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.

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.

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 431:413-414 (2004)

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

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!

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

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

Cor transgenics GH2002 GH2003 FIELD2003 GH2004 Southerns indicate most of these have 3-5 copies of transgene. Paromomycin resistance segregation is simple except for 206-4-7 which has two or more effective transgene resistance loci.

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

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.

Opiate-free poppy codeinone reductase

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, 1559-66.

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.

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.

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.

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

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.