Download Golden Rice & Golden Crops

Golden Rice & Golden Crops
Transgenic Plants for Food Security in the Context of Development
Peter Beyer
University of Freiburg
Germany
Pontifical Academy of Science, Vatican, May 2009
Nutritional Diversity
Iron, Zinc
Folate
Provit A
Vit. E
Rice (grain)
-
-
-
-
Tomato
-
-
(+)
+
Beans
+
+
-
+
Spinach
+
+
+
+
Nutritional Diversity
Iron, Zinc
Folate
Facts:
Provit A
Vit. E
Three billion
- live on-less than -2 $ per day,
1.5 billion on less than 1 $ per day and cannot afford a
Tomato
- industrially
(+) supplements
+
diversified diet or
produced
Beans
+
+
+
Rice
Spinach
Meat
+
+
+
+
Vit A +
+
+
• Millions are chronically micronutrient
malnourished
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?
Biofortification is an alternative to classical
interventions to fight micronutrient deficiencies
Improve the nutritional value of agronomically important crop
tissues through
1. Breeding
2. Recombinant DNA technologies
is all achievable through breeding ?
Simple answer: NO!!
1. Some crop plants do not show adequate trait
variability
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)
Golden Rice cannot be bred
….the application of recombinant
DNA technology is 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) UNICEF; Humphrey et al.,
1992)
• A severe public health problem in (118) countries (WHO)
HOW?
E8
Assembly-line technologies
E7
E6
E5E4E3 E2 E1
Precursor
Intermediates
Expressed genes
DNA, mRNA
Product
(HOW?)
Starting Point: Wild-type
E1
PP
IPP
PP
E2
GGPP-Synthase
E3
Phytoene-Synthase
Phytoene
Synthase
E4
Phytoene Desaturase
E5
(Cis/trans Isomerase?)
Neurosporene
E6
-Carotene Desaturase
Lycopene
E7
Lycopene cis/trans Isomerase
DMAPP
PP
GGPP
Phytoene
Phytofluene
-Carotene
E8
-Carotene
-Carotene
All missing (not expressed) in rice endosperm???
, -Lycopene Cyclase
(HOW?)
Towards a concept:
PSY (E3) transformation
E1
PP
IPP
Wild-type rice endosperm
PP
E2
GGPP-Synthase
E3
Phytoene-Synthase
Phytoene Desaturase
Phytofluene
E4
-Carotene
E5
(Cis/trans Isomerase?)
Neurosporene
E6
-Carotene Desaturase
Lycopene
E7
Lycopene cis/trans Isomerase
DMAPP
PP
GGPP
Phytoene
E8
-Carotene
, -Lycopene Cyclase
-Carotene
Wild-type endosperm can produce a precursor molecule, GGPP!
Assembly-line technologies
E8
E7
E6
E5E4E3 E2 E1
Precursor
Intermediate
Only two appeared at work!
Product
(HOW?)
E1
PP
IPP
Wild-type rice endosperm
PP
E2
GGPP-Synthase
E3
Phytoene-Synthase
Phytoene
Synthase
E4
Phytoene Desaturase
E5
(Cis/trans Isomerase?)
Neurosporene
E6
-Carotene Desaturase
Lycopene
E7
Lycopene cis/trans Isomerase
DMAPP
PP
GGPP
Phytoene
Phytofluene
-Carotene
E8
-Carotene
, -Lycopene Cyclase
-Carotene
It is a nightmare to transform six transgenes. Luckily there is CrtI !
Luckily there is CrtI
transformed
E. coli
Pantoea ananatis carotenoid gene cluster
crtE
ORF2
ORF3
ORF4
crtX
crtY
crtI
crtB
ORF6
CrtI substitutes for 4 plant genes
crtZ
ORF12
Carotene Desaturases
Complex vs. „simple“
Cyanobacteria and Plants
Bacteria
15-cis-Phytoene
E4
E5
15-cis-Phytoen
PDS
Z-ISO ??
9, 15, 9‘-tri-cis-z-Carotene
CRTI
E6
E7
ZDS
CRTISO
9, 9‘-di-cis-z-Carotene
7, 9, 9‘, 7‘-tetra-cis-Lycopene
all-trans-Lycopene
all-trans-Lycopin
The CrtI gene product provides a shortcut
B
Plant Desaturation pathway
CrtI
shortcut
A
(HOW?)
Towards a prototype:
The concept
E1
PP
IPP
Wild-type rice endosperm
PP
E2
GGPP-Synthase
E3
Phytoene-Synthase
E4
Phytoene Desaturase
E5
(Cis/trans Isomerase?)
Neurosporene
E6
-Carotene Desaturase
Lycopene
E7
Lycopene cis/trans Isomerase
DMAPP
PP
GGPP
E3
Phytoene
Phytofluene
CrtI
-Carotene
E7
E8
-Carotene
, -Lycopene Cyclase
-Carotene
´Three instead of six!!
Pathway Complementation in Rice, Co-Transformation
Gt1p
35Sp
(1)
pZPsC
E3 (PSY)
aph IV
E,4,5,6,7 (tp-CrtI)
35Sp
Gt1p
(2)
pZLcyH
E8(ß-LCY)
With the selectable marker gene only in (2), all yellow
transgenic seeds expressed lycopene-ß-cyclase.
They all contained ß-carotene; this was the expected
outcome.
Control
hpc11
But there was a second construct without lycopene cyclase !
aph IV
Gt1p
35Sp
pB19hpc
E3 (PSY)
E3,4,5,6,7 (tp-CrtI)
0.018
0.016
…Lesson learned:
no need for lycopene
ß-cyclase
0.008
-Carotene
0.010
Zeaxanthin
Lutein
0.012
0.006
0.004
ß-Carotene
Single transformant
hpc 2b
0.014
…why is Golden Rice golden
(yellow) instead of red???
0.002
0.000
-0.002
0
20
40
60
80
100
Ye et al., 2000; Science 287:303
We just need to bridge a gap!!
E1
PP
PP
IPP
E2
GGPP-Synthase
E3
Phytoene-Synthase
Phytoene
Synthase
E4
Phytoene Desaturase
E5
(Cis/trans Isomerase?)
Neurosporene
E6
-Carotene Desaturase
Lycopene
E7
Lycopene cis/trans Isomerase
DMAPP
Wild-type rice endosperm
PP
GGPP
E3
Phytoene
Phytofluene
CrtI
-Carotene
E8
-Carotene
Xanthophylls
-Carotene
, -Lycopene Cyclase
Only two transgenes are necessary!!
Schaub et al. (2005), Plant Physiol. 138: 441
Assembly-line technologies
E8
E7
E6
E5E4E3 E2 E1
Precursor
Intermediate
Only two transgenes are necessary to fill the gap!!
Product
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
Improved Golden Rice variants came in two versions
In the public and in the private sector (Syngenta - Orynova)
Gt1p
PSY (Np)
Gt1p
tp-CrtI
(from Narcissus)
No selectable marker gene (co-transformed and removed)
Almost 1000 events
Deregulation-amenable integration
CrtI controlled by an endosperm-specific promoter
In Cocodrie (Javanica)
Amount up to 6 µg/g
Three events preselected
Known as Golden Rice 1
Technology works in Indica varieties
A
B
T2 Rice grains
C
D
E
F
G
H
Hoa et al., Plant Physiol. 133, 2003
The preselected events (PS&S) underwent 2 field trials
at Louisiana State University……
…where the GR1 events showed 4,8 – 7,1 µg/g
Improvements:
The past years were
dominated by efforts
to increase the amount
of ß-carotene in GR
both, in the public
sector as well as at
Syngenta
Potential bottlenecks to higher carotenoid levels
C3-Carbon Metabolism
Precursor
shortage?
IPP/DMAPP
Phytoene synthase
(E3) activity?
GGDP
Phytoene
Desaturation
(CrtI)
activity?
Carotenoid
storage?
-Carotene
Lycopene
ß-Carotene
-Carotene
Zeaxanthin
Lutein
Inefficient
Inefficient
transgene
transgene
expression?
expression?
CrtI
Achieved!!!
But no significantly improved
ß-carotene accumulation.
Carotene desaturation is not
rate-limiting in Golden Rice
PSY
Cacar 48-67-8-7 (T3)
Western
Cacar 48-67-4-9 (T3)
pFun3 promoter change & codon optimized
pCarNew promoter change
CarNew E4-4 (T1)
GluBp Synth tp crtI
WT
PSY (Np)
+ control
PMI 35Sp
CarNew E1-19 (T1)
Improving CrtI (E4,5,6) expression
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
Carotenoid content (mg g-1 dwt)
Transformation into a japonica short-grain rice,
(Asanohikare) 20+ plants each
20
18
16
14
12
10
8
6
4
2
0
Pepper Psy
Rice and Maize PSY (E3)
Best. Proportion of
ß-carotene increased.
rice
Psy/crtI
maize
Psy/crtI
pepper
Psy/crtI
tomato
Psy/crtI
Individual transgenic plant (event)
daffodil
Psy/crtI
Assembly-line technologies
OK
E7
OK
OK
Too slow!!!
CrtI
CrtI
E1
E2
E3
CrtI
Precursor
Zwischenprodukt
Produkt
Golden Rice 2 was made for implementation
GT1pI
tp-CrtI
GT1pI
ZmPSY
ubi1p
PMI
pSYN12424
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
Improved provitamin A
Accumulation in Golden Rice
I and II
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
GR2
GR1
Wild-Type
1. Some crop plants do not show adequate trait
variability
Maize, for instance
Maize is the world´s third most important
staple crop.
In maize, the pathway proceeds beyond beta
carotene. The genetic variability for high
carotenoid levels is very substantial, but low
for provitamin A carotenoids.
A Psy-CrtI combination, as used in GR
boosts ß-carotene production in an African
white cultivar to 60 µg/g !
Breeding approaches (ongoing - lycE
polymorphisms identified) have yielded so
far ca.14 µg/g ß-carotene.
Naqvi et al., PNAS, 2009
Harjes et al., Science
2. Some crop plants show adequate trait
Variability 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 sterile
Most 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
Transient testing of the transgenes using direct Agrotransformation of banana fruits
NT
NT
Ubi-Apsy2a
Ubi-CrtI
Ubi-Apsy2a+CrtI
Preliminary HPLC data indicated increased -carotene, -carotene and lutein
2. 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 micronutrients
Provit 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, BMGF
University of Freiburg – CIAT, Harvest Plus
Cassava promoter CP2 - crtB
Line #12
…more lines epressing multiple genes coming this year
2. Some crop plants cannot be (easily) bred
Potato, for instance
potato ranks fourth, among the staple foods
of mankind, after wheat, rice and maize
pK-I
pK-BI
pK-YBI
pP-I
pP-BI
pP-YBI
35S
TP
CrtI
Nos
Pat1
TP
CrtB
Ocs
35S
CrtY
TP
35S
Pat1
Nos
Pat2
TP
Pat1
TP
Pat2
TP
CrtI
Nos
CrtB
Ocs
CrtY
CrtY
Nos
Pat2
Pat1
TP
TP
TP
TP
CrtI
Nos
CrtB
Ocs
CrtI
Nos
CrtB
Ocs
35S
Pat2
TP
TP
CrtI
CrtI
Nos
Nos
only this one:
A mini-pathway
„Golden Potato“
Diretto et al., PlosOne, 2007
All of the here-mentioned examples bear significant potential:
They represent the major staples
The 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 underway
Howe and Tanumihardjo, J. Nutr. 2006
Cassava: 3.7:1, Gerbil
Howe et al., British Journal of Nutrition (2009)
Spinach: 20:1, Human
Tang et al., Am J Clin Nutr 2005;
Very good bioavailability of ß-carotene from simple starchy food matrices
(Like banana and potato?)
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
To all our sponsors since before 1990:
Rice Teams & budgets @
•IRRI
•Phil Rice- Philippines
ETH / Swiss Federal Funds
European Commission
HarvestPlus
USAID
Syngenta Company
Syngenta Foundation
National Institutes of Health (USA)
Bill and Melinda Gates Foundation
The Golden Rice Humanitarian Board
Bayer, Mogen, Novartis, Monsanto, Orynova, Zeneca
•CLRRI- Vietnam
•DBT, IARI, DRR,TNAU- India