Golden Rice –A Humanitarian Biotechnology Project
Peter Beyer, Center for Applied Biosciences, University of Freiburg, Germany
COMM4BIOTECH Congress; 18-19 Nov. 2011
Source: C. Lupi, BATS report, I/95
Pop
ulat
ion
[milli
on]
Year
0
2000
4000
6000
8000
10000
1990 2000 2010 2020 2030 2040 2050
Industrialized Nations
Population growth
Developing Nations
WHY?
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.
Staples (rice)
Non-staples(Vegetables)Fish and otheranimal sources
Source: H. Bouis, IFPRI; HarvestPlus
Share of daily energy intake for rural Bangladesh
84 %
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.
Iron, Zinc Folate Provit A
Rice (milled grain) - - -Tomato - - (+)Beans + + -Spinach + + +
Nutritional Diversification
Iron, Zinc Folate Provit A Vit. E
Rice - - - -Tomato - - (+) +Beans + + - +Spinach + + + +Meat + + Vit A + -
Nutritional DiversityFacts:
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 micronutrientmalnourished
Intervention strategies:SupplementationIndustrial fortificationEducation
All necessary and very valuable but there are drawbacks:
Distrubution, educated medical staffCentrally processed food itemsOnly partially applicable
Economically sustainable?
Biofortification is a cost effective alternative toclassical interventions
is all achievable through breeding ?
Simple answer: NO!!
Improve the nutritional value of agronomically important croptissues through
BreedingRecombinant DNA technology
Some crops cannot be bred or breeding is very difficultTrait variability is insufficient
www.harvestplus.org
Some traits cannot be bred because there is noadequate 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)
Golden Rice cannot be bred
….the application of recombinantDNA technology is necessary
Breeding where possible
Genetic modification where necessary
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)
Xerophthalmia
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)
Global prevalence of vitamin A deficiency in populations at risk 1995–2005WHO Global Database on Vitamin A Deficiency
Assembly-line technologies
E2 E1E3E4E5E6
E7
Precursor
Product
Intermediates
HOW?
Expressed genes
E8
Phytoene-Synthase
PP
PP PP
GGPP-SynthaseIPP DMAPP
GGPP
Lycopene cis/trans Isomerase
Phytoene
Phytofluene
ζ-Carotene
Neurosporene
Lycopene
E1
E2
E3
E4
E5
E6E7
β-Carotene α-Carotene
Phytoene Synthase
ζ-Carotene Desaturase
Phytoene Desaturase
Starting Point: Wild-type
(Cis/trans Isomerase?)
All missing (not expressed) in rice endosperm???
E8 α, β-Lycopene Cyclase
HOW?
Phytoene-Synthase
PP
PP PP
GGPP-SynthaseIPP DMAPP
GGPP
Lycopene cis/trans Isomerase
Phytoene
Phytofluene
ζ-Carotene
Neurosporene
Lycopene
E1
E2
E3
E4
E5
E6E7
β-Carotene α-Carotene
Phytoene Synthase
ζ-Carotene Desaturase
Phytoene Desaturase
Wild-type rice endosperm
Cis/trans Isomerase
E8 α, β-Lycopene Cyclase
We just need to bridge a gap!!
Xanthophylls Schaub et al. (2005), Plant Physiol. 138: 441
HOW?
E2 E1E3E4E5E6
E7E8
Precursor
Product
Intermediate
Assembly-line technologies
Phytoene-Synthase
PP
PP PP
GGPP-SynthaseIPP DMAPP
GGPP
Lycopene cis/trans Isomerase
Phytoene
Phytofluene
ζ-Carotene
Neurosporene
Lycopene
E1
E2
E3
E4
E5
E6E7
β-Carotene α-Carotene
Phytoene Synthase
ζ-Carotene Desaturase
Phytoene Desaturase
Wild-type rice endosperm
Cis/trans Isomerase
E8 α, β-Lycopene Cyclase
So many transgenes?That´s not such a great idea!
Xanthophylls Schaub et al. (2005), Plant Physiol. 138: 441
Luckily there is CrtI !
HOW?
Luckily there is a bacterial gene, CrtI
ORF2 ORF3 ORF4 ORF6
crtE crtX crtY crtI crtB crtZ ORF12
transformed E. coli
CrtI substitutes for 4 plant genes
Pantoea ananatis carotenoid gene cluster
15-cis-Phytoene
Cyanobacteria and Plants
7, 9, 9‘, 7‘-tetra-cis-Lycopene
ZDS 9, 9‘-di-cis-z-Carotene
Ζ-ISO 9, 15, 9‘-tri-cis-z-Carotene
PDS
all-trans-Lycopene
CRTISO
all-trans-Lycopin
CRTI
15-cis-Phytoen
Bacteria
Carotene DesaturasesComplex vs. „simple“
E4
E5
E7
E6
The CrtI gene product provides a shortcut
A
B
Plant Desaturation pathway
CrtI shortcut
Phytoene-Synthase
PP
PP PP
GGPP-SynthaseIPP DMAPP
GGPP
Lycopene cis/trans Isomerase
Phytoene
Phytofluene
ζ-Carotene
Neurosporene
Lycopene
E1
E2
E3
E4
E5
E6E7
β-Carotene α-Carotene
Phytoene Synthase
ζ-Carotene Desaturase
Phytoene Desaturase
Wild-type rice endosperm
Cis/trans Isomerase
E8 α, β-Lycopene Cyclase
Only two transgenes needed
Xanthophylls
E3
CrtI
Schaub et al. (2005), Plant Physiol. 138: 441
Prototypes:
Happy Easter
Not apt forproduct development
Construct ill-definedIntegration ill-definedAntibiotic selectable markerLow amount of bC (1.6 µg/g)
Start from scratch include Indica ricevarieties.
Ye et al., 2000; Science 287:303
Improved Golden Rice variants came in two versions
In the public and in the private sector
Gt1p PSY (Np) tp-CrtIGt1p
(from Narcissus)
No selectable marker geneCa. 1000 events„Clean“ integration selectedCrtI controlled by an endosperm-specific promoterThree high expressing events preselectedKnown as Golden Rice 1 Technology works in Indica varieties
The preselected events underwent 2 field trialsat Louisiana State University……
…where the GR1 events showed 4,8 – 7,1 µg/g
Improvements:
Five years of researchwere dominated by efforts to increase theamount of provitamin A in GR both, in the Public Sector as well as at Syngenta
Precursor shortage?
Carotenoid storage?
Phytoene synthase(E3) activity? Inefficient
transgene expression?
Inefficient transgene
expression?
Potential bottlenecks to higher carotenoid levels
IPP/DMAPP
C3-Carbon Metabolism
GGDP
Phytoene
ζ-Carotene
Lycopene
ß-Carotene
Lutein
α-Carotene
Zeaxanthin
Desaturation(CrtI)
activity?
Daffodil Psy
Maize Psy
Rice Psy
Tomato Psy
Pepper Psy
Seed promoter Ubi promoter hygRSeed promoterCrtI
Transformation into a japonica short-grain rice, (Asanohikare) 20+ plants each
02468
101214161820
ricePsy/crtI
maizePsy/crtI
pepperPsy/crtI
tomatoPsy/crtI
daffodilPsy/crtI
Individual transgenic plant (event)
Car
oten
oid
cont
ent (μg
g-1 d
wt) 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.
Paine et al., 2005 Nat Biotechnol. 23, 482-487
E2E1
E3
E8
Precursor
Produkt
Zwischenprodukt
E3 (PSY is rate-limiting)
Too slow!!!OK
OK
CrtICrtICrtI
OK
OK
Golden Rice 2 was re-made for implementation
pSYN12424
ZmPSY ubi1p
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
PMItp-CrtIGT1pI GT1pI
Improved provitamin Aaccumulation in GoldenRice I and II
Increase in provit-A content: ca. 10-fold over GR1; about 25-fold over the prototype
9 events into 15 selected varieties (MAS), preceding event selectionIR64 & IR36: Mega-varieties with broad Asian coverage BRRI dhan 29 : The most popular boro rice variety in BangladeshPSB Rc82: The most popular rice variety in the PhilippinesOS 6561: Most popular in VietnamChehirang: Leading variety in Indonesia (with IRRC) Stacking With other micronutrients (zinc, iron vit E, lysine)Stacking With new agronomic traits (Submergence tolerance)
GR was a breeding project during the past 4 years
Partner Institutions:o IRRI (Int.), leado Philrice (Philippines)o BRRI (Bangladesh)o CLRRI (Vietnam)o IARI (India)o TNAU (India)o DRR (India)o Huazhong Univ. (China)
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)
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
The big question:
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)
Which level of provitamin A must be delivered by GR to be effective? Human bioavailability studyConducted 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
The big question:All done?
Exposure evaluationModelling analysis for intended use.
Bioavailability study.
Protein production and equivalenceExtraction from GMO plant or heterologous source
Biochemical characterisation
Function/ specificity/ mode of action.
Protein evaluationNo homology with toxins and allergens.
Rapid degredation in gastric /intestinal studies.
Heat lability
No indication of acute toxicity in rodents.
Further allergenicity assessments
Event independent studies
Regulatory dossiers can be complex and expensive
Molecular characterization and genetic stabilitySingle 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 profilingGene expression levels at key growth stages.
Evidence for seed-specific expression.
Event dependent studies
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
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 goesbeyond discovery.
(the development if GR relies on US-Philanthropy and on National funding inGR-implementing countries )
Kuiper et al., 2001 Assessment of the food safety issues related to genetically modified foods. Plant J. 2001 Sep;27(6):503-28.
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?)
Consequence:Can raise costs to unaffordable levels - delays.
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 man on foot waving a red flag andblowing 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 notrepealed until 1896
Where we are in the timeline?(Philippines - first launch)
Production of stable lines; fingerprinting(Bioavailability, biosafety & other studies)
Single location trial of elite lines (2 seasons), generation of protein data
Multi-location trials; gather agronomic and biosafety data
(2 seasons)Release
BPI application
2007
2008
2009
2010
2013/14The 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)
First Outdoor Trial of GR in Asia IR64 GR1 event 309; 20 lines
Transplanting at IRRI on April 2, 2008
April 10, 2008
May 30, 2008
Activities beyond technical and safety issues
Golden Rice:communication & social marketing
ConsumersGrowers
Focus groups: Qualitative information on consumer & grower attitudes in vitamin A-deficient areas
(+ Policy Makers, Heath Community, Regulators, Traders, NGOs, etc. )
MBA students of the Asian Institute of Management, Manila –training at AIM, IRRI. PhilRice
The Focus Group normally 10-12 people for structured open ended discussion for
2 hours, with careful recording
First social marketing research was coducted in 2009(then continued by a company)
In 4 selected islands in the PhilippinesAIM-MBA students teams from theseareas~ 720 hours interviews with 360 individuals in selected groups: gender, rice farming or not farming
Examples of the story‐board designs
Focus Group Methodology
The problem of VAD remains :GoldenRice is a potentially
significant contribution to alleviation.
It is now on its way towards registration
GR2 GR1
Wild-TypeFor additional information, see www.goldenrice.org
Philippinesgenerally high VAD, (and increasing) especially Visayas & W Mindanao
(figures are % of that population with VAD)
Source: Philippine Government Statistics 1998, 2003
Children, 6 Months to 5 Years Pregnant Women Lactating Women
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