Transcript No Slide Title - Oregon State University

Science Community Lecture.
Oregon State University Corvallis, 13 October 2005
Genetic engineering of pro-vitamin A
production in rice.
Ingo Potrykus
Professor Emeritus Plant Sciences ETH Zuerich
Vitamin A Deficiency & Rice
The problem :
Rice as major staple does not contain any provitamin A.
The consequences:
400 million rice-eating poor suffer from vitamin A-deficiency.
6‘000 die per day, 500‘000 become blind per year.
The transgenic concept:
Introduce, under endosperm-specific regulation, all genes
necessary to establish the biochemical pathway.
Why genetic engineering in addition to the traditional
interventions?
The genetic basis in the rice gene pool does not offer a basis
for a conventional approach.
In developing countries 500 000 children per year go blind and
up to 6 000 per day die from vitamin A-malnutrition.
And this will continue year by
year, if we do not complement
traditional interventions.
‚Biofortification‘ – improvement
of the micro-nutrient content of
crops on a genetic basis – can
reduce malnutrition in a costeffective and sustained manner.
The majority of the vitamin A-deficient depend upon rice
which totally lacks provitamin A and is poor in other micronutrients such as iron, zinc,and essential amino acids.
Biochemical pathway engineering from GGPP
to pro-vitamin A in rice endosperm.
Narcissus
Phytoene synthase
Narcissus
Phytoene desaturase
Narcissus
 Carotene desaturase
-
-
Narcissus
Provitamin A
Lycopene cyclase
Biochemical pathway engineering from GGPP
to pro-vitamin A in rice endosperm.
Phytoene synthase
Narcissus
Erwinia
-
Double desaturase
-
Narcissus
Provitamin A
Lycopene cyclase
LB
I-sce I
I-sce I
Kpn I
GGPP
pZPsC
Gt1 pr
psy
nos! 35S pr
crtI
nos! RB
kb
Phytoene
23.1
9.4
6.6
4.4
phytoene synthase
2.3
Restriction enzymes: a) I-sce I, b) Kpn I. Probe:
psy
kb
Lycopene
23.1
9.4
6.6
4.4
phytoene desaturase
2.3
LB 35S!
-Carotene
crt I
Spe I
I-sce I
I-sce I
Restriction enzymes: a) I-sce I, b) Kpn I. Probe:
pZcycH
aph 4
35S pr
35S!
cyc
Gt1 pr
RB
kb
23.1
9.4
6.6
4.4
lycopene cyclase
Restriction enzymes: a) I-sce I, b) Spe I. Probe:
cyc
Provitamin A producing rice: HPLC analysis
Provitamin A
Provitamin A
Provitamin A
Analysis of
Regulatory
Clean events
In Indica rice
IR64 and
Japonica rice
TP309:
Provitamin A,
-oryzanol,
Vitamin E
-carotene
-carotene
-cryptoxanthin
zeaxanthin
lutein
Carotenoid (ng/g dm)
1200
1000
800
600
400
200
0
200
180
160
140
120
100
80
60
40
20
0
-Oryzanol (µg/g dm)
25
Vit E (µg/g dm)
20
15
10
5
0
TP309
WT
IR 64
TP309
IR64
IR 64
IR 64
Wildtype
Golden Rice
Biochemical pathway engineering from GGPP
to pro-vitamin A in rice endosperm.
Narcissus
Erwinia
-
Phytoene synthase
Double desaturase
-
Provitamin A
Experiments in progress to increase and
stabilize the pro-vitamin A content in rice.
Group Peter Beyer, Freiburg, Germany.
1. Use of codon-optimized crtI
35S term
CaMV35S Promoter
CrtI-Synthetic
CaMV35S PolyA PSY
T-Border (r)
Gt-1 GluB-1
T-BorderPMI
(l)
nos
3. To raise expression with
appropriate transcription factor.
Gt1-Promoter
CaMV35S Promoter
PMI
Rap 2.2
pC1380MPI-Gt1-AtRAP2.2
5. To convert -carotene into
retinoic acid (vitamin A)
pZPDiox
2. To channel more carbon
into the pathway
Pyruvate + D-Glyceraldehyde
3-phosphate
DXS
DXR
CMT
CMK
MECPS
4. To block -carotene degrading
and converting enzymes.
At least 8 putative
carotenoid cleaving
enzymes identified in rice
genome. One of these leads
to decolorization of carotene accumulating
E.coli.
ß-carotene dioxygenase
JA Payne et al., Nature Biotechnology 23: 482-487, 2005.
Improving the nutritional value of Golden Rice through
increased pro-vitamin A content.
The level of expression can be regulated with he choice of
phytoene synthase genes. The total amount of pro-vitamin A
ranges, so far, from 0.8 to 40 g/g endosperm.
Genes from ‚proof-ofconcept experiment,
Science 2000
More than 80% of the carotenoids consist of the most
effective pro-vitamin A - -carotene.
JA Payne et al., Nature Biotechnology 23: 482-487, 2005.
Improving the nutritional value of Golden Rice through
increased pro-vitamin A content.
Wildtype
Golden Rice
„Golden Rice“ contains the genes required
to activate the biochemical pathway leading
to accumulation of provitamin A. Proof of
concept was complete in 1999.
And this was the end of public funding.
How much GoldenRice has a child to eat to prevent
vitamin A-malnutrition?
Lines used for subsequent calculation.
Wildtype
SGR 1
SGR2
Further lines
with much
higher content
of provitamin A
in the pipeline.
SGR1: 1.6g
reguatory
clean; jointly
developed by
public & private
sector.
SGR2: 16g
developed by
private sector;
donated to the
humanitarian
project!
Amount depends upon the typical diet. Example Bangladesh
Calculation from International Food Policy Institute:
(1) Share of calorie intake for rural Bangladesh.
79% energy
intake from
rice but no
provitamin A!
Provitamin A
in vegetables
and fruit
Vitamin A in
fish &
animal food
Contribution to WHO/FAO recommended nutrient intake of
provitamin A from a conventional diet in Bangladesh from
vegetables/friut, animal/fish, and ‚Golden Rice‘
GoldenRice
16 g line
32 g line is
also availale
Golden Rice could minimize vitamin A-malnutrition at no
costs, if it could replace ordinary rice.
Also populations with 40% energy from rice would be
protected because of higher provitamin A background.

Exposure evaluation
 Bioavailability study.
Event independent studies
Requirements for GoldenRice deregulation.
Golden
Rice
Modelling analysis for intended use.
Deregulation   
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 (Daffodil!)
Molecular characterization and genetic stability
 Single copy effect; marker gene at same locus.
 Simple integration; Mendelian inheritance over
Event dependent studies
Requirements for GoldenRice deregulation.
Deregulation   
three generations (minimum).
 No potential gene disruption.
 No unknown open reading frames.
 No DNA transfer beyond borders.
Golden
Rice
 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.
Golden
Expression profiling
Rice
 Gene expression levels at key growth stages.
 Evidence for seed-specific expression.
Event dependent studies - continued
Requirements for GoldenRice deregulation.
Deregulation   
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
Genetically Modified Rice Adoption: Impact for Welfare and
Poverty Aleviation. K Anderson, LA Jackson, CP Nielsen.
World Bank Policy Research Working Paper
3380, August 2004
The paper uses the ‚global economy-wide computable general
equilibrium model‘ to analyse the potential economic effects of
adopting first and second generation GMO crops in Asia.
 ‚The results suggest that farm productivity gaines could be
dwarfed by the welfare gains resulting from the potential
health-enhancing attributes of Golden Rice‘.
 Projected gaines from Golden Rice adoption by developing
Asia would amount to $ 15.2 billion per year globally.
 Enhanced productivity of Asian unskilled labor in $ billion:
China 7.209; India 2.528; Other S+SE Asia 4.140.
Iron Deficiency Aneamia
The problems :
Rice contains very little iron (1), an inhibitor of iron
resorption (2), and does not support iron uptake from a
vegetative diet (3).
The consequences:
Rice-eating poor suffer from iron deficiency. Iron deficiency
affects nearly 3 billion people. It impairs body growth,
mental & motor development, activity, intellect & emotion;
it favours infectious diseases. It is the major cause for
maternal and child death in pregancy.
The transgenic concept:
Decrease the inhibitor (1), increase iron content (2), add
uptake-promoting substances (3).
High Iron Rice: Decrease in inhibitor
Phytase activity
Phytate content in rice seeds after
PU [nmol free P/ g rice/ min]
simulated small intestine conditions
10’000
1.00
WT
8’000
4’000
IP4
IP3
0.75
non treated
%
6’000
TR
after acid
treatment
0.50
2’000
0.25
0
0.00
WT
TR
IP6
IP5
n.t. 1h, 6.5
37°C
n.t. 1h, 6.5
37°C
High Iron Rice: „Thermostability“ of the fungal phytase
residual activity of
after 20 min at 100ºC
the purified phytase [%]
with rice seeds
25
100
80
20
60
15
in the transgenic seeds
10
PU (mol free P
/ min/ mg)
PU (mol free P
/g/min)
8
TR
6
WT
50%
40
10
20
5
0
0
0 20 40 60 80 100
Temperature [ºC]
4
2
0
0
5
10
15
Minutes
20
„High-Iron“ Rice has 2-fold iron, 7-fold uptake-promoting
cystein, and high inhibitor-degrading activity. However,
the inhibitor-degrading enzyme (phytase) does not
retain its heat stability when in the apoplast; and when
active in the phytate vesicles, is provoking replacment
of degraded phytate.
Experiments are, therefore, in progress for
a) expressing the phytase in a different compartment,
b) enhancing iron uptake via excretion from the roots of
mucogenic acid, and
c) supporting transport of iron via an iron transporter.
Introgression of the GoldenRice trait into Vietnamese varieties.
High iron/high yield variety Khang Dan and High yielding/good
grain quality/ aromatic varieties.
IR1490
OM2031
CS2000
DS2001
ASS996
Jasmine 85
MTL 250
OM 3536
Engineered provitamin A
IPP/DMAPP-Isomerase
pathway in rice
endosperm.
PP
IPP
PP
DMAPP
GGPP-Synthase
PP
GGPP
Phytoene-Synthase
Phytoene
Phytoene Desaturase
-Carotene-Desaturase
Phytofluene
-Carotene
Neurosporene
Lycopene isomerase
, -Lycopene Cyclase
Golden
Rice
Science is moving towards
nutritional optimization, but
regulators will not deregulate.
Regulation is set to look at risks,
not benefits. 24‘000 death per
day are irrelevant!
other ?
 Vitamin A
Lipids ?
Vitamin E 
-Oryzanol 
Carotenoids: 
-carotene,lutein,
zeaxanthin
Lycopene
Iron & zinc bioavailability:
Ferritin, Phytase,
GoldenRice follow-up projects.
„Biofortification“- genetics-based
improvement of the nutritional value
of crops for sustained reduction of
micronutrient malnutrition.
Two international programs, using
traditional and molecular
techniques, are supporting the
concept of „biofortification“ with
support from The World Bank and
the NIH-Gates Foundation.
International, multidisciplinary.
„Grand
Challenges in
Global Health.“
Rice
Sorghum
Cassava
Banana
„HarvestPlus“.
CGIAR
 others ?
 Vitamin A
Lipids ?
Vitamin E 
Rice
-Oryzanol 
Maize
Carotenoids: 
Wheat
-carotene,lutein,
zeaxanthin
Cassava
Sweet Potato
Beans
Iron- & Zinc Bioavailability:
Ferritin, Phytase,
Acknowledgement
ETH Zürich for support of ‚proof-of-concept‘ work.
• Group Peter Beyer & Ingo Potrykus for sciene.
• Humanitarian Golden Rice Board for guidance
and decisions.
• Humanitarian Golden Rice Network for
development of varieties.
• Syngenta (Adrian Dubock) for key contributions
in product development, deregulation, strategic
advice.
• AgBiotech companies for IPR donation
• Syngenta Foundation, Rockefeller Foundation,
USAid, HarvestPlus, Gates Foundation,
Government of India for financial support.
No support from EU or EU national agencies!