Golden Rice & Golden Crops

1. Golden Rice & Golden Crops Transgenic Plants for Food Security in the Context of Development Peter Beyer University of Freiburg Germany Pontifical Academy of Science, Vatican, May 2009

2. Nutritional Diversity Iron, Zinc Folate Provit A Vit. E Rice (grain) - - - - Tomato - - (+) + Beans + + - + Spinach + + + +

3. Iron, Zinc Folate Provit A Vit. E Rice - - - - Tomato - - (+) + Beans + + - + Spinach + + + + Meat + + Vit A + - Nutritional Diversity Facts: Threebillion live on lessthan 2 $ per day, 1.5 billion on lessthan 1 $ per dayandcannotafford a diversifieddietorindustriallyproducedsupplements • Millionsarechronicallymicronutrient • malnourished

4. Intervention strategies: • Supplementation • Industrial fortification • Education All necessary and very valuable but there are drawbacks: • Distrubution, educated medical staff • Centrally processed food items • Only partially applicable Economicallysustainable?

5. Biofortificationis an alternative toclassical interventionstofightmicronutrientdeficiencies • Improvethe nutritional valueofagronomicallyimportantcrop • tissuesthrough • Breeding • Recombinant DNA technologies is all achievable through breeding ? Simple answer: NO!!

6. 1. Somecropplants do not showadequatetrait variability Rice, (polished grains) for instance Provitamin A: Germplasmscreeningdid not revealany „yellowgrains“ with β-carotene Folate: Practically absent Iron: lowvariability, ranging from 1 – 8 ppm (Final breedingtarget 14 ppm) Zinc: muchmoreimportantvariability, ranging from 16 – 28 ppm (Target: 24 ppm)

7. Golden Rice cannotbebred ….the application of recombinant DNA technology is necessary

8. Whyengineeringß-carotene (provitamin A) biosynthesisintoriceendosperm? • Milledriceisprovitamin-a-free • Symptoms of a provitamin-a-freediet • Night-blindness • Xerophthalmia • Fatal susceptibilitytochildhooddiseases (e.g. measles) andgeneralinfections (diarrhoea, respiratorydiseases) • Epidemiology • 124 millionchildrenaredeficient in vitamin A • 1-2 milliondeathsannually (1-4 years) • 0.25-0.5 milliondeaths (5-10 years) UNICEF; Humphrey et al., 1992) • A severepublichealthproblem in (118) countries (WHO)

9. Xerophthalmia

10. Whyengineeringß-carotene (provitamin A) biosynthesisintoriceendosperm? • Milledriceisprovitamin-a-free • Symptoms of a provitamin-a-freediet • Night-blindness • Xerophthalmia • Fatal susceptibilitytochildhooddiseases (e.g. measles) andgeneralinfections (diarrhoea, respiratorydiseases) • Epidemiology • 124 millionchildrenaredeficient in vitamin A • 1-2 milliondeathsannually (1-4 years) • 0.25-0.5 milliondeaths (5-10 years) UNICEF; Humphrey et al., 1992) • A severepublichealthproblem in (118) countries (WHO)

11. E7 E6 E5 E4 E3 E2 E1 Precursor Product HOW? Assembly-linetechnologies E8 Intermediates Expressed genes DNA, mRNA

12. (HOW?) Starting Point: Wild-type E1 PP PP E2 GGPP-Synthase IPP DMAPP PP GGPP E3 Phytoene Synthase Phytoene-Synthase Phytoene E4 Phytoene Desaturase Phytofluene E5 (Cis/transIsomerase?) -Carotene E6 Neurosporene z-CaroteneDesaturase E7 Lycopene Lycopene cis/trans Isomerase E8 , -Lycopene Cyclase -Carotene -Carotene All missing (not expressed) in rice endosperm???

13. Towards a concept: PSY (E3) transformation (HOW?) E1 PP PP E2 GGPP-Synthase IPP DMAPP Wild-type riceendosperm PP GGPP E3 Phytoene-Synthase Phytoene E4 Phytoene Desaturase Phytofluene E5 (Cis/transIsomerase?) -Carotene E6 Neurosporene z-CaroteneDesaturase E7 Lycopene Lycopene cis/trans Isomerase E8 , -Lycopene Cyclase -Carotene -Carotene Wild-type endosperm can produce a precursor molecule, GGPP!

14. Assembly-linetechnologies E7 E6 E5 E4 E3 E2 E1 E8 Precursor Intermediate Product Only two appeared at work!

15. (HOW?) E1 PP PP E2 GGPP-Synthase IPP DMAPP Wild-type riceendosperm PP GGPP E3 Phytoene Synthase Phytoene-Synthase Phytoene E4 Phytoene Desaturase Phytofluene E5 (Cis/transIsomerase?) -Carotene E6 Neurosporene z-CaroteneDesaturase E7 Lycopene Lycopene cis/trans Isomerase E8 , -Lycopene Cyclase -Carotene -Carotene Itis a nightmaretotransformsix transgenes. LuckilythereisCrtI !

16. LuckilythereisCrtI ORF2 ORF3 ORF4 ORF6 transformed E. coli Pantoea ananatis carotenoid gene cluster crtI crtE crtX crtY crtB crtZ ORF12 CrtIsubstitutesfor 4 plant genes

17. CaroteneDesaturases Complex vs. „simple“ Cyanobacteria and Plants Bacteria 15-cis-Phytoene 15-cis-Phytoen PDS E4 E5 9, 15, 9‘-tri-cis-z-Carotene Z-ISO ?? CRTI E6 ZDS 9, 9‘-di-cis-z-Carotene E7 7, 9, 9‘, 7‘-tetra-cis-Lycopene CRTISO all-trans-Lycopene all-trans-Lycopin

18. The CrtIgeneproductprovides a shortcut B Plant Desaturation pathway CrtI shortcut A

19. Towards a prototype: The concept (HOW?) E1 PP PP E2 GGPP-Synthase IPP DMAPP Wild-type riceendosperm PP E3 GGPP E3 Phytoene-Synthase Phytoene E4 Phytoene Desaturase Phytofluene CrtI E5 (Cis/transIsomerase?) -Carotene E6 Neurosporene z-CaroteneDesaturase E7 Lycopene Lycopene cis/trans Isomerase E7 E8 , -Lycopene Cyclase -Carotene -Carotene ´Threeinsteadofsix!!

20. PathwayComplementation in Rice, Co-Transformation Gt1p 35Sp (1) pZPsC E3 (PSY) E,4,5,6,7 (tp-CrtI) aph IV 35Sp Gt1p (2) pZLcyH E8(ß-LCY) With the selectable marker gene only in (2), all yellow transgenic seeds expressed lycopene-ß-cyclase. They all contained ß-carotene; this was the expected outcome.

21. Control

22. hpc11

23. 0.018 0.016 0.014 0.012 0.010 0.008 0.006 0.004 0.002 0.000 -0.002 0 20 40 60 80 100 But there was a secondconstructwithout lycopene cyclase ! aph IV Gt1p 35Sp pB19hpc E3 (PSY) E3,4,5,6,7 (tp-CrtI) Single transformant hpc 2b …Lesson learned: no need for lycopene ß-cyclase Lutein ß-Carotene Zeaxanthin -Carotene …why is Golden Rice golden (yellow) instead of red??? Ye et al., 2000; Science 287:303

24. We just needtobridge a gap!! E1 PP PP E2 GGPP-Synthase IPP DMAPP Wild-type riceendosperm PP E3 GGPP E3 Phytoene-Synthase Phytoene Synthase Phytoene E4 Phytoene Desaturase Phytofluene CrtI E5 (Cis/transIsomerase?) -Carotene E6 Neurosporene z-CaroteneDesaturase E7 Lycopene Lycopene cis/trans Isomerase E8 , -Lycopene Cyclase Onlytwo transgenes arenecessary!! -Carotene -Carotene Schaub et al. (2005), Plant Physiol. 138: 441 Xanthophylls

25. Assembly-linetechnologies E7 E6 E5 E4 E3 E2 E1 E8 Precursor Intermediate Product Only two transgenes are necessary to fill the gap!!

26. Prototypes: Not apt for product development Constructill-defined Integration ill-defined Antibioticselectablemarker Low amountofbC (1.6 µg/g) Start from scratch include Indica rice varieties. Happy Easter

27. ImprovedGolden Rice variantscame in twoversions In the public and in the private sector (Syngenta - Orynova) Gt1p PSY (Np) Gt1p tp-CrtI (from Narcissus) No selectable marker gene (co-transformed and removed) Almost 1000 events Deregulation-amenable integration CrtI controlled by an endosperm-specific promoter In Cocodrie (Javanica) Amount up to 6 µg/g Three events preselected Known as Golden Rice 1 Technology works in Indica varieties

28. B A T2 Rice grains D C F E H G Hoa et al., Plant Physiol. 133, 2003

29. The preselected events (PS&S) underwent 2 field trials at Louisiana State University……

30. …where the GR1 events showed 4,8 – 7,1 µg/g

31. Improvements: The past years were dominated by efforts to increase the amount of ß-carotene in GR both, in the public sector as well as at Syngenta

32. Inefficient transgene expression? Inefficient transgene expression? Potential bottlenecks to higher carotenoid levels C3-Carbon Metabolism Precursor shortage? IPP/DMAPP Phytoene synthase (E3) activity? GGDP Phytoene Desaturation (CrtI) activity? -Carotene Lycopene ß-Carotene -Carotene Carotenoid storage? Zeaxanthin Lutein

33. Cacar 48-67-8-7 (T3) Cacar 48-67-4-9 (T3) CarNew E1-19 (T1) PMI 35Sp PSY (Np) GluBp Synth tp crtI CarNew E4-4 (T1) + control WT CrtI PSY ImprovingCrtI (E4,5,6) expression pFun3 promoter change & codon optimized pCarNew promoter change Western Achieved!!! But no significantly improved ß-carotene accumulation. Carotene desaturation is not rate-limiting in Golden Rice

34. Phytoene synthase was investigatedby Rachel Drake (Syngenta) Because PSY expression is good in GR, different versionsof the PSY gene were assayed. Seed promoter CrtI Seed promoter Ubi promoter hygR Daffodil Psy Maize Psy Rice Psy Tomato Psy Transformation into a japonica short-grain rice, (Asanohikare) 20+ plants each Pepper Psy Rice and Maize PSY (E3) Best. Proportion of ß-carotene increased.

35. Assembly-linetechnologies OK OK OK Too slow!!! E7 CrtI E1 CrtI E2 E3 CrtI Precursor Zwischenprodukt Produkt

36. Golden Rice 2 was madeforimplementation GT1pI tp-CrtI GT1pI ZmPSY ubi1p PMI pSYN12424 Transform long grain rice variety (Kaybonnet) Sugar selectable marker 619 individual GM rice plants Screen for seed colour, gene copy number, fertility Select 6 “Golden Rice 2” events for further screening and development

37. Improved provitamin A Accumulation in Golden Rice I and II

38. GR 2… • ContainsthebacterialCrtIandand PSY (but frommaize) • just likethepreviousversions. Both genes areunder • endospermspecificpromotercontrol; theselectablemarker • agentismannose. • Increase in provitamin A contentisabout 10-fold over GR1 • andabout 25-fold overthe prototype • Golden Rice ismainly a breedingprojecttoday: • Philippines (IRRI, PhilRice) • Vietnam (CLDRI) • India (IARI,TNAU, DRRI) • Bangladesh (BRRI) • Introgressing 8 eventsinto 11 varieties • Event selectioncompleted, movingtowardsderegulation

39. GR2 GR1 Wild-Type

40. 1. Somecropplants do not showadequatetrait variability Maize, for instance Maize is the world´s third most important staple crop. In maize, the pathway proceeds beyond beta carotene. The genetic variability for high carotenoid levels is very substantial, but low for provitamin A carotenoids. A Psy-CrtIcombination, asused in GR boosts ß-caroteneproduction in an African whitecultivarto 60 µg/g ! Breedingapproaches (ongoing - lycE polymorphismsidentified) haveyielded so far ca.14 µg/g ß-carotene. Naqvi et al., PNAS, 2009 Harjes et al., Science

41. 2. Somecropplantsshowadequatetrait Variability but cannotbe (easily) bred Bananas, for instance • Bananas are a staple in 50 (+) countries • (Uganda; 222 kg/person year) • East Africa Highland Bananas are • very low in micronutrients • (ProvitA 2.7 µg/g; Vit E 1 µg/g; Iron 2.6 ppm, fresh weight) • Conventional breeding: extremely difficult as bananas are essentially sterile • Most current cultivars are sterile triploids selected from the wild • Have not been genetically improved for thousands of years • Huge challenges from global movement of devastating diseases James Dale, QUT Australia, Grand Challenges in Global Health

42. Transient testing of the transgenes using direct Agro-transformation of banana fruits Ubi-CrtI NT NT Ubi-Apsy2a+CrtI Ubi-Apsy2a Preliminary HPLC data indicated increased a-carotene, b-carotene and lutein

44. 2. Somecropplantscannotbe (easily) bred Cassava, for instance • 250 million sub-Saharan Africans and 600 millions globally rely on cassava as their major source of calories • Ranks 5th among crops directly consumed by humans (No. 1 in Sub-Saharan Africa). Provides food security. • Very low in micronutrients • Provit A (mostly) 1-5 µg/g; VitE, 1 µg/g; • Iron 5 ppm, Zinc 1 ppm (fresh weight) • Varietal recovery very difficult upon • breeding (vegatativelypropageted) • Very long breeding cycle Richard Sayre, Danforth Center, St. Louis, USA, Grand Challenges in Global Health, BMGF University of Freiburg – CIAT, Harvest Plus

45. Cassava promoter CP2 - crtB Line #12 …morelinesepressing multiple genes comingthisyear

46. 2. Somecropplantscannotbe (easily) bred Potato, for instance potato ranks fourth, among the staple foods of mankind, after wheat, rice and maize CrtI 35S TP Nos pK-I CrtB CrtI Pat1 TP Ocs 35S TP Nos pK-BI CrtB CrtY Ocs CrtI pK-YBI Nos TP 35S 35S TP Nos Pat1 TP Pat2 TP CrtI pP-I Nos CrtB CrtI Ocs Pat2 TP pP-BI Nos Pat1 TP onlythisone: A mini-pathway CrtY CrtB pP-YBI CrtI Pat2 TP CrtY Nos Pat1 TP Ocs Pat2 TP Nos

47. „Golden Potato“ Diretto et al., PlosOne, 2007

48. All ofthehere-mentionedexamplesbearsignificant potential: Theyrepresentthemajorstaples The provitamin A bioavailabilityisverygood! Bioavailability: Golden Rice: 3.8:1, Human (maybe even better) Tang et al., Am J Clin Nutr 2009 Maize: 3:1 Gerbil, human study is underway Howe and Tanumihardjo, J. Nutr. 2006 Cassava: 3.7:1, Gerbil Howe et al., British Journal of Nutrition (2009) Spinach: 20:1, Human Tang et al., Am J Clin Nutr 2005; Very good bioavailability of ß-carotene from simple starchy food matrices (Like banana and potato?)

49. The problem of VAD remains : GoldenRice and other “Golden Crops” are a potentially significant contribution to alleviation. Genetic modification is an indispensible tool Breeding where possible Genetic modification where necessary

50. Rice Teams & budgets @ • IRRI • Phil Rice- Philippines • CLRRI- Vietnam • DBT, IARI, DRR,TNAU- India To all our sponsors since before 1990: • ETH / Swiss Federal Funds • European Commission • HarvestPlus • USAID • Syngenta Company • Syngenta Foundation • National Institutes of Health (USA) • Bill and Melinda Gates Foundation • The Golden Rice Humanitarian Board • Bayer, Mogen, Novartis, Monsanto, Orynova, Zeneca