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The Science of Transgenics

Phil McCleanDepartment of Plant Science

North Dakota State University

The Sociological, Economic, and Ethical Impactof Transgenic Organisms Workshop

Fargo, NDFebruary 21, 2003

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Transgenics are a Biotechnology Product

How about some definitions

Biotechnology - General Definition

The application of technology to improve a biological organism

Biotechnology - Detailed Definition

The application of the technology to improve the biological function of an organism by adding genes from another organisms

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What About the TermGenetic Engineering?

Genetic engineering involves:

Isolating genes Modifying genes so they function better Preparing genes to be inserted into a new species Developing transgenes

Genetic engineering is the basic tool set of biotechnology

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What is a transgenic?

Transgene – the genetically engineered gene added to a species

Ex. – modified EPSP synthase gene (encodes a protein that functions even when plant is treated with Roundup)

Transgenic – an organism containing a transgene introduced by technological (not breeding) methods

Ex. – Roundup Ready Crops

Concept Based on the Term Transgene

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We can develop organisms that express a “novel” trait not normally found in the species

Why are transgenics important?

Extended shelf-life tomato (Flavr-Savr)

Herbicide resistant soybean (Roundup Ready)

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Agriculture Transgenics On the Market

Source: USDA

Insect resistant cotton – Bt toxin kills the cotton boll worm• transgene = Bt protein

Insect resistant corn – Bt toxin kills the European corn borer• transgene = Bt protein

Normal Transgenic

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Virus resistance - papya resistant to papaya ringspot virus• transgene = virus coat protein

Source: Monsanto

Herbicide resistant crops Now: soybean, corn, canola Coming: sugarbeet, lettuce, strawberry alfalfa, potato, wheat (2005?)• transgene = modified EPSP synthase or phosphinothricin-N-acetyltransferase

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Biotech chymosin; the enzyme used to curdle milk products• transgene = genetically engineered enzyme

bST; bovin somatotropin; used to increasemilk production• transgene = genetically engineered enzyme

Source: Rent Mother Nature

Source: Chr. Hansen

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Some Ag Biotech Products Are Discontinued

Poor Quality• FlavrSavr tomatoes (Calgene)

Negative Consumer Response• Tomato paste (Zeneca)

Negative Corporate Response• NewLeaf (Monsanto)

Universal Negative Publicity• StarLink corn (Aventis)

Why???

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Next Generation of Ag Biotech Products

Source: Minnesota Microscopy Society

Golden Rice – increased Vitamin A content (but not without controversy)transgene = three pathway enzymes

Sunflower – white mold resistancetransgene = oxalate oxidase from wheat

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Turfgrass – herbicide resistance; slower growing (= reduced mowing)

Bio Steel – spider silk expressed in goats; used to make soft-body bullet proof vests (Nexia)

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Biotechnology is Not Just on the Farm

Disease Treatment

Diagnostics 

Environmental Cleanup 

Human Applications

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Human Applications

• Pharmaceutical products New solutions to old problems • Disease diagnosis Determine what disease you have or may get 

• Gene therapy Correcting disease by introducing a corrective gene

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Biotechnology and Health

Product Use

Insulin Diabetes

Interferon Cancer

Interleukin Cancer

Human growth hormone Dwarfism

Neuroactive proteins Pain

The genes for these proteins are:

• Cloned• Inserted into bacteria• Product isolated using biofermentation

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Environmental Applications

Bioremediation - cleanup contaminated sites; uses microbes designed to degrade the pollution

Indicator bacteria – contamination can be detected in the environment

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Tooth decay – engineered Streptococcus mutans, the bacteria that destroys enamel

Future Health-related Biotech Products

Vaccines – herpes, hepatitis C, AIDS, malaria

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Edible VaccinesTransgenic Plants Serving Human Health Needs

• Works like any vaccine • A transgenic plant with a pathogen protein gene is developed• Potato, banana, and tomato are targets• Humans eat the plant • The body produces antibodies against pathogen protein• Humans are “immunized” against the pathogen• Examples:

DiarrheaHepatitis BMeasles

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A Popular Term We Need To Know

GMOs - Genetically modified organisms

• GMO - an organism that expresses traits that result from the introduction of foreign DNA

• Originally a term equivalent to transgenic organism

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Some claim any improved biological product is a GMO

They feel this will

For example, some call plant varieties biotechnology products

This is a false claim

The GMO Ruse

• ease the publics fear• pave the way for product acceptance

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Let’s Be Up Front

• Biotechnology adds traits not available in the species

 Soybean does not have a gene to breakdown Roundup The gene comes from bacteria

• Breeding Biotechnology

 Breeding only exchanges genes found in the species Breeding can transfer the transgene to other breeding materials BUT this does not make it a biotechnology procedure

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Important Plant Improvement Methods

• Breeding Crossing two individuals from the same species; produces a new, improved variety; not a biotechnology procedure

• Transformation Adding a gene from another species; the essential biotechnology procedure to produce transgenics

Source: USDA

Source: USDA

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Wheat Rye

Triticale

X

Interspecific Cross

New species, but NOT biotechnology products

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Mutagenesis

A useful procedure to produce a new trait

But the normal gene is modified

A transgene is not involved

The product of mutagenesis is not a GMO

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ATTCGA

ATTGGA

SusceptibleNormalGene

ResistantMutantGene

MutagenesisTreatment

Mutagenesis Changes the DNA Sequence

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BASF Clearfield Products

Herbicide resistance•imidazolinones

Mutant AHAS enzyme•developed by mutagenesis

Crops• Canola• Corn• Rice• Sunflower• Wheat

A Major Marketing Advantagebut lost when stacked with a transgene

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The Roundup Ready Story

• Glyphosate is a broad-spectrum herbicide• Active ingredient in Roundup herbicide • Kills all plants it come in contact with• Inhibits a key enzyme (EPSP synthase) in an amino acid pathway

• Plants die because they lack the key amino acids

• A resistant EPSP synthase gene allows crops to survive spraying

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+ Glyphosate

X

Roundup Sensitive Plants

X

X

Shikimic acid + Phosphoenol pyruvate

3-Enolpyruvyl shikimic acid-5-phosphate(EPSP)

Plant EPSP synthase

Aromaticamino acids

Without amino acids, plant dies

X

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BacterialEPSP synthase

Shikimic acid + Phosphoenol pyruvate

3-enolpyruvyl shikimic acid-5-phosphate(EPSP)

Aromaticamino acids

Roundup Resistant Plants

+ Glyphosate

With amino acids, plant lives

RoundUp has no effect;enzyme is resistant to herbicide

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The Golden Rice Story

• Vitamin A deficiency is a major health problem

• Causes blindness• Influences severity of diarrhea, measles

• >100 million children suffer from the problem

• For many countries, the infrastructure doesn’t existto deliver vitamin pills

• Improved vitamin A content in widely consumed cropsan attractive alternative

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-Carotene Pathway Problem in Plants

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Problem:Rice lacks

these enzymes

NormalVitamin A

“Deficient”Rice

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The Golden Rice Solution

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Daffodil gene

Single bacterial gene;performs both functions

Daffodil gene

-Carotene Pathway Genes Added

Vitamin APathway

is completeand functional

GoldenRice

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Introducing the Geneor

Developing Transgenics

Steps

1. Create transformation cassette

2. Introduce and select for transformants

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Transformation Cassettes

Contains

1. Gene of interest

• The coding region and its controlling elements

2. Selectable marker

• Distinguishes transformed/untransformed plants

3. Insertion sequences• Aids Agrobacterium insertion

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Transformation Steps

Prepare tissue for transformation

Introduce DNA

Culture plant tissue• Develop shoots• Root the shoots

Field test the plants

• Leaf, germinating seed, immature embryos

• Tissue must be capable of developing into normal plants

• Agrobacterium or gene gun

• Multiple sites, multiple years

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• Transformation cassettes are developed in the lab

• They are then introduced into a plant

• Two major delivery methods

Delivering the Geneto the Plant

• Agrobacterium

• Gene GunTissue culturerequired to generatetransgenic plants

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The Lab Steps

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The Next Test Is The Field

Non-transgenics

Transgenics

Herbicide Resistance

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Final Test of the TransgenicConsumer Acceptance

RoundUp Ready Corn

Before After