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Golden Rice & Golden CropsGolden Rice & Golden Crops
Peter BeyerUniversity of FreiburgGermany
Transgenic Plants for Food Security in the Context of Development
Pontifical Academy of Science, Vatican, May 2009
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Iron, Zinc Folate Provit A Vit. E
Rice (grain) - - - -Tomato - - (+) +Beans + + - +Spinach + + + +
Nutritional DiversityNutritional Diversity
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Iron, Zinc Folate Provit A Vit. E
Rice - - - -Tomato - - (+) +Beans + + - +Spinach + + + +Meat + + Vit A + -
Nutritional DiversityNutritional 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
• Millions are chronically micronutrient malnourished
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Intervention strategies: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?Economically sustainable?
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Biofortification is an alternative to classicalBiofortification is an alternative to classicalinterventions to fight micronutrient deficienciesinterventions to fight micronutrient deficiencies
is all achievable through breeding ?
Simple answer: NO!!
Improve the nutritional value of agronomically important croptissues through
1.Breeding
2.Recombinant DNA technologies
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1. Some crop plants do not show adequate trait1. Some crop plants do not show adequate trait variabilityvariability
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)
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Golden Rice cannot be bredGolden Rice cannot be bred
….the application of recombinantDNA technology is necessary
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Why engineering ß-carotene Why engineering ß-carotene (provitamin A) biosynthesis into rice (provitamin A) biosynthesis into rice
endosperm?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)
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XerophthalmiaXerophthalmia
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Why engineering ß-carotene Why engineering ß-carotene (provitamin A) biosynthesis into rice (provitamin A) biosynthesis into rice
endosperm?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)
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Assembly-line technologiesAssembly-line technologies
E2 E1E3E4E5E6
E7
Precursor
Product
Intermediates
HOW?HOW?
Expressed genesDNA, mRNA
E8
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Phytoene-Synthase
PP
PP PP
GGPP-SynthaseIPP DMAPP
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-typeStarting Point: Wild-type
(Cis/trans Isomerase?)
All missing (not expressed) in rice endosperm???
E8 , -Lycopene Cyclase
(HOW?)(HOW?)
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Phytoene-Synthase
PP
PP PP
GGPP-SynthaseIPP DMAPP
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?)(HOW?) Towards a concept: Towards a concept: PSY (E3) transformationPSY (E3) transformation
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E2 E1E3E4E5E6
E7E8
Precursor
Product
Intermediate
Assembly-line technologiesAssembly-line technologies
Only two appeared at work!
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Phytoene-Synthase
PP
PP PP
GGPP-SynthaseIPP DMAPP
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?)(HOW?)
It is a nightmare to transform six transgenes. Luckily there is CrtI ! It is a nightmare to transform six transgenes. Luckily there is CrtI !
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Luckily there is CrtI Luckily there is CrtI Luckily there is CrtI Luckily there is 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
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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 DesaturasesCarotene DesaturasesComplex vs. „simple“ Complex vs. „simple“
E4
E5
E7
E6
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The CrtI gene product provides a shortcutThe CrtI gene product provides a shortcut
A
B
Plant Desaturation pathway
CrtI shortcut
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Phytoene-Synthase
PP
PP PP
GGPP-SynthaseIPP DMAPP
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?)(HOW?)
E3
CrtI
E7
Towards a prototype:The concept
´Three instead of six!!
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Pathway Complementation in Rice, Co-TransformationPathway 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 yellowtransgenic seeds expressed lycopene-ß-cyclase.
They all contained ß-carotene; this was the expectedoutcome.
pZLcyH
pZPsC
aph IV 35Sp
35SpGt1p
Gt1p
(1)
(2)
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Control
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hpc11
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But there was a second construct without lycopene cyclase !But there was a second construct without lycopene cyclase !
pB19hpc
E3 (PSY) E3,4,5,6,7 (tp-CrtI)
aph IV 35SpGt1p
Single transformanthpc 2b
20 40 60 80 1000
0.000
0.018
0.006
0.012
0.002
0.004
0.008
0.010
0.014
0.016
-0.002
ß-C
arot
ene
Zea
xan
thin
Lu
tein
-C
arot
ene
…Lesson learned:no need for lycopene
ß-cyclase
…why is Golden Rice golden (yellow) instead of red???
Ye et al., 2000; Science 287:303
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Phytoene-Synthase
PP
PP PP
GGPP-SynthaseIPP DMAPP
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!!We just need to bridge a gap!!
Xanthophylls
E3
CrtI
Only two transgenes are necessary!!Only two transgenes are necessary!!
Schaub et al. (2005), Plant Physiol. 138: 441
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E2 E1E3E4E5E6
E7E8
Precursor
Product
Intermediate
Assembly-line technologiesAssembly-line technologies
Only two transgenes are necessary to fill the gap!!
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Prototypes: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 rice varieties.
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Improved Improved Golden Rice Golden Rice variants came in two versionsvariants came in two versions
In the public and in the private sector (Syngenta - Orynova)
Gt1p PSY (Np) tp-CrtIGt1p
(from Narcissus)
No selectable marker gene (co-transformed and removed)Almost 1000 eventsDeregulation-amenable integrationCrtI controlled by an endosperm-specific promoterIn Cocodrie (Javanica)Amount up to 6 µg/g Three events preselectedKnown as Golden Rice 1 Technology works in Indica varieties
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A B
C D
E F
G H
T2 Rice grains
Hoa et al., Plant Physiol. 133, 2003
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The preselected events (PS&S) underwent 2 field trialsat Louisiana State University……
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…where the GR1 events showed 4,8 – 7,1 µg/g
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Improvements:
The past years weredominated by effortsto increase the amountof ß-carotene in GRboth, in the public sector as well as at Syngenta
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Precursor shortage?
Carotenoid storage?
Phytoene synthase
(E3) activity?
Potential bottlenecks to higher carotenoid levelsPotential bottlenecks to higher carotenoid levels
IPP/DMAPP
C3-Carbon Metabolism
GGDP
Phytoene
-Carotene
Lycopene
ß-Carotene
Lutein
-Carotene
Zeaxanthin
Desaturation
(CrtI)
activity?
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Improving CrtI (E4,5,6) expressionImproving CrtI (E4,5,6) expression
pFun3 promoter change & codon optimizedpCarNew promoter change
GluBp Synth tp crtIPMI 35Sp PSY (Np)
WT
Car
New
E1-
19 (
T1)
Cac
ar 4
8-67
-4-9
(T
3)
Car
New
E4-
4 (T
1)
Cac
ar 4
8-67
-8-7
(T
3)
+ c
ontr
ol
CrtI
PSY
Achieved!!!But no significantly improvedß-carotene accumulation.Carotene desaturation is notrate-limiting in Golden Rice
Western
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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
02468101214161820
ricePsy/crtI
maizePsy/crtI
pepperPsy/crtI
tomatoPsy/crtI
daffodilPsy/crtI
Individual transgenic plant (event)
Caro
ten
oid
co
nte
nt
(m g
g-1 d
wt) Rice and Maize PSY (E3)
Best. Proportion of ß-carotene increased.
Phytoene synthase was investigated by Rachel Drake (Syngenta)Phytoene synthase was investigated by Rachel Drake (Syngenta)Because PSY expression is good in GR, different versions of the PSY gene were assayed.
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E2E1
E3
CrtIE7
Precursor
Produkt
Zwischenprodukt
Assembly-line technologiesAssembly-line technologies
Too slow!!!OKOK
OK
CrtICrtI
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Golden Rice 2 was made for implementationGolden Rice 2 was made for implementation
pSYN12424
ZmPSY ubi1p
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 GT1pI
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Improved provitamin AAccumulation in Golden RiceI and II
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GR 2…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
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GR2 GR1
Wild-Type
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1. Some crop plants do not show adequate trait1. Some crop plants do not show adequate trait variabilityvariability
Maize, for instance
Naqvi et al., PNAS, 2009Harjes et al., Science
A Psy-CrtI combination, as used in GR boosts ß-carotene production in an Africanwhite 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 importantstaple crop.
In maize, the pathway proceeds beyond betacarotene. The genetic variability for high carotenoid levels is very substantial, but lowfor provitamin A carotenoids.
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2. Some crop plants show adequate trait2. Some crop plants show adequate traitVariability but cannot be (easily) bredVariability 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 sterileMost 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
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Transient testing of the transgenes using direct Agro-Transient testing of the transgenes using direct Agro-transformation of banana fruitstransformation of banana fruits
Ubi-Apsy2a Ubi-Apsy2a+CrtI
Ubi-CrtINT NT
Preliminary HPLC data indicated increased -carotene, -carotene and lutein
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2. Some crop plants cannot be (easily) bred2. 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 5th among crops directly consumed by humans (No. 1 in Sub-Saharan Africa). Provides food security.
• Very low in micronutrientsProvit 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, BMGFUniversity of Freiburg – CIAT, Harvest Plus
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Cassava promoter CP2 - crtBCassava promoter CP2 - crtB
Line #12
…more lines epressing multiple genes coming this year
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Pat1 CrtBTP
35SNos TP
Pat2
Nos35S CrtITPpK-I
pK-BI
pK-YBI
pP-I
pP-BI
pP-YBI
Nos35S TPOcs
Pat1 TP Nos35S TPOcs
Pat2 CrtITP Nos
Pat1 TP Ocs Pat2 TP Nos
Pat1 TP Ocs Pat2 TP NosTP CrtY Nos
CrtI
CrtICrtBCrtY
CrtB CrtI
CrtICrtBCrtYonly this one:
A mini-pathway
2. Some crop plants cannot be (easily) bred2. Some crop plants cannot be (easily) bred
Potato, for instance
potato ranks fourth, among the staple foods of mankind, after wheat, rice and maize
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Diretto et al., PlosOne, 2007
„„Golden Potato“Golden Potato“
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All of the here-mentioned examples bear significant potential:They represent the major staplesThe 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 underwayHowe and Tanumihardjo, J. Nutr. 2006
Cassava: 3.7:1, Gerbil Howe et al., British Journal of Nutrition (2009)
Spinach: 20:1, HumanTang et al., Am J Clin Nutr 2005;
Very good bioavailability of ß-carotene from simple starchy food matrices(Like banana and potato?)
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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
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ETH / Swiss Federal Funds European CommissionHarvestPlusUSAIDSyngenta CompanySyngenta FoundationNational Institutes of Health (USA)Bill and Melinda Gates FoundationThe Golden Rice Humanitarian BoardBayer, 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: