Golden Rice –A Humanitarian Biotechnology Project COMM4BIOTECH Congress; 18-19 Nov. 2011Peter Beyer, Center for Applied Bi...
WHY?Population growth                        10000                         8000 Population [million]                      ...
Directly, or indirectly, plants provide all of               humanity’s food During the past 100 years agriculture has (of...
Share of daily energy intake   for rural Bangladesh                                  Staples (rice)                       ...
Directly, or indirectly, plants provide all of               humanity’s food During the past 100 years agriculture has (of...
Nutritional Diversification                  Iron, Zinc   Folate   Provit ARice (milled grain)   -          -         -Tom...
Nutritional Diversity               Iron, Zinc   Folate   Provit A   Vit. E                            Facts:  Rice   Thre...
Intervention strategies:       Supplementation       Industrial fortification       EducationAll necessary and very valuab...
Biofortification is a cost effective alternative to               classical interventionsImprove the nutritional value of ...
Some traits cannot be bred because there is no  adequate trait variability    Rice, (polished grains) for instanceProvitam...
Golden Rice cannot be bred….the application of recombinant  DNA technology is necessary             Breeding where possibl...
Why engineering ß-carotene(provitamin A) biosynthesis into rice           endosperm? Milled rice is provitamin-a-free Symp...
Xerophthalmia
Why engineering ß-carotene(provitamin A) biosynthesis into rice           endosperm? Milled rice is provitamin-a-free Symp...
Global prevalence of vitamin A deficiency in populations at risk 1995–2005WHO Global Database on Vitamin A Deficiency
HOW?         Assembly-line technologiesE8       E7                     E6                            E5 E3 E2 E1          ...
HOW?                   E1                            Starting Point: Wild-type                  PP        PP            IP...
We just need to bridge a gap!!   HOW?                            E1                              PP           PP          ...
Assembly-line technologiesE8   E7              E6                    E5 E3 E2 E1                      E4                  ...
So many transgenes?                                   E1  HOW?                        PP           PP                     ...
Luckily there is a bacterial gene, CrtI                                transformed                 E. coli       Pantoea a...
Carotene Desaturases                     Complex vs. „simple“     Cyanobacteria and Plants                                ...
The CrtI gene product provides a shortcut           B                     Plant Desaturation pathway  CrtIshortcutA
Only two transgenes needed                                   E1                              PP           PP              ...
Prototypes:               Not apt for               product development               Construct ill-defined               ...
Improved Golden Rice variants came in two versionsIn the public and in the private sector           Gt1p       PSY (Np)   ...
The preselected events underwent 2 field trials     at Louisiana State University……
…where the GR1 events showed 4,8 – 7,1 µg/g
Improvements:Five years of researchwere dominated byefforts to increase theamount of provitaminA in GR both, in thePublic ...
Potential bottlenecks to higher carotenoid levels                C3-Carbon Metabolism                                     ...
Phytoene synthase was investigated by Rachel Drake (Syngenta)  Because PSY expression is good in GR, different versions of...
E3 (PSY is rate-limiting)OK     OK                   OK   OK                             Too slow!!!E8     CrtI           ...
Golden Rice 2 was re-made for implementation             GT1pI        tp-CrtI        GT1pI   ZmPSY      ubi1p   PMI       ...
Improved provitamin A                                      accumulation in Golden                                      Ric...
GR was a breeding project during the past 4 years 9 events into 15 selected varieties (MAS), preceding event selection    ...
Event selection                         (All single locus intact ingtegration)  Which event(s) produce consistent levels o...
Event selection                         (All single locus intact ingtegration)  Which event(s) produce consistent levels o...
Regulatory dossiers can be complex and expensive                 Event independent studies                     Exposure ev...
Event dependent studiesMolecular characterization and genetic stability   Single copy effect; marker gene at same locus.  ...
Phenotype analysis           Field performance, typical agronomic traits, yield -                        compared to isoge...
Why in the „Public Sector“?  Some products of high general demand are not commercialy interesting.The private sector will ...
The „Danger Zone“ is in EuropeRequirements: Raised to the point where anything that appears technicallyfeasible is being r...
Overregulation of novel technologies: the „Locomotive Act“            England, 1861 (the Red Flag Act)Self-propelled vehic...
Where we are in the timeline?                    (Philippines - first launch)                            Production of sta...
First Outdoor Trial                                           of GR in Asia                                           IR64...
Activities beyond technical and safety issues                       Golden Rice:    communication & social marketing      ...
The Focus Groupnormally 10-12 people for structured open ended discussion for               2 hours, with careful recordin...
First social marketing research was coducted in 2009(then continued by a company)                                   Focus ...
The problem of VAD remains :       GoldenRice is a potentially significant contribution to alleviation.It is now on its wa...
GR2                                                     GR1                                                     Wild-TypeF...
Philippines     generally high VAD, (and increasing) especially Visayas & W Mindanao                    (figures are % of ...
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Golden Rice – A Humanitarian Biotechnology Project

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Presentation of Golden Rice Co-inventor Peter Beyer, professor at the university of Freiburg, at the Comm4Biotech conference 2011 in Strasbourg - more information on http://www.comm4biotech.eu .

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Golden Rice – A Humanitarian Biotechnology Project

  1. 1. Golden Rice –A Humanitarian Biotechnology Project COMM4BIOTECH Congress; 18-19 Nov. 2011Peter Beyer, Center for Applied Biosciences, University of Freiburg, Germany
  2. 2. WHY?Population growth 10000 8000 Population [million] 6000 4000 Developing Nations 2000 0 Industrialized Nations 1990 2000 2010 2020 2030 2040 2050 Year Source: C. Lupi, BATS report, I/95
  3. 3. Directly, or indirectly, plants provide all of humanity’s food During the past 100 years agriculture has (of necessity) focused on increasing yield. Nutrient content has largely been ignored in breeding. More of our food comes from fewer species (54% from corn, rice and wheat). We’ve lost biochemical diversity in the staples in our diets. Plants, in principle provide all macro -and micronurients required, but the latter are very unevenly distributed.
  4. 4. Share of daily energy intake for rural Bangladesh Staples (rice) Non-staples (Vegetables) 84 % Fish and other animal sources Source: H. Bouis, IFPRI; HarvestPlus
  5. 5. Directly, or indirectly, plants provide all of humanity’s food During the past 100 years agriculture has (of necessity) focused on increasing yield. Nutrient content has largely been ignored in breeding. More of our food comes from fewer species (54% from corn, rice and wheat). We’ve lost biochemical diversity in our diets. Plants provide all macro -and micronurients required, but the latter are very unevenly distributed in plant tissues.
  6. 6. Nutritional Diversification Iron, Zinc Folate Provit ARice (milled grain) - - -Tomato - - (+)Beans + + -Spinach + + +
  7. 7. Nutritional Diversity Iron, Zinc Folate Provit A Vit. E Facts: Rice Three billion live on-less than - $ per day, - 2 -1.5 billion on less than 1 $ per day and cannot afford a Tomato - - (+) +diversified diet or industrially produced supplements Beans + + - + Spinach + + + + Meat + + Vit A + - • Millions are chronically micronutrient malnourished
  8. 8. Intervention strategies: Supplementation Industrial fortification EducationAll necessary and very valuable but there are drawbacks: Distrubution, educated medical staff Centrally processed food items Only partially applicable Economically sustainable?
  9. 9. Biofortification is a cost effective alternative to classical interventionsImprove the nutritional value of agronomically important croptissues through Breeding Recombinant DNA technology is all achievable through breeding ? Simple answer: NO!! Some crops cannot be bred or breeding is very difficult Trait variability is insufficient www.harvestplus.org
  10. 10. Some traits cannot be bred because there is no adequate trait variability Rice, (polished grains) for instanceProvitamin A: No „yellow grains“ ingermplasm collectionsFolate: Practically absentIron: low variability, ranging from 1 – 8 ppm (Target 14 ppm)Zinc: much more important variability, ranging from 16 – 28 ppm(Target: 24 ppm)
  11. 11. Golden Rice cannot be bred….the application of recombinant DNA technology is necessary Breeding where possible Genetic modification where necessary
  12. 12. 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)
  13. 13. Xerophthalmia
  14. 14. 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) • A severe public health problem in (118) countries (WHO)
  15. 15. Global prevalence of vitamin A deficiency in populations at risk 1995–2005WHO Global Database on Vitamin A Deficiency
  16. 16. HOW? Assembly-line technologiesE8 E7 E6 E5 E3 E2 E1 E4 Precursor Intermediates Expressed genes Product
  17. 17. HOW? E1 Starting Point: Wild-type PP PP IPP DMAPP E2 GGPP-Synthase PP GGPP E3 Phytoene Synthase Phytoene-Synthase Phytoene Phytofluene E4 Phytoene Desaturase ζ-Carotene E5 (Cis/trans Isomerase?) Neurosporene E6 ζ-Carotene Desaturase E7 Lycopene Lycopene cis/trans Isomerase E8 α, β-Lycopene Cyclase β-Carotene α-CaroteneAll missing (not expressed) in rice endosperm???
  18. 18. We just need to bridge a gap!! HOW? E1 PP PP IPP DMAPP E2 GGPP-SynthaseWild-type rice endosperm PP GGPP E3 Phytoene Synthase Phytoene-Synthase Phytoene Phytofluene E4 Phytoene Desaturase ζ-Carotene E5 Cis/trans Isomerase Neurosporene E6 ζ-Carotene Desaturase Lycopene E7 Lycopene cis/trans Isomerase E8 α, β-Lycopene Cyclase β-Carotene α-Carotene Schaub et al. (2005), Plant Physiol. 138: 441 Xanthophylls
  19. 19. Assembly-line technologiesE8 E7 E6 E5 E3 E2 E1 E4 Precursor Intermediate Product
  20. 20. So many transgenes? E1 HOW? PP PP That´s not such a great idea! IPP DMAPP E2 GGPP-SynthaseWild-type rice endosperm PP GGPP E3 Phytoene Synthase Phytoene-Synthase Phytoene Phytofluene E4 Phytoene Desaturase ζ-Carotene E5 Cis/trans Isomerase Neurosporene E6 ζ-Carotene Desaturase Lycopene E7 Lycopene cis/trans Isomerase E8 α, β-Lycopene Cyclase β-Carotene α-Carotene Luckily there is CrtI ! Xanthophylls Schaub et al. (2005), Plant Physiol. 138: 441
  21. 21. Luckily there is a bacterial gene, CrtI transformed E. coli Pantoea ananatis carotenoid gene cluster crtE crtX crtY crtI crtB crtZ ORF12ORF2 ORF3 ORF4 ORF6 CrtI substitutes for 4 plant genes
  22. 22. Carotene Desaturases Complex vs. „simple“ Cyanobacteria and Plants Bacteria 15-cis-Phytoene 15-cis-Phytoen PDSE4E5 Ζ-ISO 9, 15, 9‘-tri-cis-z-Carotene CRTIE6 ZDS 9, 9‘-di-cis-z-CaroteneE7 CRTISO 7, 9, 9‘, 7‘-tetra-cis-Lycopene all-trans-Lycopene all-trans-Lycopin
  23. 23. The CrtI gene product provides a shortcut B Plant Desaturation pathway CrtIshortcutA
  24. 24. Only two transgenes needed E1 PP PP IPP DMAPP E2 GGPP-SynthaseWild-type rice endosperm PP E3 GGPP E3 Phytoene Synthase Phytoene-Synthase Phytoene Phytofluene E4 Phytoene Desaturase CrtI ζ-Carotene E5 Cis/trans Isomerase Neurosporene E6 ζ-Carotene Desaturase Lycopene E7 Lycopene cis/trans Isomerase E8 α, β-Lycopene Cyclase β-Carotene α-Carotene Schaub et al. (2005), Plant Physiol. 138: 441 Xanthophylls
  25. 25. Prototypes: 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.Happy Easter Ye et al., 2000; Science 287:303
  26. 26. Improved Golden Rice variants came in two versionsIn the public and in the private sector Gt1p PSY (Np) Gt1p tp-CrtI (from Narcissus) No selectable marker gene Ca. 1000 events „Clean“ integration selected CrtI controlled by an endosperm-specific promoter Three high expressing events preselected Known as Golden Rice 1 Technology works in Indica varieties
  27. 27. The preselected events underwent 2 field trials at Louisiana State University……
  28. 28. …where the GR1 events showed 4,8 – 7,1 µg/g
  29. 29. Improvements:Five years of researchwere dominated byefforts to increase theamount of provitaminA in GR both, in thePublic Sector as wellas at Syngenta
  30. 30. Potential bottlenecks to higher carotenoid levels C3-Carbon Metabolism Precursor shortage? IPP/DMAPPPhytoene synthase GGDP (E3) activity? Inefficient Inefficient transgene Phytoene transgene expression?Desaturation expression? ζ-Carotene (CrtI) activity? Lycopene ß-Carotene α-Carotene Carotenoid storage? Zeaxanthin Lutein
  31. 31. Phytoene synthase was investigated by Rachel Drake (Syngenta) Because PSY expression is good in GR, different versions of the PSY gene were assayed. Seed promoter CrtI Seed promoter Daffodil Psy Ubi promoter hygR Maize Psy Rice Psy Tomato PsyTransformation into a japonica short-grain rice,(Asanohikare) 20+ plants each Pepper Psy 20 18 Rice and Maize PSY (E3) Carotenoid content (μg g dwt) 16 14 best. Proportion of -1 12 10 8 ß-carotene increased. 6 4 2 0 rice maize pepper tomato daffodil Psy/crtI Psy/crtI Psy/crtI Psy/crtI Psy/crtI Individual transgenic plant (event) Paine et al., 2005 Nat Biotechnol. 23, 482-487
  32. 32. E3 (PSY is rate-limiting)OK OK OK OK Too slow!!!E8 CrtI E1 CrtI CrtI E3 E2 Precursor Zwischenprodukt Produkt
  33. 33. Golden Rice 2 was re-made for implementation GT1pI tp-CrtI GT1pI ZmPSY ubi1p PMI pSYN12424Transform long grain rice variety (Kaybonnet)Sugar selectable marker (PMI) 619 individual GM rice plants Screen for seed colour, gene copy number, fertility Select 6 “Golden Rice 2” events for further screening and development
  34. 34. Improved provitamin A accumulation in Golden Rice I and IIIncrease in provit-A content: ca. 10-fold over GR1; about 25-foldover the prototype
  35. 35. GR was a breeding project during the past 4 years 9 events into 15 selected varieties (MAS), preceding event selection IR64 & IR36: Mega-varieties with broad Asian coverage BRRI dhan 29 : The most popular boro rice variety in Bangladesh PSB Rc82: The most popular rice variety in the Philippines OS 6561: Most popular in Vietnam Chehirang: Leading variety in Indonesia (with IRRC) Stacking With other micronutrients (zinc, iron vit E, lysine) Stacking With new agronomic traits (Submergence tolerance) Partner Institutions: o IRRI (Int.), lead o Philrice (Philippines) o BRRI (Bangladesh) o CLRRI (Vietnam) o IARI (India) o TNAU (India) o DRR (India) o Huazhong Univ. (China)
  36. 36. Event selection (All single locus intact ingtegration) Which event(s) produce consistent levels of povitamin A across cultivars? Which event(s) reproduce consistently the characteristics of the recurrent parents? (Completed, all other events destroyed) The big question: Human bioavailability study Which level of provitamin A must be delivered by GR to be effective? Conducted at TUFTS first and then in ChinaBioconversion determined: 3.8-to-1 (!!!)Tang et al., (2009) Golden Rice is an effective source of vitamin A. Am J Clin Nutr89:1776–83
  37. 37. Event selection (All single locus intact ingtegration) Which event(s) produce consistent levels of povitamin A across cultivars? Which event(s) reproduce consistently the characteristics of the recurrent parents? (Completed, all other events destroyed) The big question: All done? Which level of provitamin A must be delivered by GR to be effective? Human bioavailability study Conducted at TUFTS first and then in ChinaBioconversion determined: 3.8-to-1 (!!!)Tang et al., (2009) Golden Rice is an effective source of vitamin A. Am J Clin Nutr89:1776–83
  38. 38. Regulatory dossiers can be complex and expensive Event independent studies Exposure evaluation Modelling analysis for intended use. Bioavailability study. Protein production and equivalence Extraction from GMO plant or heterologous source Biochemical characterisation Function/ specificity/ mode of action. Protein evaluation No homology with toxins and allergens. Rapid degredation in gastric /intestinal studies. Heat lability No indication of acute toxicity in rodents. Further allergenicity assessments
  39. 39. Event dependent studiesMolecular characterization and genetic stability Single copy effect; marker gene at same locus. Simple integration; Mendelian inheritance over three generations (minimum). No potential gene disruption. No unknown open reading frames. No DNA transfer beyond borders.No antibiotic resistance gene or origin of replication. Insert limited to the minimum necessary. Insert plus flanking plant genome sequenced. Phenotypic evidence for stability over 3 generations Biochemical evidence for stability. Unique DNA identifier for tracebility/detection. Expression profiling Gene expression levels at key growth stages. Evidence for seed-specific expression.
  40. 40. Phenotype analysis Field performance, typical agronomic traits, yield - compared to isogenic lines. Pest and disease status to be same as isogenic background. Compositional analysisData from 2 seasons x 6 locations x 3 reps. on proximates, macro and micro nutrients, antinutrients, inherent toxins and allergens. Data generated on modified and isogenic background. Environmental risk assessment Minimize potential for gene flow. Evaluate any change in insect preference – by field survey. Data submitted must be of scientific publication quality (but will seldomly result in academic recognition) Significant Costs
  41. 41. Why in the „Public Sector“? Some products of high general demand are not commercialy interesting.The private sector will not make major investments. Hence the public sector should take over. Funding is required To conduct more proof-of principle experimentation and translational research No expectations for funding in € Public research funding on transgenic crops in Europe goes into „Biosafety“ research. There is virtually no funding for research that goes beyond discovery.(the development if GR relies on US-Philanthropy and on National funding in GR-implementing countries )
  42. 42. The „Danger Zone“ is in EuropeRequirements: Raised to the point where anything that appears technicallyfeasible is being requested/offered to be applied. This is inpart driven by some public sector scientists.Justification:Genetically modified plants are little understood (while weunderstand traditionally bred or mutagenized varieties?)Kuiper et al., 2001 Assessment of the food safety issues related togenetically modified foods. Plant J. 2001 Sep;27(6):503-28.Consequence:Can raise costs to unaffordable levels - delays.
  43. 43. Overregulation of novel technologies: the „Locomotive Act“ England, 1861 (the Red Flag Act)Self-propelled vehicles on public roads in theUnited Kingdom must be preceded by a manon foot waving a red flag andblowing a horn.This effectively killed road auto developmentin the UK for most of the rest of the 19thcentury The red flag law was not repealed until 1896
  44. 44. Where we are in the timeline? (Philippines - first launch) Production of stable lines; fingerprinting 2007 (Bioavailability, biosafety & other studies) 2008 BPI application Single location trial of elite lines (2 2009 seasons), generation of protein data Multi-location trials; gather 2010 agronomic and biosafety data (2 seasons) 2013/14 ReleaseThe Philippines has experience in the regulatory review of a transgenic nutritionally-enhanced crop: LY038. 7 GMOs approved in the Philippines (http://agbios.com/dbase.php)
  45. 45. First Outdoor Trial of GR in Asia IR64 GR1 event 309; 20 lines Transplanting at IRRI on April 2, 2008 May 30, 2008April 10, 2008
  46. 46. Activities beyond technical and safety issues Golden Rice: communication & social marketing Consumers Growers (+ Policy Makers, Heath Community, Regulators, Traders, NGOs, etc. ) Focus groups: Qualitative information on consumer & grower attitudes in vitamin A-deficient areas
  47. 47. The Focus Groupnormally 10-12 people for structured open ended discussion for 2 hours, with careful recording MBA students of the Asian Institute of Management, Manila –training at AIM, IRRI. PhilRice
  48. 48. First social marketing research was coducted in 2009(then continued by a company) Focus Group Methodology In 4 selected islands in the Philippines AIM-MBA students teams from these areas ~ 720 hours interviews with 360 individuals in selected groups: gender, rice farming or not farming Examples of the story‐board designs
  49. 49. The problem of VAD remains : GoldenRice is a potentially significant contribution to alleviation.It is now on its way towards registration
  50. 50. GR2 GR1 Wild-TypeFor additional information, see www.goldenrice.org
  51. 51. Philippines generally high VAD, (and increasing) especially Visayas & W Mindanao (figures are % of that population with VAD)Children, 6 Months to 5 Years Pregnant Women Lactating Women Source: Philippine Government Statistics 1998, 2003

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