Golden Rice – A Humanitarian Biotechnology Project

Golden Rice –A Humanitarian Biotechnology Project

Peter Beyer, Center for Applied Biosciences, University of Freiburg, Germany

COMM4BIOTECH Congress; 18-19 Nov. 2011

Source: C. Lupi, BATS report, I/95

Pop

ulat

ion

[milli

on]

Year

0

2000

4000

6000

8000

10000

1990 2000 2010 2020 2030 2040 2050

Industrialized Nations

Population growth

Developing Nations

WHY?

Directly, or indirectly, plants provide all of humanity’s food

During the past 100 years agriculture has (of necessity) focused on increasing yield. Nutrient content has largely been ignored in breeding.

More of our food comes from fewer species (54% from corn, rice and wheat). We’ve lost biochemical diversity in the staples in our diets.

Plants, in principle provide all macro -and micronurients required, but the latter are very unevenly distributed.

Staples (rice)

Non-staples(Vegetables)Fish and otheranimal sources

Source: H. Bouis, IFPRI; HarvestPlus

Share of daily energy intake for rural Bangladesh

84 %

Directly, or indirectly, plants provide all of humanity’s food

During the past 100 years agriculture has (of necessity) focused on increasing yield. Nutrient content has largely been ignored in breeding.

More of our food comes from fewer species (54% from corn, rice and wheat). We’ve lost biochemical diversity in our diets.

Plants provide all macro -and micronurients required, but the latter are very unevenly distributed in plant tissues.

Iron, Zinc Folate Provit A

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

Nutritional Diversification

Iron, Zinc Folate Provit A Vit. E

Rice - - - -Tomato - - (+) +Beans + + - +Spinach + + + +Meat + + Vit A + -

Nutritional DiversityFacts:

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 micronutrientmalnourished

Intervention strategies:SupplementationIndustrial fortificationEducation

All necessary and very valuable but there are drawbacks:

Distrubution, educated medical staffCentrally processed food itemsOnly partially applicable

Economically sustainable?

Biofortification is a cost effective alternative toclassical interventions

is all achievable through breeding ?

Simple answer: NO!!

Improve the nutritional value of agronomically important croptissues through

BreedingRecombinant DNA technology

Some crops cannot be bred or breeding is very difficultTrait variability is insufficient

www.harvestplus.org

Some traits cannot be bred because there is noadequate trait variability

Rice, (polished grains) for instance

Provitamin A: No „yellow grains“ in germplasm collections

Folate: Practically absent

Iron: low variability, ranging from 1 – 8 ppm (Target 14 ppm)

Zinc: much more important variability, ranging from 16 – 28 ppm (Target: 24 ppm)

Golden Rice cannot be bred

….the application of recombinantDNA technology is necessary

Breeding where possible

Genetic modification where 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)• A severe public health problem in (118) countries (WHO)

Global prevalence of vitamin A deficiency in populations at risk 1995–2005WHO Global Database on Vitamin A Deficiency

Assembly-line technologies

E2 E1E3E4E5E6

E7

Precursor

Product

Intermediates

HOW?

Expressed genes

E8

Phytoene-Synthase

PP

PP PP

GGPP-SynthaseIPP DMAPP

GGPP

Lycopene cis/trans Isomerase

Phytoene

Phytofluene

ζ-Carotene

Neurosporene

Lycopene

E1

E2

E3

E4

E5

E6E7

β-Carotene α-Carotene

Phytoene Synthase

ζ-Carotene Desaturase

Phytoene Desaturase

Starting Point: Wild-type

(Cis/trans Isomerase?)

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

E8 α, β-Lycopene Cyclase

HOW?

Phytoene-Synthase

PP

PP PP

GGPP-SynthaseIPP DMAPP

GGPP

Lycopene cis/trans Isomerase

Phytoene

Phytofluene

ζ-Carotene

Neurosporene

Lycopene

E1

E2

E3

E4

E5

E6E7

β-Carotene α-Carotene

Phytoene Synthase

ζ-Carotene Desaturase

Phytoene Desaturase

Wild-type rice endosperm

Cis/trans Isomerase

E8 α, β-Lycopene Cyclase

We just need to bridge a gap!!

Xanthophylls Schaub et al. (2005), Plant Physiol. 138: 441

HOW?

E2 E1E3E4E5E6

E7E8

Precursor

Product

Intermediate

Assembly-line technologies

Phytoene-Synthase

PP

PP PP

GGPP-SynthaseIPP DMAPP

GGPP

Lycopene cis/trans Isomerase

Phytoene

Phytofluene

ζ-Carotene

Neurosporene

Lycopene

E1

E2

E3

E4

E5

E6E7

β-Carotene α-Carotene

Phytoene Synthase

ζ-Carotene Desaturase

Phytoene Desaturase

Wild-type rice endosperm

Cis/trans Isomerase

E8 α, β-Lycopene Cyclase

So many transgenes?That´s not such a great idea!

Xanthophylls Schaub et al. (2005), Plant Physiol. 138: 441

Luckily there is CrtI !

HOW?

Luckily there is a bacterial gene, 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

Ζ-ISO 9, 15, 9‘-tri-cis-z-Carotene

PDS

all-trans-Lycopene

CRTISO

all-trans-Lycopin

CRTI

15-cis-Phytoen

Bacteria

Carotene DesaturasesComplex vs. „simple“

E4

E5

E7

E6

The 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

ζ-Carotene

Neurosporene

Lycopene

E1

E2

E3

E4

E5

E6E7

β-Carotene α-Carotene

Phytoene Synthase

ζ-Carotene Desaturase

Phytoene Desaturase

Wild-type rice endosperm

Cis/trans Isomerase

E8 α, β-Lycopene Cyclase

Only two transgenes needed

Xanthophylls

E3

CrtI

Schaub et al. (2005), Plant Physiol. 138: 441

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 ricevarieties.

Ye et al., 2000; Science 287:303

Improved Golden Rice variants came in two versions

In the public and in the private sector

Gt1p PSY (Np) tp-CrtIGt1p

(from Narcissus)

No selectable marker geneCa. 1000 events„Clean“ integration selectedCrtI controlled by an endosperm-specific promoterThree high expressing events preselectedKnown as Golden Rice 1 Technology works in Indica varieties

The preselected events underwent 2 field trialsat Louisiana State University……

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

Improvements:

Five years of researchwere dominated by efforts to increase theamount of provitamin A in GR both, in the Public Sector as well as at Syngenta

Precursor shortage?

Carotenoid storage?

Phytoene synthase(E3) activity? Inefficient

transgene expression?

Inefficient transgene

expression?

Potential bottlenecks to higher carotenoid levels

IPP/DMAPP

C3-Carbon Metabolism

GGDP

Phytoene

ζ-Carotene

Lycopene

ß-Carotene

Lutein

α-Carotene

Zeaxanthin

Desaturation(CrtI)

activity?

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

02468

101214161820

ricePsy/crtI

maizePsy/crtI

pepperPsy/crtI

tomatoPsy/crtI

daffodilPsy/crtI

Individual transgenic plant (event)

Car

oten

oid

cont

ent (μg

g-1 d

wt) Rice and Maize PSY (E3)

best. Proportion of ß-carotene increased.

Phytoene synthase was investigated by Rachel Drake (Syngenta)Because PSY expression is good in GR, different versions of the PSY gene were assayed.

Paine et al., 2005 Nat Biotechnol. 23, 482-487

E2E1

E3

E8

Precursor

Produkt

Zwischenprodukt

E3 (PSY is rate-limiting)

Too slow!!!OK

OK

CrtICrtICrtI

OK

OK

Golden Rice 2 was re-made for implementation

pSYN12424

ZmPSY ubi1p

Transform long grain rice variety (Kaybonnet) Sugar selectable marker (PMI)

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 GoldenRice I and II

Increase in provit-A content: ca. 10-fold over GR1; about 25-fold over the prototype

9 events into 15 selected varieties (MAS), preceding event selectionIR64 & IR36: Mega-varieties with broad Asian coverage BRRI dhan 29 : The most popular boro rice variety in BangladeshPSB Rc82: The most popular rice variety in the PhilippinesOS 6561: Most popular in VietnamChehirang: Leading variety in Indonesia (with IRRC) Stacking With other micronutrients (zinc, iron vit E, lysine)Stacking With new agronomic traits (Submergence tolerance)

GR was a breeding project during the past 4 years

Partner Institutions:o IRRI (Int.), leado Philrice (Philippines)o BRRI (Bangladesh)o CLRRI (Vietnam)o IARI (India)o TNAU (India)o DRR (India)o Huazhong Univ. (China)

Event selection (All single locus intact ingtegration)

Which event(s) produce consistent levels of povitamin A across cultivars?

Which event(s) reproduce consistently the characteristics of the recurrent parents? (Completed, all other events destroyed)

Which level of provitamin A must be delivered by GR to be effective? Human bioavailability study

Conducted at TUFTS first and then in China

Bioconversion determined: 3.8-to-1 (!!!)Tang et al., (2009) Golden Rice is an effective source of vitamin A. Am J Clin Nutr89:1776–83

The big question:

Event selection (All single locus intact ingtegration)

Which event(s) produce consistent levels of povitamin A across cultivars?

Which event(s) reproduce consistently the characteristics of the recurrent parents? (Completed, all other events destroyed)

Which level of provitamin A must be delivered by GR to be effective? Human bioavailability studyConducted at TUFTS first and then in China

Bioconversion determined: 3.8-to-1 (!!!)Tang et al., (2009) Golden Rice is an effective source of vitamin A. Am J Clin Nutr89:1776–83

The big question:All done?

Exposure evaluationModelling analysis for intended use.

Bioavailability study.

Protein production and equivalenceExtraction from GMO plant or heterologous source

Biochemical characterisation

Function/ specificity/ mode of action.

Protein evaluationNo homology with toxins and allergens.

Rapid degredation in gastric /intestinal studies.

Heat lability

No indication of acute toxicity in rodents.

Further allergenicity assessments

Event independent studies

Regulatory dossiers can be complex and expensive

Molecular characterization and genetic stabilitySingle copy effect; marker gene at same locus.

Simple integration; Mendelian inheritance over

three generations (minimum).

No potential gene disruption.

No unknown open reading frames.

No DNA transfer beyond borders.

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.

Expression profilingGene expression levels at key growth stages.

Evidence for seed-specific expression.

Event dependent studies

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

(but will seldomly result in academic recognition)

Significant Costs

Why in the „Public Sector“? Some products of high general demand are not commercialy interesting.

The private sector will not make major investments. Hence the public sector should take over.

Funding is required To conduct more proof-of principle experimentation and translational

research

No expectations for funding in €Public research funding on transgenic crops in Europe goes into

„Biosafety“ research. There is virtually no funding for research that goesbeyond discovery.

(the development if GR relies on US-Philanthropy and on National funding inGR-implementing countries )

Kuiper et al., 2001 Assessment of the food safety issues related to genetically modified foods. Plant J. 2001 Sep;27(6):503-28.

The „Danger Zone“ is in Europe

Requirements:Raised to the point where anything that appears technically feasible is being requested/offered to be applied. This is in part driven by some public sector scientists.

Justification: Genetically modified plants are little understood (while we understand traditionally bred or mutagenized varieties?)

Consequence:Can raise costs to unaffordable levels - delays.

Overregulation of novel technologies: the „Locomotive Act“England, 1861 (the Red Flag Act)

Self-propelled vehicles on public roads in theUnited Kingdom must be preceded by a man on foot waving a red flag andblowing a horn.

This effectively killed road auto development in the UK for most of the rest of the 19th century

The red flag law was notrepealed until 1896

Where we are in the timeline?(Philippines - first launch)

Production of stable lines; fingerprinting(Bioavailability, biosafety & other studies)

Single location trial of elite lines (2 seasons), generation of protein data

Multi-location trials; gather agronomic and biosafety data

(2 seasons)Release

BPI application

2007

2008

2009

2010

2013/14The Philippines has experience in the regulatory review of a transgenic nutritionally-enhanced crop: LY038. 7 GMOs approved in the Philippines (http://agbios.com/dbase.php)

First Outdoor Trial of GR in Asia IR64 GR1 event 309; 20 lines

Transplanting at IRRI on April 2, 2008

April 10, 2008

May 30, 2008

Activities beyond technical and safety issues

Golden Rice:communication & social marketing

ConsumersGrowers

Focus groups: Qualitative information on consumer & grower attitudes in vitamin A-deficient areas

(+ Policy Makers, Heath Community, Regulators, Traders, NGOs, etc. )

MBA students of the Asian Institute of Management, Manila –training at AIM, IRRI. PhilRice

The Focus Group normally 10-12 people for structured open ended discussion for

2 hours, with careful recording

First social marketing research was coducted in 2009(then continued by a company)

In 4 selected islands in the PhilippinesAIM-MBA students teams from theseareas~ 720 hours interviews with 360 individuals in selected groups: gender, rice farming or not farming

Examples of the story‐board designs

Focus Group Methodology

The problem of VAD remains :GoldenRice is a potentially

significant contribution to alleviation.

It is now on its way towards registration

GR2 GR1

Wild-TypeFor additional information, see www.goldenrice.org

Philippinesgenerally high VAD, (and increasing) especially Visayas & W Mindanao

(figures are % of that population with VAD)

Source: Philippine Government Statistics 1998, 2003

Children, 6 Months to 5 Years Pregnant Women Lactating Women