Professional Documents
Culture Documents
Peter Beyer
University of Freiburg
Germany
Rice (grain) - - - -
Tomato - - (+) +
Beans + + - +
Spinach + + + +
Nutritional Diversity
Iron, Zinc Folate Provit A Vit. E
Facts:
Rice Three billion
- live on-less than -2 $ per day,
-
1.5 billion on less than 1 $ per day and cannot afford a
Tomato - industrially
diversified diet or - (+) supplements
produced +
Beans + + - +
Spinach + + + +
Meat + + Vit A + -
• Millions are chronically micronutrient
malnourished
Intervention strategies:
Supplementation
Industrial fortification
Education
Economically sustainable?
Biofortification is an alternative to classical
interventions to fight micronutrient deficiencies
1. Breeding
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
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
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? Assembly-line technologies
E8 E7
E6
E5E4E3 E2 E1
Precursor
Intermediates
Expressed genes
DNA, mRNA
Product
(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
Neurosporene
E6 ζ-Carotene Desaturase
Lycopene
E7 Lycopene cis/trans Isomerase
α, β-Lycopene Cyclase
E8
β-Carotene α-Carotene
E3 Phytoene-Synthase
Phytoene
Phytoene Desaturase
Phytofluene
E4
Neurosporene
E6 ζ-Carotene Desaturase
Lycopene
E7 Lycopene cis/trans Isomerase
α, β-Lycopene Cyclase
E8
β-Carotene α-Carotene
E8 E7
E6
E5E4E3 E2 E1
Precursor
Intermediate
E3 Phytoene-Synthase
Phytoene Synthase
Phytoene
Phytofluene
E4 Phytoene Desaturase
Neurosporene
E6 ζ-Carotene Desaturase
Lycopene
E7 Lycopene cis/trans Isomerase
α, β-Lycopene Cyclase
E8
β-Carotene α-Carotene
transformed E. coli
15-cis-Phytoene 15-cis-Phytoen
PDS
E4
CRTI
E6 ZDS 9, 9‘-di-cis-z-Carotene
all-trans-Lycopene
all-trans-Lycopin
The CrtI gene product provides a shortcut
CrtI
shortcut
A
(HOW?) Towards a prototype:
E1 The concept
PP PP
IPP DMAPP
E2 GGPP-Synthase
Wild-type rice endosperm PP
GGPP
E3 E3 Phytoene-Synthase
Phytoene
Phytofluene
E4 Phytoene Desaturase
Neurosporene
E6 ζ-Carotene Desaturase
Lycopene
E7 Lycopene cis/trans Isomerase
E7 α, β-Lycopene Cyclase
E8
β-Carotene α-Carotene
(1)
pZPsC
(2)
pZLcyH
E8(ß-LCY)
pB19hpc
0.018
0.016
Single transformant
0.014
hpc 2b
…Lesson learned:
0.012
ß-Carotene no need for lycopene
Zeaxanthin
Lutein
0.010
ß-cyclase
α-Carotene
0.008
0.006
0.004
0.002 …why is Golden Rice golden
0.000 (yellow) instead of red???
-0.002
Phytofluene
E4 Phytoene Desaturase
CrtI ζ-Carotene
E5 (Cis/trans Isomerase?)
Lycopene
E7 Lycopene cis/trans Isomerase
E8 α, β-Lycopene Cyclase
β-Carotene α-Carotene
Only two transgenes are necessary!!
Schaub et al. (2005), Plant Physiol. 138: 441
Xanthophylls
Assembly-line technologies
E8 E7
E6
E5E4E3 E2 E1
Precursor
Intermediate
Construct ill-defined
Integration ill-defined
Antibiotic selectable marker
Low amount of bC (1.6 µg/g)
(from Narcissus)
T2 Rice grains
C D
E F
G H
ß-Carotene α-Carotene
Carotenoid
storage? Zeaxanthin Lutein
Cacar 48-67-8-7 (T3)
Cacar 48-67-4-9 (T3)
CarNew E1-19 (T1)
Improving CrtI (E4,5,6) expression
+ control
WT
pFun3 promoter change & codon optimized
pCarNew promoter change
Western
CrtI
Achieved!!!
But no significantly improved
ß-carotene accumulation.
Carotene desaturation is not
rate-limiting in Golden Rice
PSY
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-1 dwt)
16
14
12
Best. Proportion of
10
8
ß-carotene increased.
6
4
2
0
rice maize pepper tomato daffodil
Psy/crtI Psy/crtI Psy/crtI Psy/crtI Psy/crtI
E7 CrtI
E1
CrtI E3 E2
CrtI
Precursor
Zwischenprodukt
Produkt
Golden Rice 2 was made for implementation
GT1pI tp-CrtI GT1pI ZmPSY ubi1p PMI
pSYN12424
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.
NT
NT Ubi-CrtI
Ubi-Apsy2a Ubi-Apsy2a+CrtI
• 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.
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
pK-YBI Nos CrtY TP 35S Pat1 TP CrtB Ocs 35S TP CrtI Nos
Suspicion
...Mimicry
… strange places
… strong opposition...
CP crtB Field
100
80
60
40
20
0
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007
Increase of 12%, 12.3 million hectares (30 million acres), between 2006 and 2007.
Source: Clive James, 2007.
Developing countries have already
benefited from modern agricultural
biotechnology products
Performance Advantage of Insect Resistant over Conventional Cotton
(expressed as a percentage)
Argentina China India Mexico S. Africa
Yield 33 19 34 11 65
Revenue 34 23 33 9 65
Pesticide costs -47 -67 -41 -77 -58
Seed costs 530 95 17 165 89
Profit 31 340 69 12 299
Adapted from: Raney. T. (2006) Economic impact of transgenic crops in developing countries.
Current Opinion in Biotechnology 17:1–5