1. Opium poppy, Papaver somniferum Alternative products – alternative uses.
Phil Larkin
CSIRO, Canberra
Poppy is erect annual plant. Large flowers, various petal colour. Sessile leaves. Fruit is a capsule, bearing thousands of seeds.

2. Australian poppy industry
>50% of world’s legally traded opiates
Demonstrated the power of genetics to transform the industry to new products
Thebaine poppies (top1 mutation)
Thebaine feedstock for high value painkillers, such as buprenorphine, oxycodone.

5. Top1 mutation CO52 Thebaine Control mutant Zero morphine and codeine.
Proprietary mutation of Tasmanian Alkaloids
Morphine-pathway block in top1 poppies.
Millgate, Pogson, Wilson, Kutchan, Zenk, Gerlach, Fist, Larkin.
Nature 431: (2004)

6. Transgenic poppies
Biotechnology is another strategy capable of transforming the industry:
Increasing morphinan content
Novel products

7. Transformation of Poppy
Hypocotyl explants
Agrobacterium
Black primary callus
White embryogenic callus
Somatic embryos
Poppy (Papaver somniferum) belongs to Papaveraceae; little in the literature for this genus, or any other in the Family, to indicate whether it would be easy to transform.
Thanks largely to Julie Chitty, we were able to achieve transgenic plants quite quickly even though there were a number of unusual features to the behaviour in culture – eg. initial callus goes black!

9. Metabolic Engineering of Opium Poppy
Increasing alkaloid yield
thebaine
morphine
Engineering novel products
reticuline
morphine
hpRNAi
anticancer
and
antimalarial
activities
analgesics

10. COR gene transcript is increased
Transgenic over-expression of COR (codeinone reductase) increases yield
COR gene transcript is increased
Morphinan alkaloid content is increased …..
About 10 fold increase in transcript

11. Cor transgenics GH2002 GH2003 FIELD2003 GH2004
Southerns indicate most of these have 3-5 copies of transgene.
Paromomycin resistance segregation is simple except for which has two or more effective transgene resistance loci.

12. COR over-expression – Field Trial
% DW
Morphine
Codeine
Thebaine
Total
CO58-34
control
2.83
0.19
0.21
3.25 1a
3.43 *
0.24
0.22
3.91 1b
3.32
0.31
0.32
3.97 2
3.44
0.38
4.17 ** 3
3.31
0.30
0.28
3.95 4
3.38
0.27
0.26
3.92 7
3.45
0.25
0.18
3.89 8
3.16
3.60
* , P<0.05
**, P<0.01
Control n=18, 20 plants per replicate
Transgenics n=6, 20 plants per replicate

13. Metabolic engineering for alternative products
Zero opium poppy by genetic engineering
CSIRO gene silencing technology to shut down all opiates.
Hairpin RNAi
E.g. Target gene: codeinone reductase
Accumulation of alternative non-opiate product, reticuline.

14. Opiate-free poppy
codeinone reductase

15. Potential feedstock for other bioactives
Opiate-free poppy
Potential feedstock for other bioactives
Allen, RS et al. (2004) RNAi-mediated replacement of morphine with nonnarcotic alkaloid reticuline in opium poppy.
Nature Biotechnology 22,

16. Opiate-free poppy
Transgenic
poppies
Control
poppies
standards
Morphine, codeine and thebaine replaced by reticuline, codamine, laudanine and laudanosine. Ls Ln Cm R T O C M
This example is with a enzyme encoded by a multigene family. Two hairpin cassettes were designed which together should silence all six members of the family.
Alternative intermediates did accumulate in every one of nearly 30 transgenics.
The new chemical phenotype was inherited with the transgenes.

17. Proposal for Afghan Poppy Industry
Phased transformation to a legal non-opiate industry:
Phase 1. Biodiesel from poppy seed oil.
Phase 2. Higher value “soft” biodiesel.
Phase 3. Industrial and pharmaceutical products.

18. Poppyseed oil for biodiesel.
Current world diesel consumption 1087 billion litres p.a.
Biodiesel currently accounts for 0.4%.
Poppy yields 1.8 tonnes/ha seed (cf. 2.4 for canola)
45-50% oil content (cf. 40% in canola)
0.8 tonnes oil per ha.
Afghan poppy area in 2004 was 131,000 ha
<2 % of arable land
Would produce 100,000 tonnes of oil
= 2.5% of current world biodiesel consumption.
Scope to expand production greatly.
Protein “meal” byproduct as food and feed.

19. Phase 1. Biodiesel from poppy seed oil.
Replacement seed of opiate-free poppies mutation
Mutant cv. Sujata, opiate-free, opium-free poppy
(CSIR Lucknow, India)
Village-scale oil press and biodiesel processing
Local energy supply
Transport fuel
Electricity generators
Basis of a carbon-neutral export biodiesel industry.
Low tech and high volume
Requiring minimal adjustment from the poppy growing culture and expertise.
Begins the process of legalising the industry:
Agronomic support
Potential Carbon subsidies
Breeding support
Trade support

21. Phase 2. Higher value “soft” biodiesel.
Value adding to poppy oil with biotechnology
Increased oil yield
Overexpressing DAGAT2 (boost transfer of last acyl to glycerol)
High oleic biodiesel.
Polyunsaturates can form oxidised polymers which reduce engine performance.
High oleic oils (monounsaturates) are preferred.
Well attested CSIRO technology used to produce high oleic oils in soybean, canola, flax and cottonseed oils:
RNAi silencing 12-desaturase (high oleic)
RNAi silencing FATB thioesterase (eliminate palmitic)
High oleic poppy oil also a healthier food oil.