Plant Biotechnology:
from classical breeding to
genetic engineering
Plant Uses
2
Why We Can’t do Without Plants
• Produce oxygen for the atmosphere
• Produce lumber for building
• Provide homes and food for many organisms
• Prevent erosion
• Used for food
3
More Reasons We Can’t do
Without Plants
• Produce wood pulp for paper products
• Source of many medicines
• Ornamental and shade for yards
• Fibers such as cotton for fabric
• Dyes
4
250,000 plant species
3 crop species (rice, wheat and maize) provide
60% of all callories and 54% of all proteins
Plant Agriculture
• Crop production and protection
– Genetically engineered (transgenic) crops
– Using biological methods to protect crops
– Exploiting cooperative relationships in nature
• Nutritional value of crops
– Improving food quality and safety
– Healthier cooking oils by decreasing the conc. of
saturated fatty acids in vegetable oils
– Functional foods: foods containing significant levels
of biologically active components that impart health
benefits
Plant Biotechnology
Plant Biotechnology
◼ Manipulating plants for the benefit of mankind
◼ A process to produce a genetically modified
plant by removing genetic information from an
organism, manipulating it in the laboratory and
then transferring it into a plant to change
certain of its characteristics
◼ Technology:
◼Tissue culture
◼Plant transformation
Traditional Plant Breeding
▪ Cross breading of
similar plants to produce
new varieties with
different traits.
▪ Take many
generations to achieve
desired results
Breeding the traditional way
1. Identify source of a trait by phenotypic
screening of germ plasm collections.
2. Cross source with elite cultivars.
3. Select desirable progeny.
4. Evaluate desirable lines for other
Wild Clove Pink in flower, Turkey
agronomic traits.
5. Repeat crossing and selection until
quality reaches variety level.
Classical
Plant Biotechnology
Plant Breeding
Genetic modification
Genetic modification offollowing
a plant introduction
cell by of
large amounts
introduction of undefined
of defined DNA
DNA(s) from
from a a
genetically different
genetically similar source.
source.
Goals of Plant Biotechnology
Disease control
Insect control
Processing improvements Weed control
Protein
Quality improvements
Climatic tolerance
Removal Addition
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
History of genetic engineering
and food
• 1983 First transformed plants
• 1990 Recombinant chymosing replaces rennet in cheese
production
• 1994 Flavrsavr tomato approved for US grocery stores
• 1995 Genetically modified soybeans introduced on the
market
• 1997 18 GM crops approved in US
• 1999 Golden Rice developed
Plant Biotech
Products
• Flavr savr tomato
• Gene knockout
(antisense expression)
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
• Bt Corn
• Gene
overexpression
(constitutive)
Bt corn
The European corn borer, Ostrinia nubilalis, destroys corn crops by burrowing into the stem, causing the plant to fall over.
Development of insect resistance in crops such as maize by
incorporating a gene from Bacillus thuringiensis.
Plant Biotech
Products
• Roundup Ready
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
A.
(Error Bars Indicate Standard Deviation)
14
12
TSC (m g/g dry w eight)
10 Root
8 Leaf
Stem
6
0
Control pVINV
Genotype
Plant Biotech
Products
Recombinant miraculin -
tomatos
leaves (102.5) &
Fruits(90.7) μg/g fresh weight
Plant Biotech
Products
Medical hypothesis, 2006
Tomatoes comes in many varieties, colors and shapes
Transgenic tomatoes - expressing different malarial antigens
Current Opinion in Plant Biology 2007, 10:283–28
Normal and mutant tomato fruit
high-pigment 1 (hp1/hp1), high-pigment 2 (hp2/hp2), Never-ripe (Nr/Nr),
Green-ripe (Gr/Gr), Colorless non-ripening (Cnr/Cnr) &
ripening-inhibitor (rin/rin) mutations
Plant Biotech
Products
Delivery of a corn-based
edible vaccine
Transgenic corn kernels (a)
Corn snack (b) or
Embryo or germ cells (c)
Plant Biotech
Products Tearless Onion
http://www.sciencedaily.com/images/2008/02/080202115345.jpg
Dr Eady
Crop & Food Research in New
Zealand and his collaborators in
Japan
Plant Biotech
Products
http://www.dailymail.co.uk/news/article-514799/The-orange-purple-green-
cauliflowers-scientists-claim-healthier-you.html
Plant Biotech
Purple tomatoes high in
Products anthocyanins
http://news.bbc.co.uk/2/hi/health/7688310.
stm
High anthocyanin purple tomato
and red wild-type tomato
Plant Biotech
ProductsProf Cathie Martin from the John Innes Centre
Snapdragon is an annual flower planted for winter colors. Children love this winter flower.
Anthocyanins offer protection against certain cancers, cardiovascular
disease and age-related degenerative diseases. Anthocyanins also
have anti-inflammatory activity, promote visual acuity and hinder obesity
and diabetes.
Tomatoes already contain high levels of the antioxidant lycopene. Highly
processed tomatoes are the best source, or tomatoes cooked in a little
oil, which helps to release the lycopene from cells.
Flavonoids meanwhile are soluble in water, and foods containing both
water soluble and fat-dissolved antioxidants are considered to offer the
best protection against disease.
In this study the scientists expressed two genes from snapdragon that
induce the production of anthocyanins in snapdragon flowers. The
genes were turned on in tomato fruit.
Anthocyanins accumulated in tomatoes at higher levels than anything
previously reported for metabolic engineering in both the peel and flesh
of the fruit. The fruit are an intense purple colour.
http://www.seedquest.com/News/releases/2008/october/24091.htm
The Rose
Creation of blue rose was
achieved introducing blue
color-related enzyme gene
from pansy.
Plant-made pharmaceuticals
aka
Molecular pharming
Duckweed
Grow in lab
Genetic Protein
Engineering Purification
Or field
corn
Oral vaccine– eat the fruit
Corn
BioPharm
Pharmaceuticals made in plants
• Protein Based Drugs
– Expensive: high processing cost
– Hard to make in large quantities
• Produced in seeds, leaves and tubers
• Benefits
– Low capital, scaleable production
– Small acreage, high profit for growers
• Risks
– Pollen, Co-Mingling, Exposure, Environment
http://www.colostate.edu/programs/lifesciences/TransgenicCrops/index.html
BioPharm
Product Definition Examples
Antibodies Proteins for immune defense Specific antibodies developed to
responses fight cancer, treat inflammation,
and fight viral and bacterial
dieseses.
Antigens Stimulate production of Vaccines for protection against
(vaccines) antibodies that protect against cholera, diarrhea (Norwalk virus),
disease and hepatitis B
Enzymes Proteins that catalyze Enzymes used to treat and to
biochemical reactions diagnose disease.
Hormones Chemical messengers Insulin for diabetics
Structural Proteins for structural support Collagen is a structural protein
proteins to cells or tissues found in animal connective
tissues and used in cosmetics
Anti-disease Variety of proteins The anti-infection agents
agents interferon and lactoferrin, and
aprotinin have been engineered
in plants
http://www.colostate.edu/programs/lifesciences/TransgenicCrops/index.html
Phytoremediation example
How to remediate mercury in soil
www.uga.genetics.edu/rmblab
Phytosensor example:
plants to detect landmines
No TNT induction +TNT
Using inducible
promoter/GFP
fusions
Facts on GMOs
www.ers.usda.gov/D
ata/BiotechCrops/
Facts on GMOs
Global area of transgenic crops
Source: Gerhard
Wenzel, 2006
Facts on GMOs
Source: Gerhard
Wenzel, 2006
Facts on GMOs
Time scale of
genetically modified
characters in crops
Source: Gerhard
Wenzel, 2006
Constraints of GE
• Isolation of the gene of interest and the understanding
of the biochemical pathways and knowledge in the field
of metabolomics
• Reluctance towards gene technology
• The presence of marker genes may complicate future
commercialization (antibiotic resistance markers)
•From model plants (e.g. A. tumefaciens) to crops.
•Growth in the open environment is legally controlled
and substantially restricted (gene flow).
•Cultural problems (e.g. Golden Rice)
GM debate
Environment
• Loss of biodiversity
• Cross-pollination
• Emergence of superweeds and superbugs
• Potential increase in use of herbicides
Human health
• Fear of unknown allergens
Socio-economic concerns
• Centralised control of food production
Ethical objections to genetically modified foods
Gary Comstock
GM debate
Two Types of Regulations
1. Regulations to protect the public from the risks
of biotechnology
2. Regulations to protect biotechnology from the
fears of the public
BT crops - Benefits
• Reduced application of chemical pesticides.
• Potentially dangerous pesticides may be
eliminated.
• Efficacy of the treatment is independent of the
weather.
• These controls are in place throughout the
growing season.
• Treatment can be designed to be truly systemic,
reaching all parts of the plant that pesticides
may not penetrate.
BT crops - Benefits
• The treatment can be designed to be in place when the
insect is at its most sensitive stage of its life cycle, when
it is easier to control.
• The only insects that are affected are those that actually
feed on the plant.
• The active agent is usually a biodegradable protein, so
there is little potential for ground water contamination or
other environmental problems.
• There is widespread consumer acceptance of this
technology, compared to the use of pesticides.
• There are no human toxicology problems with Bt toxin
proteins.
BT crops- What are current
debates?
What are current debates?
– Anti Bt crop
• Is Bt crop really safe to eat?
– Nobody knows, that‘s why we don‘t eat
• Uninteded gene transfer
– Transfer of antibiotic-resistance genes
– Transfer of allergen and toxin
• Affect gene flow
– Exchange of genes between different (usually related)
populations
– Usually transferred by pollens
• Bt crop may kill non-target animals
– Monarch butterfly
Effect of Bt-transgenic
corn on Monarch
butterfly larvae feeding
on milkweed?
X
What are current debates
Bt crop supporters
• Is Bt crop really safe to eat?
– Millions of peoples eat GMO (corn soybean, oilseed rape) but
no adverse effects have been observed
– Lack of evidences
• Conventional plant breeding cause same problems
– Some potato cultivars contain excessive levels on naturally
occuring toxin
• Crops made by conventional breeding have not been
subjected to regulatory controls
– Transfer of genes between wild and domesticated plants are
similar to GMOs
• Safer than pesticides
– Many dangerous pesticides are registered
Genetically Modified Organisms
(GMO’s)
• All creatures are genetically modified
• Genetic modification is natural
– Viruses
– Bacteria
– Radiation
Genetically modified foods
60-plus plant species
▪Tomatoes ▪Canola
▪Potatoes ▪Soybeans
▪Corn ▪Sugarbeets
▪Rice ▪Sugarcane
Most major crops have been genetically modified,
…and the list is growing.
How do you make
transgenic plants?
• Plants can be regenerated from a single cell
• Add the new gene
– Biolistics: Fire DNA gold particle into the cell
– Vector transport: Agrobacterium
• Bacteria “drops” the DNA into the cell
• Select a cell with the gene
– Marker genes for selection
• Protects cells that have it.
How Plants are Transformed?
– Functional genomics
– Recombinant DNA techniques
– Plant transformation
– Tissue culture
– Screening and isolation of transformants
– Field and laboratory testing
Functional Genomics
Functional genomics can be roughly defined as using
the emerging knowledge about genomes to understand
the gene and their product functions and interactions,
and most importantly of all, how all this makes
organisms to function the way they do.
Plant Genome Projects
• Initiative began in 1998.
• Arabidopsis was the first model plant.
– Small genome size.
– Little repetitive DNA.
– Short life cycle.
The Plant Researchers’ pet:
Arabidopsis thaliana (thale cress)
• A model plant
• Best-studied plant genome
• Entire genome sequenced (2000)
• Genome size: ~100,000 kb (~ 20,000 genes)
• 5 Chromosomes
• Rapid life cycle (about 6-8 weeks from germination
to mature seed)
• Prolific seed production and easy cultivation
• Easy to generate transgenic plants (A. tumefaciens)
• Large number of mutant lines and genomic
resources
• Multinational research community of academic,
government and industry laboratories
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
DNA Micro-Array Technology
• Complete organism
genome on microarray
• 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 Antibiotic
Antibiotic Resistance
• Modular structure Resistance Gene
• Parts can be cut Gene pVINV
and pasted with Antibiotic
great precision Resistance
Gene
• Composed of DNA
from many sources
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 *
Plant
Transformation
Agrobacterium Particle gun
Tissue
Culture
• The big picture
• Regeneration
from single cells
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?
