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

2. Iron, ZincFolateProvit AVit. E Rice (grain) ---- Tomato -- (+)+ Beans ++ - + Spinach ++++ Nutritional Diversity

3. Iron, ZincFolateProvit AVit. E Rice ---- Tomato -- (+)+ Beans ++ - + Spinach ++++ Meat ++ Vit A + - Nutritional Diversity Facts: Three billion live on less than 2 $ per day, 1.5 billion on less than 1 $ per day and cannot afford a diversified diet or industrially produced supplements Facts: Three billion live on less than 2 $ per day, 1.5 billion on less than 1 $ per day and cannot afford a diversified diet or industrially produced supplements Millions are chronically micronutrient malnourished Millions are chronically micronutrient 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 Economically sustainable?

5. Biofortification is an alternative to classical interventions to fight micronutrient deficiencies is all achievable through breeding ? Simple answer: NO!! Improve the nutritional value of agronomically important crop tissues through 1.Breeding 2.Recombinant DNA technologies

6. 1. Some crop plants do not show adequate trait variability variability Rice, (polished grains) for instance Provitamin A: Germplasm screening did not reveal any „yellow grains“ with β-carotene Folate: Practically absent Iron: low variability, ranging from 1 – 8 ppm (Final breeding target 14 ppm) Zinc: much more important variability, ranging from 16 – 28 ppm (Target: 24 ppm)

7. Golden Rice cannot be bred ….the application of recombinant DNA technology is necessary

8. Why engineering ß-carotene (provitamin A) biosynthesis into rice endosperm?  Milled rice is provitamin-a-free  Symptoms of a provitamin-a-free diet Night-blindness Xerophthalmia Fatal susceptibility to childhood diseases (e.g. measles) and general infections (diarrhoea, respiratory diseases)  Epidemiology 124 million children are deficient in vitamin A 1-2 million deaths annually (1-4 years) 0.25-0.5 million deaths (5-10 years) UNICEF; Humphrey et al., 1992) A severe public health problem in (118) countries (WHO)

9. Xerophthalmia

10. Why engineering ß-carotene (provitamin A) biosynthesis into rice endosperm?  Milled rice is provitamin-a-free  Symptoms of a provitamin-a-free diet Night-blindness Xerophthalmia Fatal susceptibility to childhood diseases (e.g. measles) and general infections (diarrhoea, respiratory diseases)  Epidemiology 124 million children are deficient in vitamin A 1-2 million deaths annually (1-4 years) 0.25-0.5 million deaths (5-10 years) UNICEF; Humphrey et al., 1992) A severe public health problem in (118) countries (WHO)

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

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

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

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

15. Phytoene-Synthase PP GGPP-Synthase IPPDMAPP GGPP Lycopene cis/trans Isomerase Phytoene Phytofluene  -Carotene Neurosporene Lycopene E1 E2 E3 E4 E5 E6 E7  -Carotene  -Carotene Phytoene Synthase  -Carotene Desaturase Phytoene Desaturase Wild-type rice endosperm (Cis/trans Isomerase?) E8 ,  -Lycopene Cyclase (HOW?) It is a nightmare to transform six transgenes. Luckily there is CrtI !

16. Luckily there is CrtI ORF2ORF3ORF4ORF6 crtE crtXcrtY crtI crtBcrtZORF12 transformed E. coli CrtI substitutes for 4 plant genes Pantoea ananatis carotenoid gene cluster

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

18. The CrtI gene product provides a shortcut A B Plant Desaturation pathway CrtI shortcut

19. Phytoene-Synthase PP GGPP-Synthase IPPDMAPP GGPP Lycopene cis/trans Isomerase Phytoene Phytofluene  -Carotene Neurosporene Lycopene E1 E2 E3 E4 E5 E6 E7  -Carotene  -Carotene  -Carotene Desaturase Phytoene Desaturase Wild-type rice endosperm (Cis/trans Isomerase?) E8 ,  -Lycopene Cyclase (HOW?) E3 CrtI E7 Towards a prototype: The concept ´Three instead of six!!

20. Pathway Complementation in Rice, Co-Transformation E3 (PSY) E,4,5,6,7 (tp-CrtI) 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. pZLcyH pZPsC aph IV 35Sp Gt1p (1) (2)

21. Control

22. hpc11

23. But there was a second construct without lycopene cyclase ! pB19hpc E3 (PSY)E3,4,5,6,7 (tp-CrtI) aph IV35SpGt1p Single transformant hpc 2b 20406080100 0 0.000 0.018 0.006 0.012 0.002 0.004 0.008 0.010 0.014 0.016 -0.002 ß-Carotene Zeaxanthin Lutein  -Carotene …Lesson learned: no need for lycopene ß-cyclase …why is Golden Rice golden (yellow) instead of red??? Ye et al., 2000; Science 287:303

24. Phytoene-Synthase PP GGPP-Synthase IPPDMAPP GGPP Lycopene cis/trans Isomerase Phytoene Phytofluene  -Carotene Neurosporene Lycopene E1 E2 E3 E4 E5 E6 E7  -Carotene  -Carotene Phytoene Synthase  -Carotene Desaturase Phytoene Desaturase Wild-type rice endosperm (Cis/trans Isomerase?) E8 ,  -Lycopene Cyclase We just need to bridge a gap!! Xanthophylls E3 CrtI Only two transgenes are necessary!! Schaub et al. (2005), Plant Physiol. 138: 441

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

26. Prototypes: Happy Easter Not apt for product development Construct ill-defined Integration ill-defined Antibiotic selectable marker Low amount of bC (1.6 µg/g) Start from scratch include Indica rice varieties.

27. Improved Golden Rice variants came in two versions In the public and in the private sector (Syngenta - Orynova) Gt1pPSY (Np)tp-CrtIGt1p (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. A B C D E F G H T 2 Rice grains 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. Precursor shortage? Carotenoid storage? Phytoene synthase (E3) activity? Inefficient transgene expression? Potential bottlenecks to higher carotenoid levels IPP/DMAPP C3-Carbon Metabolism GGDP Phytoene  -Carotene Lycopene ß-Carotene Lutein  -Carotene Zeaxanthin Desaturation (CrtI) activity?

33. Improving CrtI (E4,5,6) expression pFun3 promoter change & codon optimized pCarNew promoter change GluBpSynth tp crtIPMI35SpPSY (Np) WT CarNew E1-19 (T1) Cacar 48-67-4-9 (T3) CarNew E4-4 (T1) Cacar 48-67-8-7 (T3) + control CrtI PSY Achieved!!! But no significantly improved ß-carotene accumulation. Carotene desaturation is not rate-limiting in Golden Rice Western

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

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

36. Golden Rice 2 was made for implementation pSYN12424 ZmPSYubi1p 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 PMItp-CrtIGT1pI

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

38. GR 2… Contains the bacterial CrtI and and PSY (but from maize) just like the previous versions. Both genes are under endosperm specific promoter control; the selectable marker agent is mannose. Increase in provitamin A content is about 10-fold over GR1 and about 25-fold over the prototype Golden Rice is mainly a breeding project today: Philippines (IRRI, PhilRice) Vietnam (CLDRI) India (IARI,TNAU, DRRI) Bangladesh (BRRI) Introgressing 8 events into 11 varieties Event selection completed, moving towards deregulation

39. GR2 GR1 Wild-Type

40. 1. Some crop plants do not show adequate trait variability variability Maize, for instance Naqvi et al., PNAS, 2009 Harjes et al., Science A Psy-CrtI combination, as used in GR boosts ß-carotene production in an African white cultivar to 60 µg/g ! Breeding approaches (ongoing - lycE polymorphisms identified) have yielded so far ca.14 µg/g ß-carotene. 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.

41. 2. Some crop plants show adequate trait 2. Some crop plants show adequate trait Variability but cannot be (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-Apsy2a Ubi-Apsy2a+CrtI Ubi-CrtI NT Preliminary HPLC data indicated increased  -carotene,  -carotene and lutein

44. 2. Some crop plants cannot be (easily) bred 2. Some crop plants cannot be (easily) bred Cassava, for instance 250 million sub-Saharan Africans and 600 millions globally rely on cassava as their major source of calories Ranks 5 th 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 (vegatatively propageted) 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 …more lines epressing multiple genes coming this year

46. Pat1 CrtB TP 35SNosTP Pat2 Nos35S CrtI TP pK-I pK-BI pK-YBI pP-I pP-BI pP-YBI Nos35STPOcs Pat1TP Nos35STP Ocs Pat2 CrtI TP Nos Pat1TP OcsPat2TP Nos Pat1TP OcsPat2TP NosTPCrtYNos CrtI CrtB CrtY CrtBCrtI CrtBCrtY only this one: A mini-pathway 2. Some crop plants cannot be (easily) bred 2. Some crop plants cannot be (easily) bred Potato, for instance potato ranks fourth, among the staple foods of mankind, after wheat, rice and maize

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

48. All of the here-mentioned examples bear significant potential: They represent the major staples The provitamin A bioavailability is very good! 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. 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 Rice Teams & budgets @ IRRI Phil Rice- Philippines CLRRI- Vietnam DBT, IARI, DRR,TNAU- India To all our sponsors since before 1990: