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 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

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? 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

ζ-Carotene E5 (Cis/trans Isomerase?)

Neurosporene
E6 ζ-Carotene Desaturase

Lycopene
E7 Lycopene cis/trans Isomerase

α, β-Lycopene Cyclase
E8
β-Carotene α-Carotene

All missing (not expressed) in rice endosperm???

 (HOW?) Towards a concept:
E1 PSY (E3) transformation
PP PP
IPP DMAPP
E2 GGPP-Synthase
Wild-type rice endosperm PP
GGPP

E3 Phytoene-Synthase
Phytoene

Phytoene Desaturase
Phytofluene
E4

ζ-Carotene E5 (Cis/trans Isomerase?)

Neurosporene
E6 ζ-Carotene Desaturase

Lycopene
E7 Lycopene cis/trans Isomerase

α, β-Lycopene Cyclase
E8
β-Carotene α-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 PP
IPP DMAPP
E2 GGPP-Synthase
Wild-type rice endosperm PP
GGPP

E3 Phytoene-Synthase
Phytoene Synthase
Phytoene

Phytofluene
E4 Phytoene Desaturase

ζ-Carotene E5 (Cis/trans Isomerase?)

Neurosporene
E6 ζ-Carotene Desaturase

Lycopene
E7 Lycopene cis/trans Isomerase

α, β-Lycopene Cyclase
E8
β-Carotene α-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 crtX crtY crtI crtB crtZ ORF12

ORF2 ORF3 ORF4 ORF6

CrtI substitutes for 4 plant genes

 Carotene Desaturases
Complex vs. „simple“
Cyanobacteria and Plants Bacteria

15-cis-Phytoene 15-cis-Phytoen

PDS
E4

E5 Ζ-ISO ?? 9, 15, 9‘-tri-cis-z-Carotene

CRTI

E6 ZDS 9, 9‘-di-cis-z-Carotene

E7 CRTISO 7, 9, 9‘, 7‘-tetra-cis-Lycopene

all-trans-Lycopene
all-trans-Lycopin
 The CrtI gene product provides a shortcut

Plant Desaturation pathway

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

CrtI ζ-Carotene E5 (Cis/trans Isomerase?)

Neurosporene
E6 ζ-Carotene Desaturase

Lycopene
E7 Lycopene cis/trans Isomerase
E7 α, β-Lycopene Cyclase
E8
β-Carotene α-Carotene

´Three instead of six!!

Pathway Complementation in Rice, Co-Transformation
Gt1p 35Sp

(1)
pZPsC

E3 (PSY) E,4,5,6,7 (tp-CrtI)

aph IV 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
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

0 20 40 60 80 100 Ye et al., 2000; Science 287:303

 We just need to bridge a gap!!
E1
PP PP
IPP DMAPP
E2 GGPP-Synthase
Wild-type rice endosperm PP
GGPP
E3 E3 Phytoene-Synthase
Phytoene Synthase
Phytoene

Phytofluene
E4 Phytoene Desaturase

CrtI ζ-Carotene
E5 (Cis/trans Isomerase?)

Neurosporene E6 ζ-Carotene Desaturase

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

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
Inefficient
Inefficient
transgene
Phytoene transgene
expression?
Desaturation expression?
ζ-Carotene
(CrtI)
activity?
Lycopene

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

CarNew E4-4 (T1)

PMI 35Sp PSY (Np) GluBp Synth tp crtI

+ 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

Individual transgenic plant (event)

 Assembly-line technologies
OK
OK
OK
Too slow!!!

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

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 Naqvi et al., PNAS, 2009
far ca.14 µg/g ß-carotene. 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 Agro-
transformation of banana fruits

NT
NT Ubi-CrtI

Ubi-Apsy2a Ubi-Apsy2a+CrtI

Preliminary HPLC data indicated increased α-carotene, β-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 35S TP CrtI Nos

pK-BI Pat1 TP CrtB Ocs 35S TP CrtI Nos

pK-YBI Nos CrtY TP 35S Pat1 TP CrtB Ocs 35S TP CrtI Nos

pP-I Pat2 TP CrtI Nos

pP-BI Pat1 TP CrtB Ocs Pat2 TP CrtI Nos

pP-YBI CrtY CrtB CrtI

Pat2 TP CrtY Nos Pat1 TP Nos
Ocs Pat2 TP 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
 Rice Teams & budgets @
To all our sponsors since before 1990:
•IRRI
•Phil Rice- Philippines
ETH / Swiss Federal Funds •CLRRI- Vietnam
European Commission •DBT, IARI, DRR,TNAU- India
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
On our way we meet...

Suspicion
...Mimicry
… strange places
… strong opposition...

GoldenRice Field Trial

(one week before harvest!)

Hurricane “Ivan” September 2004

Hurricane “Katrina” August 2005

Hurricane “Rita” September 2005

Typhoon Fengshen June 2008

Promoter Gene Current status

CP crtB Field

Patatin crtB-crtI-crtY* Field, Greenhouse

Extensin crtB Transformed cell lines

crtB-crtI-crtY
Yam crtB Transformed cell lines
crtB-crtI-crtY Under construction
Sugar beet crtB
crtB-crtI-crtY Transformed cell lines
 GLOBAL AREA OF BIOTECH CROPS
Million Hectares (1996 to 2007)

Total 23 Biotech Crop Countries

140 Industrial
Developing
120

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

Based on peer-reviewed studies of 2-3 seasons of commercial farm production

Adapted from: Raney. T. (2006) Economic impact of transgenic crops in developing countries.
Current Opinion in Biotechnology 17:1–5