 4.4 g L “1 , glucose 18 g L “1 , sucrose 30 g L “1 , acetosyringone 10 ⁇ , pH 5.2) and the OD 6 oo was adjusted between 0.5 to 1.5.
• the calli were transferred to co-cultivation medium (MS 4.4 g L “1 , glucose 10 g L “1 , sucrose 30 g L “1 , casein 300 mg L “1 , myoinositol 100 mg L “1 acetosyringone 10 ⁇ , 2,4-D 1 mg L “1 , kinetin 0.1 mg L “1 , ascorbic acid 15 mg L “1 , pH 5.2).
• the culture plates with transformed calli were incubated in dark in the Sanyo versatile environmental chamber (MLR-351 ) at 20°C D/N for 3 d.
• the washed calli were blotted dry on sterile filter paper and transferred to regeneration medium
• the regeneration medium consists of 3 ⁇ 4 MS, sucrose 30 g L "1 , Phytagel 0.3%, pH 5.7, after autoclaving this medium was supplemented with filter sterilised stock solutions of naphthalene acetic- acid- 1 mg L "1 , benzylaminepurine-0.5 mg L “1 , cefotaxime- 400 mg L “1 and appropriate concentration of the selection agent (Table 4).
• the calli were maintained under selection (see Figures 1 1 D, E and F) and subcultured every two weeks on fresh medium until shoot regeneration was observed.
• the concentrations of selection agents determined by performing kill curve experiments on all three varieties of Papaver spp.
• Figure 1 1 provides photos evidencing callus induction and plant regeneration of P. bracteatum var PB-1.
• Regenerated P. bracteatum plantlets can be screened for the presence and/or expression of the transgene using standard techniques known in the art (e.g. PCR, Q- PCR, Southern blot, Northern blot). However, this is not entirely necessary given that identification of the presence of codeine, oripavine and/or morphine in the transgenic plants will in effect represent confirmation that the transgene is present and is being expressed.
• transformed P. bracteatum plants were grown for approximately three months, together with wild-type "control" P. bracteatum plants.
• Five small immature plants (approx. 0.10 g in total) from each group were collected and combined, and the combined plant material was macerated using a mortar and pestle.
• the alkaloid was extracted from the combined plant in 5 ml of 10% acetic acid solution.
• the liquid was separated from the plant matter, with the extracted liquid sample being used for the subsequent alkaloid content analysis.
• control plants i.e. plants lacking the T60DM and CODM genes
• the control plants do however produce thebaine as expected.
• Plants transformed with the T60DM construct were shown to produce the alkaloid codeine, thereby establishing that the pathway from thebaine to codeine had been restored in this transformant.
• Plants transformed with the CODM construct were shown to produce oripavine, thereby establishing that the pathway from thebaine to oripavine had been restored in this transformant.
• plants transformed with the CODM and T60DM constructs were able to produce all four alkaloids confirming that the pathway from thebaine to morphine (see Figure 1 ) had been restored.
• the above alkaloid analysis procedure can also be performed on more mature plants with root tissue, leaf tissue, latex and/or capsules being assayed for alkaloid content.

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