challenges and opportunities in plant biotechnology

Challenges and Challenges and Opportunities in Plant Opportunities in Plant

BiotechnologyBiotechnology

Dave A. VadnaisDave A. Vadnais

Lecture OutlineLecture Outline

Applications in Plant BiotechnologyApplications in Plant Biotechnology How Plants are TransformedHow Plants are Transformed

– Functional GenomicsFunctional Genomics– Recombinant DNA TechniquesRecombinant DNA Techniques– Plant TransformationPlant Transformation– Tissue CultureTissue Culture– Screening and Isolation of TransformantsScreening and Isolation of Transformants– Field and Laboratory TestingField and Laboratory Testing

Lecture Outline (cont.)Lecture Outline (cont.)

Gene Transfer to Commercial Gene Transfer to Commercial CultivarsCultivars

Product Commercialization Product Commercialization Plant Transformation Issues Plant Transformation Issues

– Regulatory and Safety IssuesRegulatory and Safety Issues– Societal IssuesSocietal Issues

Applications in Plant Applications in Plant BiotechnologyBiotechnology

Agronomic trait improvementAgronomic trait improvement– improved winter hardiness and stand improved winter hardiness and stand

persistencepersistence– insect resistanceinsect resistance– pathogen tolerancepathogen tolerance– herbicide toleranceherbicide tolerance– nitrogen fixationnitrogen fixation– drought tolerancedrought tolerance


Nutritional ImprovementNutritional Improvement– increasing genistein content (soybeans)increasing genistein content (soybeans)– oil quality (soybeans & canola)oil quality (soybeans & canola)– changing amino acid ratios (corn)changing amino acid ratios (corn)– forage digestibility (alfalfa)forage digestibility (alfalfa)

Improved BreedingImproved Breeding– QTL cloning for grain productivity and QTL cloning for grain productivity and

plant heightplant height


Green FactoriesGreen Factories– biodegradable plastics from plantsbiodegradable plastics from plants– pharmaceuticals from plantspharmaceuticals from plants

Avian influenza vaccines (alfalfa)Avian influenza vaccines (alfalfa) Large scale pharmaceutical protein Large scale pharmaceutical protein

productionproduction

– improved bioenergy sourcesimproved bioenergy sources Biobutanol – closer to gasoline than Biobutanol – closer to gasoline than

ethanolethanol

Genetically Modified Genetically Modified OrganismsOrganisms

(GMO’s)(GMO’s)

All creatures are genetically All creatures are genetically modifiedmodified

Genetic modification is naturalGenetic modification is natural– VirusesViruses– BacteriaBacteria– RadiationRadiation

Genetically Modified Genetically Modified OrganismsOrganisms

(GMO’s)(GMO’s)

Methods used by people to Methods used by people to genetically modify organismsgenetically modify organisms– Chemical mutagens (Methyl Bromide)Chemical mutagens (Methyl Bromide)– Radiation (gamma radiation)Radiation (gamma radiation)– Genetic engineeringGenetic engineering

DNA to ProteinDNA to Protein

A Short IntroductionA Short Introduction

Protein StructureProtein Structure

Primary/Secondary StructurePrimary/Secondary Structure

Tertiary StructureTertiary Structure Quaternary StructureQuaternary Structure

Protein StructureProtein Structure

Three polypeptides make up one multimeric proteinThree polypeptides make up one multimeric protein

Ala Lys Arg Cys Tyr Asn SerAla Lys Arg Cys Tyr Asn SerProtein Protein

GCGCUU AA AAAA CG CGUU UG UGUU UA UAUU AA AAUU UC UCUU mRNAmRNA

GCGCC C AAAAGG CG CGCC UG UGCC UA UACC AA AACC UC UCCC mRNAmRNA

DNA Code is DegenerateDNA Code is Degenerate

GCT AAA CGT TGT TAT AAT TCTGCT AAA CGT TGT TAT AAT TCTsensesenseDNADNA

antisenseantisenseCGA TTT GCA ACA ATA TTA AGACGA TTT GCA ACA ATA TTA AGA

From DNA to ProteinFrom DNA to Protein

Poly A

Transcription

Exon ExonIntronPromoter DNA

Exon ExonIntron RNA

Poly AExon ExonIntron RNA

Splicing

Poly AExon Exon mRNA

Translation

From DNA to ProteinFrom DNA to Protein

ProteinTransit Peptide

The Plant CellThe Plant Cell

Transit Transit peptides direct peptides direct proteins to proteins to organellesorganelles– mitochondriamitochondria– chloroplastchloroplast– vacuolevacuole– cell wallcell wall

How Plants are How Plants are TransformedTransformed

What are the requirements?What are the requirements?

Functional GenomicsFunctional Genomics Three major areas of studyThree major areas of study

– MetabolomicsMetabolomics Understanding of products and the metabolic Understanding of products and the metabolic

pathways used to their creationpathways used to their creation

– ProteomicsProteomics Understanding the role of proteins in the life and Understanding the role of proteins in the life and

regulation of living organismsregulation of living organisms

– GenomicsGenomics Understanding the genetic control of various Understanding the genetic control of various

metabolic and proteomic pathwaysmetabolic and proteomic pathways

Functional GenomicsFunctional Genomics Gene identification and isolationGene identification and isolation

– micro-array technologiesmicro-array technologies– cloningcloning– sequencingsequencing– knowledge of regulatory elementsknowledge of regulatory elements

DNA Micro-Array DNA Micro-Array TechnologyTechnology

Complete Complete organism genome organism genome on micro arrayon micro array

Used to Used to determine gene determine gene activityactivity

Helps Helps understanding of understanding of genetic pathwaysgenetic pathways

Recombinant DNA Recombinant DNA TechnologiesTechnologies

Cut DNA from Cut DNA from DonorDonor

Paste DNA into Paste DNA into plasmidplasmid

Insert plasmid Insert plasmid into Host into Host BacteriumBacterium

Transform PlantsTransform Plants

Yeast Invertase Gene PatatinTransit Peptide

The Binary VectorThe Binary Vector Multiplied in Multiplied in E. coliE. coli

and and AgrobacteriumAgrobacterium Modular structureModular structure Parts can be cut Parts can be cut

and pasted with and pasted with great precisiongreat precision

Composed of DNA Composed of DNA from many sourcesfrom many sources

AntibioticResistanceGene



Viral 35S promoter

pVINV

Bacterial SelectionBacterial Selection

Bacteria are plated Bacteria are plated onto selection mediaonto selection media– antibioticsantibiotics

E. coli E. coli – DNA isolated for DNA isolated for

Particle BombardmentParticle Bombardment AgrobacteriumAgrobacterium

– Used to infect plant Used to infect plant materialmaterial

Plant Plant TransformationTransformation

Four methods used Four methods used today:today:– ElectroporationElectroporation– Micro-injectionMicro-injection– Particle Particle

Bombardment Bombardment **– Agrobacterium Agrobacterium **

CytosolCytosol

NucleusNucleus

Plant CellPlant Cell

AgrobacteriumAgrobacterium TransformationTransformation

Biolistic Plant Biolistic Plant TransformationTransformation

Biolistic gunBiolistic gun Patented by Patented by

DupontDupont One of the two One of the two

main main transformation transformation methods usedmethods used

Used highly Used highly compressed aircompressed air

Biolistic Plant Biolistic Plant TransformationTransformation

Uses DNA coated Uses DNA coated gold or tungsten gold or tungsten beadsbeads

Compressed air Compressed air blasts beads into blasts beads into the cells or tissuethe cells or tissue

Cells take up the Cells take up the foreign DNAforeign DNA

Tissue Tissue CultureCulture

The Big The Big Picture Picture

RegeneratioRegeneration from n from single cellssingle cells

Plant Tissue CulturePlant Tissue Culture

Somatic Cell EmbryogenesisSomatic Cell Embryogenesis Cell Suspension Culture Cell Suspension Culture Protoplast RegenerationProtoplast Regeneration Anther CultureAnther Culture Organogenesis Organogenesis

Somatic Somatic EmbryogenesisEmbryogenesis

Donor plant and Donor plant and explants explants

Sterilization Sterilization Co-cultivation Co-cultivation BombardmentBombardment

Somatic EmbryogenesisSomatic Embryogenesis

Somatic Embryos Somatic Embryos OriginOrigin

Somatic Somatic EmbryogenesiEmbryogenesiss

InductionInduction


Embryo Embryo DevelopmentDevelopment


GerminationGermination


Plant Plant DevelopmentDevelopment

Somatic Somatic EmbryogenesisEmbryogenesis

Screening and Isolation of Screening and Isolation of TransformantsTransformants

Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)– is the gene present?is the gene present?

Southern AnalysisSouthern Analysis– gene copy number gene copy number

Northern AnalysisNorthern Analysis– gene expression patternsgene expression patterns

Enzyme AssaysEnzyme Assays– gene product activitygene product activity

Polymerase Chain Polymerase Chain ReactionReaction

(PCR)(PCR)

Only Foreign Only Foreign DNA targetedDNA targeted

Only specific Only specific regions of regions of DNA amplifiedDNA amplified

Easy and Easy and quick to quick to screen 100’s screen 100’s of plantsof plants

Mar

ker

Mar

ker

Non

-Tra

ns

Non

-Tra

nsPl

asm

id

Plas

mid

Tran

s 3

Tran

s 3

Tran

s 4

Tran

s 4

Tran

s 2

Tran

s 2

Tran

s 1

Tran

s 1

Southern AnalysisSouthern Analysis Determines gene copy numberDetermines gene copy number Detects only foreign DNADetects only foreign DNA

Laboratory Testing of Laboratory Testing of Transgenic PlantsTransgenic Plants

Laboratory and Greenhouse Laboratory and Greenhouse TestingTesting– Is the plant producing the required Is the plant producing the required

material? material? – Is it healthy? Is it healthy? – Will it survive?Will it survive?

Field Testing of Transgenic Field Testing of Transgenic PlantsPlants

Field TestingField Testing– Allows evaluation of the Allows evaluation of the

new plant in the real new plant in the real worldworld

– Will the plant survive Will the plant survive real world stresses?real world stresses?

– Is the new trait Is the new trait inherited?inherited?

– Will the trait transfer to Will the trait transfer to commercial varieties?commercial varieties?

Plant Biotech Plant Biotech ProductsProducts

Golden RiceGolden Rice Gene insertion Gene insertion

(ProVitamin A and (ProVitamin A and Phytase)Phytase)

Enhanced beta-Enhanced beta-carotene (vitamin A) carotene (vitamin A) and Iron absorptionand Iron absorption

Helps prevent Vitamin Helps prevent Vitamin A deficiency (VAD) A deficiency (VAD) and Iron Deficiency and Iron Deficiency Anemia (IDA)Anemia (IDA)


Flavr savr tomatoFlavr savr tomato Gene knockout Gene knockout

(antisense (antisense expression)expression)


Bt CornBt Corn Gene Gene

overexpressiooverexpression n (constitutive)(constitutive)


RR SoybeanRR Soybean Gene Gene

overexpression overexpression (constitutive)(constitutive)


Salt tolerant tomatoSalt tolerant tomato Gene insertionGene insertion Gives ability to grow Gives ability to grow

on salt contaminated on salt contaminated soilssoils

Ability to accumulate Ability to accumulate salt from the soilsalt from the soil

Zhang and Blumwald (2001) Zhang and Blumwald (2001) Nature Biotechnology 19Nature Biotechnology 19

Plant Plant Biotech Biotech ProductsProducts High Energy AlfalfaHigh Energy Alfalfa Gene targetingGene targeting

– Yeast invertase to vacuoleYeast invertase to vacuole– using patatin transit using patatin transit

peptidepeptideMean Total Soluble Carbohydrate per Plant Part(Error Bars Indicate Standard Deviation)

0

2

4

6

8

10

12

14

Control pVINV

Genotype

TS

C (

mg

/g d

ry w

eig

ht)

Root

Leaf

Stem

A.

Commercialization IssuesCommercialization Issues

Freedom To Operate Freedom To Operate – Many of the enabling technologies Many of the enabling technologies

patented patented – May require permission and/or May require permission and/or

payment of licensing feespayment of licensing fees– Can cause many problems in getting Can cause many problems in getting

the product to the marketthe product to the market– Could force researchers to re-invent Could force researchers to re-invent

the wheelthe wheel

Major Concerns About Plant Major Concerns About Plant BiotechnologyBiotechnology

Imprecise technologyImprecise technology Food and product safetyFood and product safety Cross-species transfer (horizontal Cross-species transfer (horizontal

gene transfer)gene transfer)– Genetic pollutionGenetic pollution

New weedsNew weeds

Imprecise TechnologyImprecise Technology

Scientists can now place a single Scientists can now place a single copy of a gene precisely in the copy of a gene precisely in the genome genome – Matrix Attachment RegionsMatrix Attachment Regions

More stable expression of transgenes More stable expression of transgenes

– Cre-Lox and FLP/FRT systemCre-Lox and FLP/FRT system Enables precise placement and removal of Enables precise placement and removal of

transgenestransgenes– Antibiotic resistance genesAntibiotic resistance genes– Herbicide resistance genesHerbicide resistance genes– Transgenic trait genesTransgenic trait genes

Ow and Srivastava, 2004 Trends in Biotech 22 (12): Ow and Srivastava, 2004 Trends in Biotech 22 (12): 627-629627-629

Food and Product SafetyFood and Product Safety

Canadian Food Inspection Agency Canadian Food Inspection Agency RolesRoles– Regulate transgenic field trialsRegulate transgenic field trials– Ensures field trials conducted in Ensures field trials conducted in

reproductive isolationreproductive isolation– Environmental Protection Environmental Protection

prevent gene pollution (gene transfer to non-prevent gene pollution (gene transfer to non-target species)target species)

prevent the environmental release of toxinsprevent the environmental release of toxins prevent the production of prevent the production of Super-weedsSuper-weeds


Canadian Food Inspection Agency Canadian Food Inspection Agency RolesRoles– Ensure Food SafetyEnsure Food Safety

product is non-toxic to humans or product is non-toxic to humans or animalsanimals

must be equivalent to current food must be equivalent to current food productsproducts

must be non-allergenic must be non-allergenic


Codex Alimentarius CommissionCodex Alimentarius Commission– Established in 1963 by WHO and FAO of Established in 1963 by WHO and FAO of

the UNthe UN– 2003 - established risk/safety assessment 2003 - established risk/safety assessment

guidelines for food derived from guidelines for food derived from genetically modified plants and micro-genetically modified plants and micro-organismsorganisms

– Serves as a reference for international Serves as a reference for international trade disputes over the safety of trade disputes over the safety of internationally traded foodsinternationally traded foods

Questions?Questions?

challenges and opportunities in plant biotechnology

Documents

plant heightapplications

plant biotechnologydave

coli dna

degeneratefrom dna

donorpaste dna

plant biotechnologyhow

proteinpoly afrom dna

great precisioncomposed