GENET archive


2-Plants: Finally: blue GE roses

                                  PART I
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TITLE:  Roses are red, and now blue... with the help of CSIRO technology
SOURCE: CSIRO, Australia
DATE:   30 Mar 2005

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Roses are red, and now blue... with the help of CSIRO technology

In July 2004, Melbourne biotechnology company Florigene and its partner,
Japan's Suntory corporation, announced the realisation of rose breeding's
impossible dream: a truly blue rose.

A truly blue rose has been the Holy Grail of rose breeders since 1840,
when the horticultural societies of Britain and Belgium offered a prize
of 500,000 francs to the first person to produce a blue rose.

Molecular geneticists with Florigene and Suntory achieved the prize that
had long eluded conventional rose breeders by combining something old,
something new, something borrowed, and something blue.

Roses are very old garden subjects - a favourite for some 5,000 years.
The 'something blue' was the delphinidin gene that Florigene's
geneticists cloned from a pansy, to direct pigment synthesis in the rose
into the 'blue' pathway. The 'something borrowed' was an iris gene for an
enzyme, DFR, required to complete the delphinidin-synthesis reaction.

And the 'something new' was a man-made gene, designed by Suntory
geneticists, that exploited a powerful new CSIRO-developed technology to
switch off a rose gene that had frustrated Florigene's efforts to
activate the delphinidin pathway in roses for nearly a decade.

Suntory's scientists created the 'silencer' gene to exploit a cellular
phenomenon called RNA interference (RNAi). RNAi technology is
revolutionizing research and development across the biological and
medical sciences, and has been hailed as the most important new research
tool to emerge in the past 25 years.

Dr Peter Waterhouse's research team at CSIRO Plant Industry in Canberra
pioneered the use of RNAi as a high-precision tool for exploring and
manipulating gene function in plants.

The Florigene-Suntory rose is doubly historic: although the prototype is
pale mauve, it is the first rose in the world with the genetic potential
to produce 'true blue' roses, spanning the spectrum from palest blue to
Mediterranean blue, or even navy blue.

And as potentially the first commercial plant in the world to exploit
RNAi technology, Florigene's blue rose is a harbinger of the
extraordinary future of plant breeding in the 21st century.

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

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.

How RNAi works

By the early 1990s, plant molecular geneticists suspected that plant
cells possess a mechanism that somehow detects and destroys the genetic
blueprints of invading viruses.

Dr Waterhouse's CSIRO team confirmed the existence of such a mechanism in
an historic experiment in 1997.

For decades, conventional plant breeders have laboured to develop virus-
resistant crops, using naturally occurring genes from resistant cultivars
or their wild relatives. Waterhouse and his colleagues constructed a
synthetic gene, containing a segment of genetic code copied from Potato
Virus Y (PVY), a virulent virus that infects tobacco and other members of
the potato-tomato-nightshade family.

They inserted the synthetic gene into laboratory tobacco plants known to
be susceptible to PVY infection. Seeds from these transgenic plants
produced seedlings that were completely resistant to infection.

The historic CSIRO experiment opened the way for molecular geneticists to
use RNAi technology to create synthetic resistance genes that can protect
any crop plant against any pathogenic virus, or to explore and manipulate
the function of genes in plants.

The cells of all complex organisms - animals, plants, algae and
protozoans like the malaria parasite - are now known to contain tiny
'nanomachines' called RISCs (RNA-induced silencing complexes).

As a prelude to replicating in the cells of their hosts, viruses make a
double-stranded RNA (dsRNA) copy of their genomes.

The host's own genes make single-stranded RNA copies of their own genetic
code, which serve as 'recipes' for the assembly of the proteins encoded
by the genes. The cell's protein-synthesis machinery cannot process
double-stranded viral RNAs; when the cell detects a double-stranded viral
RNA, it activates its RISCs, which contain special RNA-cleaving enzymes,
to destroy them.

The RISCs 'remember' the genetic code of any virus they encounter, and
rapidly reactivate should they encounter that virus again.

By making a double-stranded RNA copy of a unique segment of genetic code
from a known virus, and incorporating it into a synthetic gene, molecular
geneticists can now make any crop plant resistant to any known virus - a
plant could be protected against several viruses by a single RNAi gene,
armed with code sequences from each virus.

Dr Waterhouse's research team, and others around the world, have also
shown that RISC complexes can also be programmed with short sequences of
code, copied from the plant or animal cell's own genes, to suppress the
activity of any gene, with extraordinary precision. Knockout of a gene's
activity provides clues to its function.

Europe's Agrikola research consortium has used a Hellsgate RNAi vector -
a plug-and-play molecular cassette developed by CSIRO's Dr Chris
Helliwell, to develop more than 20,000 knockout lines of the plant
geneticist's 'green rat', Arabidopsis thaliana - one for almost every
gene in the Arabidopsis genome.

Arabidopsis is a model for the world's crop plants. Knowing the function
of every gene will allow crop geneticists to modify crop plants to
improve yields, adapt them to challenging environments - including saline
or acid soils, or drought, or to develop plants to yield new food or
industrial products, including plastics.

CSIRO's Dr Allan Green is applying an RNAi package that will selectively
silence genes for enzymes involved in fatty acid synthesis in oilseed
crops like cotton, linseed/flax and canola.

By adding other enzyme genes, Dr Green's team plans to modify today's
oilseed crops to produce oils rich in omega-3 fatty acids, which prevent
heart disease and have other health-giving properties - among other
things, they are important to brain development and function.

Currently, omega-3 oils are extracted from marine fish and added to
foods; RNAi technology can be used to engineer ultra low saturated oils
for the introduction of omega 3 fatty acids and create a sustainable
resource to take pressure off wild fish stocks.

                                  PART II
-------------------------------- GENET-news -------------------------------

TITLE:  World's first blue rose
SOURCE: CSIRO, Australia
        grafic attached: PI_bluerose_diagram.jpg
DATE:   29 Mar 2005

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World's first blue rose

Breeding a blue rose has been the 'Holy Grail' of rose breeding for
centuries, but roses have proven a particularly difficult candidate to
turn blue. That has all now changed with an Australian based company,
Florigene, part of the Japanese Suntory company, successfully using
CSIRO's gene silencing technology to help create the world's first blue rose.

Roses are famous for their beautiful colours including red, pink, orange,
yellow and even white. These colours have been developed through
traditional breeding but never has a blue rose successfully been bred.

Some mauve roses have been bred but as it turns out these colours are
actually produced by variations of red pigment not by the production of
blue pigment.

To develop the world's first blue rose with blue pigment three steps had
to be achieved:

1. Turn off the production of red pigment;

2. Open the 'door' to production of blue pigment; and then

3. Produce blue pigment.

One gene involved in flower colour, is the dihydroflavonol reductase
(DFR) gene. The DFR gene makes the enzyme dihydroflavonol reductase (DFR)
which turns on the manufacturing process in the plant that produces
pigment that in turn colours flowers.

In roses the DFR gene is very good at producing red pigment and hence the
range of commonly seen rose colours. However, the rose DFR gene is
particularly bad at producing blue pigment, hence the difficulty in
breeding a blue rose.

The first critical step in producing a blue rose was to stop the rose DFR
gene making red pigment.

Preventing red pigment

CSIRO first developed gene silencing, or hairpin RNAi, in 1997. It was a
significant breakthrough allowing scientists to turn down or switch off
completely the activity of genes.

Gene silencing uses a natural mechanism that degrades RNA - the courier
that delivers the gene's instructions to make proteins, like the enzyme DFR.

Florigene used CSIRO's gene silencing technology to turn off the activity
of the rose DFR gene so that it didn't produce red pigment.

Gene silencing has been used in a number of research applications to
determine gene function and in the development of experimental plants
with favourable properties. Its use in the development of the blue rose
is likely to be its first commercial application.

Opening the blue door

The second step towards a blue rose was to open the 'door' to allow for
blue pigment to be produced.

The production process of colouring flowers is like a pathway. In roses
the pathway to producing red pigment is open, but the blue pathway is closed.

Florigene inserted a gene commonly called a delphinidin gene from pansy
that opened the door to the production of blue pigment in the rose flowers.

Importing the blue colour

With the red pigment production turned off using CSIRO's gene silencing
and the door open to the production of blue pigment, Florigene's final
task was to find a DFR gene good at producing blue and placing it in the rose.

Florigene decided to replace the rose DFR gene with a DFR gene from an
iris, which is excellent at producing blue pigment. The iris DFR gene was
inserted into the rose and subsequently a rose with a blue flower was

A bluer rose

While the prototype blue rose made by Florigene is in fact a pale mauve-
blue colour it is the first rose of this colour that comes from blue
pigment. The colour of other 'blue' roses currently on the market is only
a modification of red pigment.

Even bluer flowers should be achievable if rose petals can be made less
acidic, as acidity inhibits blue pigment.

Florigene and Suntory researchers are searching for genes that affect
petal acidity or that affect petal colour in other ways, to breed a bluer

Commercial availability

Florigene has already successfully created blue carnations using gene
technology and these have been available in Australia since 1996.

It will be at least 3 years before blue roses will be commercially
available in Australia, pending approval from the Office of the Gene
Technology Regulator for their commercial release.


European NGO Network on Genetic Engineering

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