The making of the blue rose

A class of plant molecules called anthocyanins gives rise to the rich variety of colours seen in flowers, fruits and other plant tissues. The major floral pigments derive from anthocyanins, with some contributions from yellow carotenoids.

The anthocyanin dihydrokaempferol (DHK) is the precursor for all three primary plant pigments: cyanidin, pelargonidin and delphinidin.

The cyanidin gene codes for an enzyme that modifies DHK, directing it into the cyanidin pigment pathway, which produces deep red, pink and lilac-mauve hues. The delphinidin gene - not present in roses - codes for a closely related enzyme that modifies DHK to direct pigment synthesis into the delphinidin pathway.

Another enzyme, dihydroflavinol reductase (DFR) further modifies the precursor pigments in all three pathways. Up to this point, all precursor pigment molecules are colourless, so any mutation that disrupts the DFR gene results in white flowers. Florigene geneticists selected a white DFR-mutant carnation to develop the company's Moonseries carnations.

Like roses, carnations lack the delphinidin gene. Florigene introduced a delphinidin gene from petunia, coupling it with the petunia DFR gene, to replace the mutant carnation DFR gene.

Florigene's new lilac- and mauve-hued carnations, with names like 'Moondust' and 'Moonglow', now dominate the North and South American carnation cut-flower markets; the European Union has yet to approve their release.

During the 20th century, rose hybridists created an extraordinary range of novel floral hues. They included lilac and grey roses, which were hailed as a step toward truly blue roses. However, they are now known to be unusual variants from the cyanidin pathway. It is now clear that the conventional hybridization could not have produced a blue rose, because roses are genetically incapable of producing delphinidin.

Founded in 1986 as Calgene Pacific, Florigene's major commercial goal was to use gene technology to create the world's first truly blue rose. It acquired Dutch rival Florigene in 1994 and adopted its name.

Florigene's scientists took a giant step by cloning the delphinidin gene from a petunia in 1991. By the mid-1990s they had perfected techniques for genetically transforming roses and regenerating plants from transformed cell lines in tissue culture.

It enabled Florigene to create the first roses with delphinidin. By the mid-1990s, Florigene had high level expression of delphinidin in an old red variety, 'Cardinal'.

The combination of cyanidin and delphinidin yielded a very attractive dark burgundy rose. It wasn't blue, but technically it was a major advance.

To create a blue rose, Florigene researchers needed a white rose in which the DFR gene was inactivated. But they were unable to identify a DFR-knockout white rose ready-made for cut flower production - breeding one from scratch would have added years to the project.

Florigene researchers regularly consulted Dr Waterhouse's team at CSIRO Plant Industry. In 2001 Dr Waterhouse discussed how RNAi technology could be used to switch off one gene in such a way that it could be replaced by a related gene. Florigene saw the advantage of using RNAi to switch off the DFR gene in a red rose, to block the cyanidin pathway, and then install the delphinidin gene - plus a new DFR gene to complete delphinidin synthesis.

Suntory's researchers had the same idea - they used RNAi to create a synthetic gene to suppress the DFR gene in a shapely pink rose.

They cloned a new version of the delphinidin gene, from pansy, and, on a hunch, teamed it with a DFR gene from iris.

The rose and iris genes are quite similar, and share much of their DNA code, but RNAi is so exquisitely precise that they were able to design a RNAi 'hairpin' gene targeting a DNA sequence exclusive to the rose DFR gene, so the 'knockout' had no effect on the imported iris DFR gene.

The three-gene package (pansy delphinidin, iris DFR, anti-rose DFR) package worked: Suntory's transgenic rose produced very high levels of delphinidin in its petals, and a small residue of cyanidin.

The new rose is an attractive shade of mauve, similar to the current generation of mauve-lilac roses like 'Blue Moon' and 'Vol de Nuit'. But where these cultivars express cyanidin, and are thus incapable of yielding blue flowers, the new rose, with further 'tweaking', has the genetic potential to be truly blue.

Blue shades should be achievable if Florigene and Suntory researchers can make the rose's petals less acidic. Rose petals are moderately acidic, with a pH around 4.5, while carnation petals are less so, with a pH of 5.5.

Florigene and Suntory researchers have 'fished around' for roses with higher petal pH, but the low-acidity trait appears to be genetically limited in roses. Researchers are now using RNAi gene-knockout technology to identify genes that influence petal acidity, or that modulate petal colour in other ways.

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