View
66
Download
0
Category
Tags:
Preview:
DESCRIPTION
Challenges and Opportunities in Plant Biotechnology. Dave A. Vadnais. Lecture Outline. Applications in Plant Biotechnology How Plants are Transformed Functional Genomics Recombinant DNA Techniques Plant Transformation Tissue Culture Screening and Isolation of Transformants - PowerPoint PPT Presentation
Citation preview
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
Applications in Plant Applications in Plant BiotechnologyBiotechnology
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
Applications in Plant Applications in Plant BiotechnologyBiotechnology
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
AntibioticResistanceGene
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
Somatic EmbryogenesisSomatic Embryogenesis
Embryo Embryo DevelopmentDevelopment
Somatic EmbryogenesisSomatic Embryogenesis
GerminationGermination
Somatic EmbryogenesisSomatic Embryogenesis
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)
Plant Biotech Plant Biotech ProductsProducts
Flavr savr tomatoFlavr savr tomato Gene knockout Gene knockout
(antisense (antisense expression)expression)
Plant Biotech Plant Biotech ProductsProducts
Bt CornBt Corn Gene Gene
overexpressiooverexpression n (constitutive)(constitutive)
Plant Biotech Plant Biotech ProductsProducts
RR SoybeanRR Soybean Gene Gene
overexpression overexpression (constitutive)(constitutive)
Plant Biotech Plant Biotech ProductsProducts
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
Food and Product SafetyFood and Product Safety
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
Food and Product SafetyFood and Product Safety
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?
Recommended