FLORIGENE RESEARCH
The company’s research activity remains focussed on colour modification using genes of the anthocyanin biosynthesis pathway and the “blue rose” program remains high on the company’s list of objectives. Florigene maintains its research facility in the Melbourne suburb of Bundoora, and its R&D team has over the years, in collaboration with its Japanese partner Suntory, accumulated a significant patent portfolio - primarily in the area of anthocyanin biosynthesis (see Intellectual Property, below). It is through manipulation of the anthocyanin biosynthetic pathway present in most flowers that Florigene has created new varieties of carnation. These products are protected using a combination of patents, plant breeder’s rights (PBR) and trade marks. More recently Florigene and Suntory have developed a novel coloured rose which is scheduled for marketing in 2-3 years.

Flower Colour Modification
Anthocyanins are the most prevalent class of pigment compounds in plants and in flowers these are deposited in the large vacuoles of petal epidermal cells where most flower colour is localised. Anthocyanins are relatively simple, water soluble, structures with the hydroxylation pattern of the B ring at positions 3’, 4’ and 5’ being a key colour determinant. Further modifications to the molecule are common and beyond imparting small changes to max lend stability to molecular complexes involving anthocyanin molecules, copigment molecules (typically flavonols, a product of the same pathway) and in notable cases metal ions. Such complexes are further influenced by petal epidermal vacuolar pH. All of these components impact directly on the observed colour.

A key branch point of the anthocyanin biosynthetic pathway is centred around the intermediate DHK (dihydrokaempferol). The enzymes FLS (flavonol synthase), F3’H (flavonoid 3’ hydroxylase), F3’5’H (flavonoid 3’5’ hydroxylase) and DFR (dihydroflavonol reductase) all utilise this substrate suggesting that this region of the pathway is a key determinant of anthocyanin biosynthesis and thus flower colour.

Florigene R&D has cloned and protected nearly all genes in the anthocyanin/flavonoid pathway from petunia and numerous homologues from a diverse number of species in close collaboration with Suntory Research in Japan. These genes are the centrepoint of Florigene Intellectual Property (see below) and our capability in the manipulation of anthocyanin-based flower colour modification.

A schematic of a key section of the anthocyanin biosynthetic pathway showing dihydrokaempferol and the three pathway branches leading to production of different colours.

INTELLECTUAL PROPERTY
Patents Florigene and Suntory, under the IFD (International Flower Developments) banner, have been successful in pursuing broad patent claim coverage in many countries around the world. These patents provide Florigene with a strong position in the Company’s strategic area of technological competence namely manipulation of anthocyanin-based flower colour. The Table below provides a list of PCT application numbers from which Florigene’s national phase patents are derived.

IFD/Florigene PCT Applications
PCT Numbers Title
PCT/AU92/00334-Genetic sequences encoding flavonoid pathway enzymes & uses therefore
PCT/AU94/00265- Transgenic flowering plants
PCTAU96/00296- Transgenic plants exhibiting altered flower color & methods for producing same
PCT/AU93/00127- Genetic sequences encoding flavonoid pathway enzymes & uses therefore
PCT/AU97/00124- Genetic sequences encoding flavonoid pathway enzymes & uses therefore
PCT/AU93/00387- Genetic sequences encoding glycosyltransferase enzymes & uses therefore
PCT/AU93/00400- Genetic sequences encoding flavonol synthase enzyme & uses therefore
PCT/US92/02612- Carnation plants & methods for their production & genetic transformation
PCT/US91/04412- Rose plants & methods for their production & genetic transformation
PCT/US91/05805- Methods for the transformation & production of chrysanthemum plants
PCT/AU01/00358- Plant anthocyanidin rutinoside aromatic acyl transferases
PCT/JP01/04677- New Aliphatic Acyltransferase Gene
PCT/GB02/00928- Cell visual characteristic-modifying sequences
PCT/AU03/00079- Genetic sequences & uses therefore
PCT/AU03/01111- Genetic sequences & uses therefore

Trade Marks
Florigene’s series of floral product branding affords recognition of our novel varieties with their chosen trade name and moreover an association between these varieties and Florigene.

Plant Breeder’s Rights To complement Florigene’s patent and trade mark portfolios, Plant Breeders Rights (PBRs) have been sought in key territories especially those important to our breeding partners.

List of Relevant Publications
Brugliera, F., Holton, T.A., Stevenson, T.W., Farcy, E., Lu, C-Y. and Cornish, E.C. (1994) Isolation and characterization of a cDNA clone corresponding to the Rt locus of Petunia hybrida. Plant J. 5, 81-92.

Brugliera, F., Barri-Rewell, G., Holton, T. and Mason, J. (1999) Isolation and characterization of a flavonoid 3’-hydroxylase cDNA clone corresponding to the Ht1 locus of Petunia hybrida. Plant J. 19, 441-451.

Chandler SF (2003) Commercialization of genetically modified ornamental plants. J. Plant Biotech. 5: 69-77

Davies KM and Shwinn KE (1997) Biotechnology of ornamental plants. In: Geneve RL., Preece JE and Markle SA (eds) Biotechnology of Ornamental Plants )pp259-294). CAB International, Wallingford

Forkmann G and Martens S (2001) Metabolic engineering and applications of flavonoids. Curr. Opin. Biotechnol. 12: 155-160.

Forkmann G and Heller W (1999) Biosynthesis of flavonoids. In: Sankawa U (ed) Polyketides and other secondary metabolites including fatty acid and their derivatives (pp 713-748). Elsevier, Amsterdam.

Holton, T.A., Brugliera, F. Lester, D.R., Tanaka, Y. Hyland, C.D. Menting, J.G.T., Lu, C., Farcy, E., Stevenson, T.W. and Cornish, E.C. (1993) Cloning and expression of cytochrome P450 genes controlling flower colour. Nature, 366, 276-279.

Holton, T.A., Brugliera, F. and Tanaka, Y. (1993) Cloning and expression of flavonol synthase from Petunia hybrida. Plant J. 4, 1003-1010.

Holton, T.A. and Cornish, E.C. (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell, 7, 1071-1083.

Mol J, Cornish E, Mason J and Koes R (1999) Novel coloured flowers. Curr. Opin. Biotechnol. 10: 198-201

Tanaka Y and Mason J (2003) Manipulation of flower colour by genetic engineering. In: Singh RP and Jaiwal PK (eds) Plant Genetic Engineering (pp 361-385). SCI Tech Publishing, Houston

Tanaka Y, Tsuda S and Kusumi T (1998) Metabolic engineering to modify flower colour. Plant Cell Physiol 39: 1119-1126

Tanaka Y, Tsuda S and Kusumi T (1999) Application of recombinant DNA to floriculture. In Chopra VL, Malik VS and Bhat SR (eds) Applied Plant Biotechnology (pp. 177-231). Oxford and IBH, New Dehli

Tanaka Y, Katsumoto Y, Brugliera F and Mason J (2004) Genetic engineering in floriculture. Plant Cell Tissue and Organ Culture (in press).

Winkel-Shirley B. (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 126:485-493.

Winkel-Shirley B. (2001) It takes a garden. How work on diverse plant species has contributed to an understanding of flavonoid metabolism. Plant Physiol. 127(4):1399-404.

Winkel-Shirley B (2002) Biosynthesis of flavonoids and effects of stress. Curr Opin. Plant Biol. 5: 218-223.

Licensing
Florigene IP is available for limited licensing. For all inquiries please send correspondence marked to the attention of the R&D Manager, Florigene Pty Ltd, 1 Park Drive, Bundoora,Victoria - 3083, Australia or via email to jmason@florigene.com.au.