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 .

1. Golden Rice –
A Humanitarian Biotechnology Project
COMM4BIOTECH Congress; 18-19 Nov. 2011
Peter Beyer, Center for Applied Biosciences, University of Freiburg, Germany

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. 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. 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. 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. Nutritional Diversification
Iron, Zinc Folate Provit A
Rice (milled grain) - - -
Tomato - - (+)
Beans + + -
Spinach + + +

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. 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?

9. Biofortification is a cost effective alternative to
classical interventions
Improve the nutritional value of agronomically important crop
tissues 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. Some traits cannot be bred because there is no
adequate trait variability
Rice, (polished grains) for instance
Provitamin A: No „yellow grains“ in
germplasm collections
Folate: Practically absent
Iron: low variability, ranging from 1 – 8 ppm (Target 14 ppm)
Zinc: much more important variability, ranging from 16 – 28 ppm
(Target: 24 ppm)

11. Golden Rice cannot be bred
….the application of recombinant
DNA technology is necessary
Breeding where possible
Genetic modification where necessary

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. Xerophthalmia

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. Global prevalence of vitamin A deficiency in populations at risk 1995–2005
WHO Global Database on Vitamin A Deficiency

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

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-Carotene
All missing (not expressed) in rice endosperm???

18. We just need to bridge a gap!!
HOW? E1
PP PP
IPP DMAPP
E2 GGPP-Synthase
Wild-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. Assembly-line technologies
E8 E7
E6
E5 E3 E2 E1
E4
Precursor
Intermediate
Product

20. So many transgenes?
E1
HOW? PP PP
That´s not such a great idea!
IPP DMAPP
E2 GGPP-Synthase
Wild-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. Luckily there is a bacterial gene, CrtI
transformed E. coli
Pantoea ananatis carotenoid gene cluster
crtE crtX crtY crtI crtB crtZ ORF12
ORF2 ORF3 ORF4 ORF6
CrtI substitutes for 4 plant genes

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

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

24. Only two transgenes needed
E1
PP PP
IPP DMAPP
E2 GGPP-Synthase
Wild-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. 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. Improved Golden Rice variants came in two versions
In 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. The preselected events underwent 2 field trials
at Louisiana State University……

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

29. Improvements:
Five years of research
were dominated by
efforts to increase the
amount of provitamin
A in GR both, in the
Public Sector as well
as at Syngenta

30. Potential bottlenecks to higher carotenoid levels
C3-Carbon Metabolism
Precursor
shortage?
IPP/DMAPP
Phytoene 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. 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 Psy
Transformation 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. E3 (PSY is rate-limiting)
OK
OK
OK OK
Too slow!!!
E8
CrtI E1
CrtI CrtI E3 E2
Precursor
Zwischenprodukt
Produkt

33. Golden Rice 2 was re-made for implementation
GT1pI tp-CrtI GT1pI ZmPSY ubi1p PMI
pSYN12424
Transform 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. Improved provitamin A
accumulation in Golden
Rice I and II
Increase in provit-A content: ca. 10-fold over GR1; about 25-fold
over the prototype

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. 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 China
Bioconversion 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. 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 China
Bioconversion 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. 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. Event dependent studies
Molecular 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. Phenotype analysis
Field performance, typical agronomic traits, yield -
compared to isogenic lines.
Pest and disease status to be same as isogenic background.
Compositional analysis
Data 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. 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. The „Danger Zone“ is in Europe
Requirements:
Raised to the point where anything that appears technically
feasible is being requested/offered to be applied. This is in
part driven by some public sector scientists.
Justification:
Genetically modified plants are little understood (while we
understand traditionally bred or mutagenized varieties?)
Kuiper et al., 2001 Assessment of the food safety issues related to
genetically modified foods. Plant J. 2001 Sep;27(6):503-28.
Consequence:
Can raise costs to unaffordable levels - delays.

43. Overregulation of novel technologies: the „Locomotive Act“
England, 1861 (the Red Flag Act)
Self-propelled vehicles on public roads in the
United Kingdom must be preceded by a man
on foot waving a red flag and
blowing a horn.
This effectively killed road auto development
in the UK for most of the rest of the 19th
century
The red flag law was not
repealed until 1896

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 Release
The 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. First Outdoor Trial
of GR in Asia
IR64 GR1 event 309; 20 lines
Transplanting at IRRI on April 2, 2008 May 30, 2008
April 10, 2008

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. The Focus Group
normally 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. 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. The problem of VAD remains :
GoldenRice is a potentially
significant contribution to alleviation.
It is now on its way towards registration

50. GR2 GR1
Wild-Type
For additional information, see www.goldenrice.org

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