Better health through nutritionally enhanced transgenic crops

Multivitamin maize through genetic engineering

CYTALIA XIV April 22, 2009

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The Drivers

Why we do what we do?

How we do what we do?

Applied Plant Biotechnology Laboratory

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Structure of the talk

Food insecurity and nutritionRole of Biotechnology and its contribution to poverty alleviation and nutritional improvement in the developing worldThe biofortified corn (maize) paradigmThe politics and regulation of GE cropsWhat does the future hold?

Millennium Development Goals 2008

Eradicate extreme poverty and hunger

Achieve universal primary education

Promote gender equality and empower women

Reduce child mortality

Improve maternal health

Combat HIV/AIDS, malaria and other diseases

Ensure environmental sustainability

Develop a global partnership for development

Copenhagen Consensus 2008 ranking, based on the costs and benefits of the solutions-Ranked in descending order of desirability

ChallengeSolution1Malnutrition Micronutrient supplements for children (vitamin A and zinc) 2Trade The Doha development agenda 3Malnutrition Micronutrient fortification (iron and salt iodization) 4Diseases Expanded immunization coverage for children 5Malnutrition Biofortification 6Malnutrition/EducationDeworming and other nutrition programs at school 7Education Lowering the price of schooling 8WomenIncrease and improve girls schooling 9Malnutrition Community-based nutrition promotion 10WomenProvide support for womens reproductive role

Interventions to alleviate malnutrition

Food fortification (dairy products and salt)Supplementation (pills or mineral solutions)Biofortification-Agronomic interventionsBiofortification-Plant breedingBiofortification-Genetic engineering

Predicted changes in population

Year 1995 2025

Population 5702 8122

(millions)

Nature 2008

The 15 hungriest countries (% of population undernourished)

1979-81

1996-98

80%

60%

40%

20%

10%

0%

Som Afg Bur Erit Hait Cong Moz NKor Ethi Lib Nig Mong Zam SLeo Ang

Somalia

Afganistan

Burundi

Eritrea

Haiti

Congo DR

Mozambique

North Korea

Ethiopia

Liberia

Niger

Mongolia

Zambia

Sierra Leone

Angola

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In the developing world 840 million people are chronically undernourished

Many more people, perhaps half of the worlds population, suffer from diseases

caused by dietary deficiencies and inadequate supplies of vitamins and minerals

Despite the prevalence of hunger and malnutrition, global food production has

outpaced population growth over the last 40 years thanks mainly to the successes

of the Green Revolution

Today's food insecurity is caused not by insufficient food production, but by poverty,

with nearly 1.3 billion people living on less than $US1 per day and another

2 billion only marginally better off

Projected world cereal demand
N. Borlaug, April 2001

Production1999, Mt)Needed 2025, MtYield t/ha1999Yield t/ha2025 NeededWheat5859002.73.8Rice6079003.14.3Maize60510004.15.9All cereals(includingminor crops)207431002.94.1

80% of all nutritional calories come from 20 crops.

70% from five.

40% from two

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If all Chinese ate five more
Big-Macs a year, this would
wipe out the entire US feed
corn production

Jerry Coldwell, CEO Mycogen

The Corn Question

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Evolution in Food & Feed Production

domestication of plants and animals

animal power

mechanical power

genetics (traditional breeding)

chemical assistance

biotechnology

Traditional Breeding and Genetic Engineering (GE not GM!)

humanity has been shaping its environment for milleniawheat, rice and corn are all the product of breedingGE permits introduction of desirable traitsall our staple crops are GE through plant breedingtraditional breeding is at its limitGE gives us new opportunities1302.unknown

Increased

yields

Manipulation of

plant architecture

Diversion of biomass

to edible organs

Protection against

pests and pathogens

Reduced losses due to

disease and infestation

Tolerance towards

abiotic stresses

Increased use

of marginal soils

Increased

planting density

Increase in levels

of utilizable

carbohydrate,

protein or fat

Fortification with

vitamins and

minerals

Manipulation of

plant development

Shorter generation intervals

Multiple production cycles

Eliminate

anti-nutritional

factors

Enhanced

photosynthesis

and nutrient uptake

Increased

accumulation

of biomass

Higher

nutritional

value

Remove constraints

Increase potential

HUNGER/MALNUTRITION

Modification of

carbohydrate,

protein or fat content

Bioremediation

Tolerance of

extreme weather

Impact of Transgenic Plants on Food Security

Plant Biotechnology-product development timeline

time

product launch

resistances to

viruses, insects

herbicides

male
sterility

disease

resistance

nematode

resistance

improved quality

health food,

nutraceuticals

improved
yields

1996 1998 2000 20052007 2010

therapeutics

vaccines

diagnostics

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Plant Genetic Engineering

Nature at work

Creation of the first transgenic plant

Agrobacterium tumefaciens

Nature, 303: 209 1983

NOS-OCS i NOS-CAT

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Two alternative methods to create transgenic plants

Argobacterium tumefaciensDirect DNA transfer through particle bombardment1293.psd

Plant transformation vector

GENE OF INTERST

targeting

signal

tag, anchor,

retention

ATG

start

TAA

stop

5UTR

3UTR

PROMOTOR

Term.

Seed-specific expression

Constitutive

marker genes
gusA, GFP, Luciferase, DsRED

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Donor plant

Transgenic plant

Corn transformation using

direct DNA transfer

Corn seeds = 14 dap

Immature embryo

(2,4 D)

Immature embryo derived callus

Callus ready

for bombardment

3 weeks

Callus tissue under phosphinothricin selection

Callus regenerating shoots

3-4 weeks darkness

To the light

In the dark

3-4 weeks

3-4 cm tall

Plant on rooting media

1292.bin

Agronomic properties focusing on insect and herbicide resistance

Durability, sustainability and environmental friendliness

Bacillus thuringensis (Bt)

First generation of transgenic plants

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Antama,

Spain

Antama,

Spain

Lleida January 2007

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Effect on chemical use

30-40% reduction in herbicide use with herbicide-resistant plants, because farmer can evaluate weed pressure before applicationMarket share shifting from narrow to broad-spectrum herbicidesUp to 80% reduction in insecticide use with insect-resistant plants

Conclusion: transgenic plants lead to

ecology friendly agriculture!

Multi-gene engineering is a significant hurdle in complex pathway analysis due to the diminishing rate of return as more transgenes are introduced simultaneously into target plants

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RECONSTRUCTION AND EXTENSION OF THE CAROTENOID PATHWAY IN MAIZE THROUGH COMBINATORIAL NUCLEAR TRANSFORMATION*

* Relies on the mechanism of co-integration of multiple

independent transgenes via direct DNA transfer into

one genetic locus

Carotenoids are naturally occurring biologically active compounds with exquisite health promoting properties

Experimental system: South African elite white maize inbred M37W, which lacks carotenoids in the endosperm due to the absence of the enzyme phytoene synthase (PSY1)

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WHITE vs YELLOW MAIZE

M37W

White endosperm Total carotenoids: 1.1g/g DW Lutein : Zeaxanthin 0.5 : 0.27 g/g DW

A632

Yellow endosperm Total carotenoids: 28 g/g DW Lutein : Zeaxanthin 15.61 : 7.77 g/g DW

WHITE vs YELLOW MAIZE: A Comparison

RT-PCR

psy1

psy2

pds

zds

crtISO

lyc

lyc

bch 1

Actin

M37W

A632

bch 2

RNA extracted from endosperm tissues psy1 is not expressed in M37W psy2, pds, zds, crtISO and bch exhibit similar levels of

expression in both genotypes

Phytoene synthase 2 is the only enzyme responsible for

phytoene synthesis in white maize (leaves)

Lyc and lyc expression is lower in M37W as compared to A632

mRNA blot analysis

M37W

A632

M37W

A632

psy1

lyc

M37W

A632

bch

Expression of endogenous genes in the carotenoid biosynthetic pathway

psy1 (Zea mays - yellow) U32636

crtI (Erwinia uredovora) D90087

lyc (Gentiana lutea) D017367

bch (Gentiana lutea) AB027187

crtW (Paracoccus sp.) D58420

For ketocarotenoids (Astaxanthin)

EXPERIMENTAL STRATEGY

M37W (white endosperm)

The resulting combinatorial population can be mined for phenotypes corresponding to the production of specific carotenoids, which in turn correlate with specific transgene expression and metabolic profiles

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psy1+crtI

psy1+crtI+lycb+bch+crtW

Endosperm specific expression

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mRNA ANALYSIS

HPLC PROFILES (carotenoid phenotypes)

CAROTENOID COMPOSITION

M37W-WT

A632-WT

Phenotype 1

Phenotype 2

Phenotype 3

Phenotype 4

Phenotype 5

Phenotype 6

Phenotype 7

Psy1 + crtI

Astaxanthin

Adonixanthin

Epoxy-Lutein

Lutein

Zeaxanthin

OH-Echinenone

-cryptoxanthin

-cryptoxanthin

Echinenone

Lycopene

-carotene

-zeaxanthin

-carotene

-carotene

INCREASE IN TOTAL CAROTENOIDS

43-fold

69-fold

60-fold

93-fold

132-fold

2.4-fold

150-fold

PhenotypesTransgenes1psy12crtI3psy1+crtI4psy1+crtI+lycb5psy1+crtI+bch+crtW6psy1+crtI+lycb+bch+crtWpsy1+crtI+lycb+bch+crtW7Chart1M37WA6321234567g/g DWTotal Carotenoids (g/g DW)1.12847.42.69165.476.1966.66103.5146.1Sheet1g/g DWM37W1.1A63228147.422.693165.4476.19566.666103.57146.12.15S3.992.16S4.962.3S102.13S202.7S44.0811.9117.394.692.95000.710.764.5947.052.28S33.39.9918.253.682.49000.90.936.1747.41.20S300.530.8600000001.41.25S301.561.1200000002.698.8915.64.274.412.261.0801.8415.9668.491.6S2.10006.6916.836.255.156.341.5503.6426.3976.191.10S33.768.7214.719.4904.741.7501.321.120.771.324.0858.052.14S108.9515.2723.1304.32.8502.251.290.71.415.3166.662.4-S21.7M37W012.0913.2919.1236.763.146.931.991.440.83.3631.57103.5A6321.1816.5210.6115.922.3613.281.635.9129.031.1604.7340.98146.120.0212.2712.664.9443.664.641.146.4159.61165.412.7921.774.675.8623.771.910.852.834.71157.40.50.270.070.090.141.1estimatedtotal 0.7815.617.771.991.090.3328Sheet1000000000g/g DWTotal Carotenoids (g/g DW)Sheet2Sheet3

The approach provides a unique and surprisingly straightforward

strategy for pathway analysis and multi-gene engineering in plants

It involves the introduction and coordinated expression of multiple

transgenes followed by the selection of stable lines expressing the

specific combination of transgenes required for particular metabolic

outputs

Individual lines, producing specific metabolites, can be goals in

themselves if the aim is to engineer particular molecules. However,

by examining the entire diverse population of plants, it becomes

possible to dissect the pathway and subsequently reconstruct it

either in its original form or with modifications

This provides a basis for understanding and subsequently

engineering the synthesis of novel metabolites

Transgene combinations Independent transformantsexhibiting the same phenotype1psy12 (Ph-1)2psy1+crtI3 (Ph-2)3crtI4 (Ph-3)4lycb35crtI+lycb56psy1+crtI+lycb4 (Ph-4)7bch28crtW39psy1+crtI+bch+crtW2 (Ph-5)10psy1+crtI+lycb+crtW3 (Ph-6)11psy1+crtI+lycb+bch+crtW5 (Ph-7)12crtI+lycb+bch+crtW2

High Astaxanthin corn

psy1+crtI+lycb+bch+crtW

Powerful biological antioxidant (100 times the strength of Vitamin E)

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PSY1 is the rate limiting enzyme in M37W (white maize) endosperm

Over-expression of PSY1 in transgenic M37W plants increased total carotenoids

Conversion of lycopene to -carotene (lyc) is a second limiting step in white maize expressing PSY1 Combinatorial Genetic Transformation: A novel technique to produce mutants that provide useful data to study a complex biosynthetic pathway The present study provides a platform to understand the carotenoid biosynthesis in maize

Ph 3

Ph 4

Ph 7

Enhanced levels of carotenoids in maize endosperm (lycopene, -carotene, zeaxanthin, ketocarotenoids) New strategies for carotenoid production in maize can be optimized on the basis of the mutant profiles

The approach is much simpler than traditional methods for the modification of the carotenoid or other complex pathways

it relies on probability and random sampling to generate a library of metabolic variants and a rapid visual selection to identify lines of interest

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The approach is analogous to standard mutagenesis screens although the mutants are generated not by random mutagenesis to create loss-of-function phenotypes, but by random multiplex transgene insertion to create partially reconstructed pathways

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High provitamin A corn

Golden Rice 1 beta-carotene 1.6g/g DW

Golden Rice 2 beta-carotene 37g/g DW

57.35

Multi vitamin corn

D-Man-P

GDP-D-Man

GDP-L-Gal

L-Gal -P

L-Gal

L-GalL

L-Ascorbate

GDP-L-Gul

L-Gul-P

L-Gul

L-GulL

GDP-L-Gul

UDP-D-galacturonate

D-galacturonate

L-Galactonate

L-GulLoxidase

D-Glucuronate

L-Gulonate

myo-Inositol

myo-Inositol

oxidase

Monodehydroascorbate

Dehydroascorbate

Dehydroascorbate

reductase

Ascorbate

oxidase

Dehydroascorbate reductase DHAR

Vitamin C

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Folate biosynthesis in plants

ADC: aminodeoxychorismate
DHN: dihydroneopterin
DHNTP: dihydroneopterin triphosphate
GCHI: GTP cyclohydrolase I

HMDHP: hydroxymethyldihydropterin

glu: glutamate
-PP: pyrophosphate

pABA: p-aminobenzoate

folE

Multi-vitamin corn

-carotene 60 g/g DW (PSY1+CrtI)

200 g/g DW folate (folE)

110 g/g FW ascorbate (DHAR)

WT-M37W

L-1 (PSY1+CrtI)

Plus folE and dhar

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100-200 g of grain provides full RDI of -carotene (as a sole source of vitamin A), more than enough folate, and about 20% of the RDI of ascorbate

Harvest Plus

7-10 years to create high vitA corn in locally adapted varieties

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Multi vitamin x yellow corn

Food security vs politics

The Politics of Plant Biotechnology

Some personal thoughts

and reflections

Key words

Poverty

Food Security

Developing countries

Agriculture

World trait

Protectionism

Big business

Politics

Biotechnology

Genetic modification

Public perception

Hostile press

Safety

Environmental impact

Social impact

Intellectual Property

Regulation

EU vs US trade

wars

Economics

Research funding

National policies

Globalisation

GATT

Cartagena protocol

Urbanisation

Migration

Regional conflicts

Government corruption

Overpopulation

Birth control

Clean water

Has it occurred to you how astonishing the culture of Western society really is?

Industrialized nations provide their citizens with unprecedented SAFETY, HEALTH and COMFORTAverage life spans increased 50% in the last centuryYet modern people live in abject fearThey are afraid of strangers, of disease, of crime, of the environmentThey are afraid of the homes they live in, the food they eat, the technology that surrounds them

28 agosto 2003

Zaragoza Espaa

Accin de Greenpeace en un campo de maz transgnico en Zaragoza

Mad Potato Disease

Aspects of the political dimension of transgenic plants

GREENPEACE

The precautionary approach as applied to Geneticaly Enhanced products in Europe

Regulators do not need to show scientifically that a biotech crop or product is unsafe but rather that it has not been proven harmless

Applying the precautionary principle (approach?) and its implications on developing countries

Research is being slowed down without any scientific valid reason and if this situation continues, the worlds poor will be the ones to suffer

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In the 35 years since the environmental movement came into existence, science has undergone a major revolution

This revolution has brought new understanding of nonlinear dynamics, complex systems, chaos theory, catastrophy theoryIt has transformed the way one should be thinking about evolution and ecologyYet these no-longer-new ideas have hardly penetrated the thinking of environmental activists, which seems oddly fixed in the concepts and rhetoric of the 1970s

We need a new environmental movement, with new goals and new organisations

We need more people working in the field, in the actual environment, and fewer people behind computer screensWe need more scientists, many few politicians and even fewer lawyers

And that is why the intermixing of science and politics is a bad combination, with a bad history

We must remember the history, and be certain that what we present to the world as knowledge is disinterested and honest

Alston Chase

When the search for truth is confused with political correctness and advocacy, the pursuit of knowledge is reduced to the quest for power

Chemical X

An abundunt chemical found in the environement in most parts of the worldIt is found in lakes and riversIt remains on fruits and vegetables after they are washedIt makes you sweatIt is responsible for the deaths of thousands of people every year in developing and industrialised countries alike

Even if the precautionary principle were to be applied mildly, this chemical should be banned

In fact banning the chemical through legislation at the EU level is what 59% of Europeans said in a survey in the UK, Germany, France, Austria and Scandinavia

83% of the same people expressed very strong views about the need to

control global warming, objected totally to the use of any animals for

medical research and of course they were overwhelmingly anti-biotech!

Dihydrogen monoxide

Otherwise known as

Water

Jettisoning scientific risk-benefit assessment and replacing it with a precautionary approach will result in arbitrary and politically motivated decisions that will decide the fate of Genetically Enhanced crops and products

The Cartagena biodiversity protocol is the single most

important threat to transgenic crop development

This is because the original focus of the protocol has

been highjacked for politial and economic reasons

aiming solely towards protectionism and restoration of

trait barriers under the pretext of biosafety!

Plant Biotechnology has a huge potential to contribute substantially to food security and poverty alleviation, in addition to creating a better and healthier environment

The major issues we now have to address are political and economic in nature

and this is where scientists have a role to play in putting the record straight!

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European objections to transgenic crops and food derived from them are aiming to protect the CAP and NOTHING ELSE!

Transgenic plant releases and commercialization are governed by Draconian rules

unparalleled elsewhere in any other sector. The European Union in a report following

a 15 year study (1985-2000) involving 400 public research institutions, to the cost of

70 million Euros stated genetically modified plants and products derived from them

present no risk to human health or the environmentthese crops and products are

even safer than plants and products generated through conventional processes

(EC Research, 2001).

The claim that antibiotic resistance genes in transgenic plants

will escape into natural bacterial populations that will subsequently become resistant

to them, thus creating super-bacteria is at best odd, as these genes are already

present in bacterial population in nature. It is worth remembering that the selectable

marker genes were isolated from these very naturally occurring bacteria in the first

place, for use in the laboratory!

EC Research (2001) EC-Sponsored Research on Safety of Genetically

Modified Organisms: A review of results:

http://europa.eu.int/comm/research/quality-of-life/gmo/

A degree of realism on the part of regulatory agencies is necessary

to assure that over-regulation stops being undully cumbersome and

prohibitively expensive.

Regulatory agencies need to divorce themselves from environmental

and political activists that threaten to put a stop transgenic plants.

Safety and efficacy should be paramount but they need to be

realistic and proportionate, and need to consider risk-benefit ratios

Image by permission of Fotosearch.com

Biotech Crop Countries and Mega-Countries, 2007

2008/9

Create super-nutritious biofortified rice and maize seeds which will

represent a quantum leap in current efforts to address issues

of poverty alleviation and food insecurity, through biotechnology

in the developing world with durability and sustainability in mind,

mostly by limiting key agronomic inputs imposed by the multitude of

insects and noxious/parasitic weeds in marginal environments,

focusing on Sub-Saharan Africa and the Indian subcontinent

Simultaneous multi-pathway engineering in crop plants through

combinatorial genetic transformation: creating complete biofortified

cereal grains for food security (BIOFORCE)

BIOFORCE targets

Vitamines A, C, folate (B9), EMinerals Fe, Zn, Se, CaInsect resistance and eliminate Striga

European

Research

Council

Vitamin A deficiency is prevalent in the developing world, and is probably responsible

for the death of 2 million children every year.

In surviving children, vitamin A deficiency is a leading, but avoidable cause of blindness. Effects of vitamin A deficiency are manifested as xerophthalmia

(visual impairment), blindness, and increased mortality due to increased severity

of children diseases such as measles, diarrhoea, and increased maternal

transmission of virus such as HIV.

Humans can synthesize vitamin A if provided with the precursor molecule

beta-carotene (also known as provitamin A). Endosperms of food crops, such as maize

and wheat, are low in provitamin A (1-10%) as compared with non-provitamin A

carotenoid.

Teresa Capell

SPAIN

Changfu Zhu

CHINA

Koreen Ramessar

(PhD student-South Africa)

Sonia Gomez

(PhD student-Spain)

Ariadna Peremarti (PhD student-Spain)

Svetlana Dashevskaya

(PhD student-Israel)

Gemma Farr

(PhD student-Spain)

Maite Sabalza

(PhD student-Spain)

Dawie Yuan

(PhD student-China)

Ludovic Bassie

FRANCE

Bruna Miralpeix

(Final year project student

Spain)

Shaista Naqvi

(PhD studen-Pakistan)

MICINN

Generalitat de Catalunya

UdL

ICREA

EU FP 6 & 7

ERA NET

Bill & Melinda Gates Fd

G. Sandmann, Uni Frankfurt

G. Ross, Univ Murcia

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If you desire peace, cultivate justice, but.

Norman E. Borlaug, Oslo, Norway, December 11, 1970

Nobel lecture

.at the same time cultivate the fields

to produce more bread.

otherwise there will be no peace

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M37W

A632

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Total Carotenoids (g/g DW)