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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
– Field and Laboratory Testing
Lecture Outline (cont.)
Gene Transfer to Commercial Cultivars
 Product Commercialization
 Plant Transformation Issues

– Regulatory and Safety Issues
– Societal Issues
Applications in Plant
Biotechnology

Agronomic trait improvement
– improved winter hardiness and stand
persistence
– insect resistance
– pathogen tolerance
– herbicide tolerance
– nitrogen fixation
– drought tolerance
Applications in Plant
Biotechnology

Nutritional Improvement
– increasing genistein content (soybeans)
– oil quality (soybeans & canola)
– changing amino acid ratios (corn)
– forage digestibility (alfalfa)

Improved Breeding
– QTL cloning for grain productivity and plant
height
Applications in Plant
Biotechnology

Green Factories
– biodegradable plastics from plants
– pharmaceuticals from plants


Avian influenza vaccines (alfalfa)
Large scale pharmaceutical protein production
– improved bioenergy sources

Biobutanol – closer to gasoline than ethanol
Genetically Modified Organisms
(GMO’s)
All creatures are genetically modified
 Genetic modification is natural

– Viruses
– Bacteria
– Radiation
Genetically Modified Organisms
(GMO’s)

Methods used by people to genetically
modify organisms
– Chemical mutagens (Methyl Bromide)
– Radiation (gamma radiation)
– Genetic engineering
DNA to Protein
A Short Introduction
Protein Structure
Primary/Secondary Structure
Tertiary Structure
Quaternary Structure
Protein Structure
Three polypeptides make up one multimeric protein
DNA Code is Degenerate
sense
antisense
GCT AAA CGT TGT TAT AAT TCT
CGA TTT GCA ACA ATA TTA AGA
GCU AAA CGU UGU UAU AAU UCU
DNA
mRNA
Ala Lys Arg Cys Tyr Asn Ser Protein
GCC AAG CGC UGC UAC AAC UCC
mRNA
From DNA to Protein
Promoter
Exon
Intron
DNA
Exon
Transcription
Exon
Intron
Exon
Poly A
RNA
From DNA to Protein
Exon
Intron
Exon
Poly A
RNA
Splicing
Exon
Exon
Translation
Transit
Peptide
Protein
Poly A
mRNA
The Plant Cell

Transit peptides
direct proteins to
organelles
–
–
–
–
mitochondria
chloroplast
vacuole
cell wall
How Plants are Transformed
What are the requirements?
Functional Genomics

Three major areas of study
– Metabolomics

Understanding of products and the metabolic pathways
used to their creation
– Proteomics

Understanding the role of proteins in the life and
regulation of living organisms
– Genomics

Understanding the genetic control of various metabolic
and proteomic pathways
Functional Genomics

Gene identification and isolation
– micro-array technologies
– cloning
– sequencing
– knowledge of regulatory elements
DNA Micro-Array Technology



Complete organism
genome on micro
array
Used to determine
gene activity
Helps understanding
of genetic pathways
Recombinant DNA
Technologies




Cut DNA from Donor
Paste DNA into
plasmid
Insert plasmid into
Host Bacterium
Transform Plants
The Binary Vector




Multiplied in E. coli
and Agrobacterium
Modular structure
Parts can be cut and
pasted with great
precision
Composed of DNA
from many sources
Antibiotic
Resistance
Gene
Antibiotic
Resistance
Gene
pVINV
Antibiotic
Resistance
Gene
Viral 35S
promoter
Yeast Invertase Gene
Patatin
Transit Peptide
Bacterial Selection

Bacteria are plated
onto selection media
– antibiotics

E. coli
– DNA isolated for
Particle Bombardment

Agrobacterium
– Used to infect plant
material
Plant
Transformation

Four methods used
today:
–
–
–
–
Electroporation
Micro-injection
Particle Bombardment *
Agrobacterium *
Agrobacterium
Transformation
Plant Cell
Cytosol
Nucleus
Biolistic Plant Transformation




Biolistic gun
Patented by Dupont
One of the two main
transformation
methods used
Used highly
compressed air
Biolistic Plant Transformation



Uses DNA coated
gold or tungsten
beads
Compressed air
blasts beads into the
cells or tissue
Cells take up the
foreign DNA
Tissue
Culture


The Big
Picture
Regeneration
from single
cells
Plant Tissue Culture
Somatic Cell Embryogenesis
 Cell Suspension Culture
 Protoplast Regeneration
 Anther Culture
 Organogenesis

Somatic
Embryogenesis

Donor plant and
explants
Somatic Embryogenesis



Sterilization
Co-cultivation
Bombardment
Somatic
Embryogenesis

Somatic Embryos
Origin
Somatic Embryogenesis

Induction
Somatic Embryogenesis

Embryo
Development
Somatic Embryogenesis

Germination
Somatic
Embryogenesis

Plant Development
Screening and Isolation of
Transformants

Polymerase Chain Reaction (PCR)
– is the gene present?

Southern Analysis
– gene copy number

Northern Analysis
– gene expression patterns

Enzyme Assays
– gene product activity
Polymerase Chain Reaction
(PCR)



Only Foreign
DNA targeted
Only specific
regions of DNA
amplified
Easy and quick
to screen 100’s
of plants
Southern Analysis


Determines gene copy number
Detects only foreign DNA
Laboratory Testing of
Transgenic Plants

Laboratory and Greenhouse Testing
– Is the plant producing the required
material?
– Is it healthy?
– Will it survive?
Field Testing of Transgenic
Plants

Field Testing
– Allows evaluation of the
new plant in the real world
– Will the plant survive real
world stresses?
– Is the new trait inherited?
– Will the trait transfer to
commercial varieties?
Plant Biotech
Products




Golden Rice
Gene insertion
(ProVitamin A and
Phytase)
Enhanced beta-carotene
(vitamin A) and Iron
absorption
Helps prevent Vitamin A
deficiency (VAD) and
Iron Deficiency Anemia
(IDA)
Plant Biotech
Products


Flavr savr tomato
Gene knockout
(antisense expression)
Plant Biotech
Products


Bt Corn
Gene
overexpression
(constitutive)
Plant Biotech
Products
RR Soybean
 Gene overexpression
(constitutive)

Plant Biotech
Products




Salt tolerant tomato
Gene insertion
Gives ability to grow
on salt contaminated
soils
Ability to accumulate
salt from the soil
Zhang and Blumwald (2001)
Nature Biotechnology 19
Plant Biotech
Products
High Energy Alfalfa
 Gene targeting

– Yeast invertase to vacuole
– using patatin transit peptide
Mean Total Soluble Carbohydrate per Plant Part
(Error Bars Indicate Standard Deviation)
A.
TSC (m g/g dry w eight)
14
12
10
Root
8
Leaf
Stem
6
4
2
0
Control
pVINV
Genotype
Commercialization Issues

Freedom To Operate
– Many of the enabling technologies
patented
– May require permission and/or payment of
licensing fees
– Can cause many problems in getting the
product to the market
– Could force researchers to re-invent the
wheel
Major Concerns About Plant
Biotechnology
Imprecise technology
 Food and product safety
 Cross-species transfer (horizontal gene
transfer)

– Genetic pollution

New weeds
Imprecise Technology

Scientists can now place a single copy
of a gene precisely in the genome
– Matrix Attachment Regions

More stable expression of transgenes
– Cre-Lox and FLP/FRT system

Enables precise placement and removal of
transgenes
– Antibiotic resistance genes
– Herbicide resistance genes
– Transgenic trait genes
Ow and Srivastava, 2004 Trends in Biotech 22 (12): 627-629
Food and Product Safety

Canadian Food Inspection Agency Roles
– Regulate transgenic field trials
– Ensures field trials conducted in
reproductive isolation
– Environmental Protection



prevent gene pollution (gene transfer to nontarget species)
prevent the environmental release of toxins
prevent the production of Super-weeds
Food and Product Safety

Canadian Food Inspection Agency Roles
– Ensure Food Safety



product is non-toxic to humans or animals
must be equivalent to current food products
must be non-allergenic
Food and Product Safety

Codex Alimentarius Commission
– Established in 1963 by WHO and FAO of
the UN
– 2003 - established risk/safety assessment
guidelines for food derived from genetically
modified plants and micro-organisms
– Serves as a reference for international
trade disputes over the safety of
internationally traded foods
Questions?