golden rice & golden crops peter beyer university of freiburg germany transgenic plants for food...

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

  Iron, Zinc Folate Provit A Vit. E

Rice (grain) - - - -Tomato - - (+) +Beans + + - +Spinach + + + +

Nutritional DiversityNutritional Diversity

  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

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?

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

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)

Golden Rice cannot be bredGolden Rice cannot be bred

….the application of recombinantDNA technology is necessary

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)

XerophthalmiaXerophthalmia

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)

Assembly-line technologiesAssembly-line technologies

E2 E1E3E4E5E6

E7

Precursor

Product

Intermediates

HOW?HOW?

Expressed genesDNA, mRNA

E8

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?)

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

E2 E1E3E4E5E6

E7E8

Precursor

Product

Intermediate

Assembly-line technologiesAssembly-line technologies

Only two appeared at work!

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 !

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

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

The CrtI gene product provides a shortcutThe CrtI gene product provides a shortcut

A

B

Plant Desaturation pathway

CrtI shortcut

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!!

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)

Control

hpc11

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

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

E2 E1E3E4E5E6

E7E8

Precursor

Product

Intermediate

Assembly-line technologiesAssembly-line technologies

Only two transgenes are necessary to fill the gap!!

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.

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

A B

C D

E F

G H

T2 Rice grains

Hoa et al., Plant Physiol. 133, 2003

The preselected events (PS&S) underwent 2 field trialsat Louisiana State University……

…where the GR1 events showed 4,8 – 7,1 µg/g

Improvements:

The past years weredominated by effortsto increase the amountof ß-carotene in GRboth, in the public sector as well as at Syngenta

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?

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

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.

E2E1

E3

CrtIE7

Precursor

Produkt

Zwischenprodukt

Assembly-line technologiesAssembly-line technologies

Too slow!!!OKOK

OK

CrtICrtI

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

Improved provitamin AAccumulation in Golden RiceI and II

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

GR2 GR1

Wild-Type

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.

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

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

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

Cassava promoter CP2 - crtBCassava promoter CP2 - crtB

Line #12

…more lines epressing multiple genes coming this year

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

Diretto et al., PlosOne, 2007

„„Golden Potato“Golden Potato“

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?)

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

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: