Transcript No Slide Title

TRANSGENIC TECHNOLOGY
Traits that plant breeders
would like in plants





High primary
productivity
High crop yield
High nutritional
quality
Adaptation to intercropping
Nitrogen Fixation





Drought resistance
Pest resistance
Adaptation to
mechanised farming
Insensitivity to
photo-period
Elimination of toxic
compounds
Plant transformation
getting DNA into a cell
getting it stably integrated
getting a plant back from the cell
Requirement
1. a suitable transformation method
2. a means of screening for transformants
3. an efficient regeneration system
4. genes/constructs

Vectors

Promoter/terminator

reporter genes

selectable marker genes

‘genes of interest’
Transformation methods
DNA must be introduced into plant cells
Indirect
- Agrobacterium tumefaciens
Direct
- microprojectile bombardment
- electroporation
- Polyethylene glycol (PEG)-mediated
- glass-beads
- silicon carbide whiskers
Method depends on plant type, cost, application
Agrobacterium-mediated
transformation


A natural genetic
engineer
2 species
• A.tumefaciens
(produces a gall)
• A. rhizogenes
(produces roots)

Oncogenes (for
auxin and cytokinin
synthesis) + Opines

In the presence of
exudates (e.g.
acetosyringone) from
wounded plants,
Virulence (Vir) genes
are activated and cause
the t-DNA to be
transferred to plants.
Everything between the
left and right border is
transferred.
BACTERIAL GALL DISEASES

Galls:
overgrowth or proliferation of tissue, primarily due
to increased cell division (hyperplasia) and
increased cell size (hypertrophy).

Bacterial Galls:
induced by bacteria in 3 different genera.
• Agrobacterium
• Pseudomonas
• Clavibacter

Genes for plant hormone production found on
bacterial plasmids!
Crown Gall Disease:
Agrobacterium tumefaciens



Gram Dicots
Worldwide
Disease Cycle
Agrobacterium tumefaciens

Characteristics
• Plant parasite that causes Crown Gall Disease
• Encodes a large (~250kbp) plasmid called
Tumor-inducing (Ti) plasmid

Portion of the Ti plasmid is transferred between
bacterial cells and plant cells  T-DNA (Tumor
DNA)
Agrobacterium tumefaciens
T-DNA integrates stably into plant genome
Single stranded T-DNA fragment is
converted to dsDNA fragment by plant cell
Then integrated into plant genome
 2 x 23bp direct repeats play an important role in
the excision and integration process

Agrobacterium tumefaciens



Tumor formation = hyperplasia
Hormone imbalance
Caused by A. tumefaciens
• Lives in intercellular spaces of the plant
• Plasmid contains genes responsible for the
disease
 Part of plasmid is inserted into plant DNA
 Wound = entry point  10-14 days later,
tumor forms
Agrobacterium tumefaciens

What is naturally encoded in T-DNA?
• Enzymes for auxin and cytokinin synthesis


Causing hormone imbalance  tumor
formation/undifferentiated callus
Mutants in enzymes have been characterized
• Opine synthesis genes (e.g. octopine or nopaline)


Carbon and nitrogen source for A. tumefaciens growth
Insertion genes
• Virulence (vir) genes
• Allow excision and integration into plant genome
Ti plasmid of A. tumefaciens
1. Auxin, cytokinin, opine
synthetic genes
transferred to plant
2. Plant makes all 3
compounds
3. Auxins and cytokines
cause gall formation
4. Opines provide unique
carbon/nitrogen
source only A.
tumefaciens can use!
Agrobacterium tumefaciens

How is T-DNA modified to allow genes of
interest to be inserted?
• In vitro modification of Ti plasmid








T-DNA tumor causing genes are deleted and replaced with
desirable genes (under proper regulatory control)
Insertion genes are retained (vir genes)
Selectable marker gene added to track plant cells
successfully rendered transgenic [antibiotic resistance
gene  geneticin (G418) or hygromycin]
Ti plasmid is reintroduced into A. tumefaciens
A. tumefaciens is co-cultured with plant leaf disks under
hormone conditions favoring callus development
(undifferentiated)
Antibacterial agents (e.g. chloramphenicol) added to kill A.
tumefaciens
G418 or hygromycin added to kill non-transgenic plant cells
Surviving cells = transgenic plant cells
Agrobacterium and genetic engineering:
Engineering the Ti plasmid
Co-integrative and binary vectors
LB
RB
Co-integrative
Binary vector
Agrobacterium-mediated transformation
Agrobacterium tumefaciens
cause ‘Crown gall’ disease
Agrobacterium is a ‘natural genetic engineer’
i.e. it transfers some of its DNA to plants
Expose wounded plant cells to transformed
agro strain
Electroporate TDNA vector into
Agrobacterium
and select for tetr
Induce plant regeneration and select
for Kanr cell growth
Microprojectile bombardment
• uses a ‘gene gun’
• DNA is coated onto
gold (or tungsten)
particles (inert)
• gold is propelled by
helium
into plant
cells
• if DNA goes into the
nucleus
it can be
integrated into the
plant chromosomes
• cells can be
regenerated
to
whole plants


In the "biolistic" (a cross between biology and ballistics )or
"gene gun" method, microscopic gold beads are coated with
the gene of interest and shot into the plant cell with a
pulse of helium.
Once inside the cell, the gene comes off the bead and
integrates into the cell's genome.

Model from BioRad:
Biorad's Helios Gene
Gun
How do we get plants back from cells?
We use tissue culture techniques to
regenerate whole plants from single cells
getting a plant back from a single cell is
important so that every cell has the new DNA
Regeneration
Plant tissue culture uses growth regulators and
nutrients to regenerate plants in vitro
Regeneration of shoots from leaf protoplasts in
Arabidopsis thaliana
Somatic embryogenesis in peanut
Screening Technique
Not all cells take up DNA & not all cells can
regenerate so
Need an efficient regeneration system and
transformation system i.e. lots of cells
take up DNA and lots of cells regenerate
into a plant
to maximize chance of both happening
regenerable cells
Transformed cells
Cells containing new DNA that are able
to regenerate into a new plant
There are many thousands of cells in a leaf disc or
callus clump - only a proportion of these will have
taken up the DNA
therefore can get hundreds of plants back - maybe
only 1% will be transformed
How do we know which plants have taken up the
DNA?
Could test each plant - slow, costly
Or use reporter genes & selectable marker genes
Selection


Transformation frequency is low (Max 3% of all cells)
and unless there is a selective advantage for
transformed cells, these will be overgrown by nontransformed.
Usual to use a positive selective agent like antibiotic
resistance. The NptII gene encoding Neomycin
phospho-transferase II phosphorylates kanamycin
group antibiotics and is commonly used.
Reporter genes
most common
- easy to visualise or assay
- ß-glucuronidase (GUS) (E.coli)
- green fluorescent protein (GFP)
(jellyfish)
- luciferase (firefly, bacterial,
jellyfish etc)
GUS
Cells that are transformed with GUS will form a
blue precipitate when tissue is soaked in the
GUS substrate and incubated at 37oC
this is a destructive assay (cells die)
The UidA gene encoding activity is commonly used.
Gives a blue colour from a colourless substrate (X-glu)
for a qualitative assay. Also causes fluorescence from
Methyl Umbelliferyl Glucuronide (MUG) for a
quantitative assay.
GUS
Bombardment of GUS gene
- transient expression
Stable expression of
GUS in moss
Phloem-limited expression of GUS
HAESA gene encodes a receptor protein kinase that
controls floral organ abscission. (A) transgenic plant
expressing a HAESA::GUS fusion. It is expressed in
the floral abscission zone at the base of an Arabidopsis
flower.
Transgenic plants that harbor the
AGL12::GUS fusions show rootspecific expression.
GFP (Green Fluorescent Protein)
 Fluoresces green under UV illumination
 Problems with a cryptic intron now resolved.
 Has been used for selection on its own.
GFP glows bright green when irradiated by
blue or UV light
This is a nondestructive assay so the same
cells can be monitored all the way through
GFP
protoplast
colony derived
from protoplast
mass of callus
regenerated plant
Selectable marker genes - let you kill cells that
haven’t taken up DNA- usually genes that confer
resistance to a phytotoxic substance
Most common:
antibiotic resistance - e.g. kanamycin, hygromycin
[ only phytotoxic antibiotics can be used]
herbicide resistance - e.g. phosphinothricin (PPT);
glyphosate
Only those cells that have
taken up the DNA can
grow on media containing
the selection agent
APPLICATIONS
transfer of exogenous
genes
Pathogen resistance
Herbicide resistance
Bioreactors/molecular
farming
Delivery systems
Plant improvement
manipulation of
endogenous genes
Gene silencing/
downregulation
Gene silencing/ downregulation of endogenous
genes
Antisense RNA – delayed ripening; FlavR SavR
tomatoes
- modified flower colour (paler
flowers)
Post-transcriptional gene silencing
induces cytoplasmic RNA degradation system
induced by dsRNA
highly sequence specific
Applications of Plant Biotechnology
A.
B.
Crop Improvement
1. The following traits are potentially useful to plant genetic
engineering: controlling insects, manipulating petal color,
production of industrially important compounds, and plant
growth in harsh conditions.
Genetically Engineered Traits: The Big Six.
1. Herbicide Resistance
a)
Herbicides are a huge industry, with herbicide use
quadrupling between 1966 and 1991, so plants that resist
chemicals that kill them are a growing need.
b)
Critics claim that genetically engineered plants will lead
to more chemical use, and possible development of weeds
resistant to the chemicals.
Applications of Plant Biotechnology
c)
d)
Glyphosate Resistance
i. Marketed under the name Roundup, glyphosate inhibits the
enzyme EPSPS, makes aromatic amino acids.
ii.The gene encoding EPSPS has been transferred from
glyphosate-resistant E. coli into plants, allowing plants to be
resistant.
Glufosinate Resistance
i. Glufosinate (the active ingredient being phosphinothricin)
mimics the structure of the amino acid glutamine, which blocks
the enzyme glutamate synthase.
ii.Plants receive a gene from the bacterium Streptomyces that
produce a protein that inactivates the herbicide.
Applications of Plant Biotechnology
e)
f)
Bromoxynil Resistance
i. A gene encoding the enzyme bromoxynil nitrilase (BXN) is
transferred from Klebsiella pneumoniae bacteria to plants.
ii.Nitrilase inactivates the Bromoxynil before it kills the plant.
Sulfonylurea.
i. Kills plants by blocking an enzyme needed for synthesis of
the amino acids valine, leucine, and isoleucine.
ii.Resistance generated by mutating a gene in tobacco plants,
and transferring the mutated gene into crop plants.
Applications of Plant Biotechnology
2.
Insect Resistance
a)
The Bt toxin isolated from Bacillus thuringiensis has been
used in plants. The gene has been placed in corn, cotton,
and potato, and has been marketed.
b)
Plant protease inhibitors have been explored since the
1990s:
i. Naturally produced by plants, are produced in response
to wounding.
ii. They inhibit insect digestive enzymes after insects
ingest them, causing starvation.
iii. Tobacco, potato, and peas have been engineered to
resist insects such as weevils that damage crops while
they are in storage
iv. Results have not been as promising as with Bt toxin,
because it is believed that insects evolved resistance
to protease inhibitors.
Applications of Plant Biotechnology
3.
Virus Resistance
a)
Chemicals are used to control the insect vectors of
viruses, but controlling the disease itself is difficult
because the disease spreads quickly.
b)
Plants may be engineered with genes for resistance to
viruses, bacteria, and fungi.
c)
Virus-resistant plants have a viral protein coat gene that
is overproduced, preventing the virus from reproducing in
the host cell, because the plant shuts off the virus’
protein coat gene in response to the overproduction.
d)
Coat protein genes are involved in resistance to diseases
such as cucumber mosaic virus, tobacco rattle virus, and
potato virus X.
Applications of Plant Biotechnology
e)
f)
g)
h)
Resistance genes for diseases such as fungal rust disease and
tobacco mosaic virus have been isolated from plants and may be
transferred to crop plants.
Yellow Squash and Zucchini
i. Seeds are available that are resistant to watermelon mottle
virus, zucchini yellow mosaic virus, and cucumber mosaic virus.
Potato.
a)Monsanto developed potatoes resistant to potato leaf roll
virus and potato virus X, which also contained a Bt toxin gene
as a pesticide.
b)hain restaurants do not use genetically engineered potatoes
due to public pressures.
Papaya.
a)Varieties resistant to papaya ring spot virus have been
developed.
Applications of Plant Biotechnology
4.Altered Oil Content
a) Done in plants by modifying an enzyme in the fatty acid
synthesis pathway (oils are lipids, which fatty acids are a part
of).
b) Varieties of canola and soybean plants have been genetically
engineered to produce oils with better cooking and nutritional
properties.
c) Genetically engineered plants may also be able to produce oils
that are used in detergents, soaps, cosmetics, lubricants, and
paints.
5.Delayed Fruit Ripening
a) Allow for crops, such as tomatoes, to have a higher shelf life.
b) Tomatoes generally ripen and become soft during shipment to a
store.
c) Tomatoes are usually picked and sprayed with the plant
hormone ethylene to induce ripening, although this does not
improve taste.
Applications of Plant Biotechnology
d)
e)
Tomatoes have been engineered to produce less ethylene so they
can develop more taste before ripening, and shipment to markets.
What happened to the Flavr Savr tomato?
i. Produced by Calgene by blocking the polygalacturonase (PG) gene,
which is involved in spoilage. PG is an enzyme that breaks down
pectin, which is found in plant cell walls.
ii.Plants were transformed with the anti-sense PG gene, which is
mRNA that base pair with mRNA that the plant produces,
essentially blocking the gene from translation.
iii.First genetically modified organism to be approved by the FDA,
in 1994.
iv.Tomatoes were delicate, did not grow well in Florida, and cost
much more than regular tomatoes.
v.Calgene was sold to Monsanto after Monsanto filed a patentinfringement lawsuit against Calgene, and the Flavr Savr tomato
left the market.
The Flavr SavrTM Tomato
(First transgenic Plant Product)
DNA
Summary of Antisense
mechanism:
How enzyme is made?
PRODUCED
What Happens When A Cloned Antisense DNA Is
Added To The Original DNA?
When A Cloned
Antisense DNA Is Added
To The Original DNA:
Applications of Plant Biotechnology
6.
Pollen Control
a)
Hybrid crops are created by crossing two distantly
related varieties of the same crop plant.
b)
The method may generate plants with favorable traits,
such as tall soybean plants that make more seeds and are
resistant to environmental pressures.
c)
For success, plant pollination must be controlled. This is
usually done by removing the male flower parts by hand
before pollen is released. Also, sterilized plants have
been genetically engineered with a gene from the
bacteria Bacillus amyloliqueifaciens.
Applications of Plant Biotechnology
C.
Biotech Revolution: Cold and Drought Tolerance and
WeatherGard Genes.
1. Plants such fruits are subject to frost damage at low
temperatures, as well as from loss of water. They can be
genetically engineered to resist these conditions, and
increase crop yields as a result.
2. To resist cold weather, cold-regulated (COR) genes are also
called “antifreeze genes,”, which encode proteins that
protect plant cells from frost damage.
3. A transcription factor for a group of COR genes called “CBF”
was patented as WeatherGard in 1997 by a group at Michigan
State University. The genes also provide drought tolerance
and tolerance to high-salt soils.
4. All major crop species, including corn, soybean, and rice
contain CBF genes.
5. Genetically engineering plants with CBF genes survive
temperatures as much as 4 to 50C lower than non-engineered
plants.
Applications of Plant Biotechnology
D.
Genetically Engineered Foods.
1. More than 60% of processed foods in the United States
contain ingredients from genetically engineered organisms.
2. 12 different genetically engineered plants have been
approved in the United States, with many variations of each
plant, some approved and some not.
3. Soybeans.
a)
Soybean has been modified to be resistant to broadspectrum herbicides.
b)
Scientists in 2003 removed an antigen from soybean
called P34 that can cause a severe allergic response.
4. Corn
a)
Bt insect resistance is the most common use of engineered
corn, but herbicide resistance is also a desired trait.
Applications of Plant Biotechnology
Products include corn oil, corn syrup, corn flour, baking
powder, and alcohol.
c)
By 2002 about 32% of field corn in the United States was
engineered.
5. Canola.
a)
More than 60% of the crop in 2002 was genetically
engineered; it is found in many processed foods, and is
also a common cooking oil.
6. Cotton.
a)
More than 71% of the cotton crop in 2002 was engineered.
b)
Engineered cottonseed oil is found in pastries, snack
foods, fried foods, and peanut butter.
7. Other Crops
a)
Other engineered plants include papaya, rice, tomato,
sugar beet, and red heart chicory.
b)
Applications of Plant Biotechnology
E.
Nutritionally Enhanced Plants—Golden Rice: An International
Effort.
1. More than one third of the world’s population relies on rice as
a food staple, so rice is an attractive target for
enhancement.
2. Golden Rice was genetically engineered to produce high levels
of beta-carotene, which is a precursor to vitamin A. Vitamin
A is needed for proper eyesight.
3. Golden Rice was developed by Ingo Potrykus and Peter Beyer,
and several agencies are attempting to distribute the rice
worldwide.
4. Biotechnology company Syngenta, who owns the rights to
Golden Rice, is exploring commercial opportunities in the
United States and Japan. Monsanto will provide licenses to
Golden Rice technology royalty-free.
5. Other enhanced crops include iron-enriched rice and
tomatoes with three times the normal amount of betacarotene
Applications of Plant Biotechnology
6.
Cause for Concern? The Case of StarLink Corn.
a)
StarLink corn had been approved for animal consumption, but in
2000 ended up in Taco Bell taco shells. The shells were
immediately recalled.
b)
Aventis CropScience believed that precautions regarding the
corn were in place, but some farmers did not know the corn was
not for humans.
c)
Engineered and non-engineered corn was mixed in mills,
contaminating food.
d)
StarLink contained two new genes:
i. Resistance to butterfly and moth caterpillars by a modified
Bt toxin gene called Cry9c.
ii. Resistance to herbicides such as Basta and Liberty.
e)
StarLink was approved for animals because the Cry9c protein
could be an allergen in humans because it was more stable to
heat and in the stomach.
Applications of Plant Biotechnology
Currently, no cases of allergic reactions have been
reported, and the EPA ruled in 2001 that StarLink was
not safe for humans.
7. Cause for Concern? Genetically Engineered Foods and Public
Concerns.
a)
The release of the Flavr Savr tomato generated much
discussion over the potential risks of genetically
engineered food:
i. The primary public fear was that genetically
engineering a plant may produce unexpected results,
such as allergic reactions or even shock.
ii. Genetically engineered food may also raise concerns
about the selection of food if, for example, an apple
has a gene from an animal.
iii. The use of antibiotic resistance markers may possibly
inactivate antibiotics, leading to scientists trying to
find ways to remove markers from plants.
f)
Applications of Plant Biotechnology
iv. Another concern is that deleting genes may bring about
side effects when ingested, such as secondary metabolites
that may protect people from compounds that would
normally be broken down by the plant.
v. Uncharacterized DNA included along with the gene of
interest may produce unexpected, harmful side effects in
the plant.
vi. Crops may spread the trait to other plants through
pollination, which may damage ecosystems. Male-sterile
plants may deal with this problem.
Applications of Plant Biotechnology
F.
Molecular Farming
1. A new field where plants and animals are
genetically engineered to produce important
pharmaceuticals, vaccines, and other valuable
compounds.
2. Plants may possibly be used as bioreactors to
mass-produce chemicals that can accumulate
within the cells until they are harvested.
3. Soybeans have been used to produce monoclonal
antibodies with therapeutic value for the
treatment of colon cancer. Clot-busting drugs
can also be produced in rice, corn, and tobacco
plants.
Applications of Plant Biotechnology
4. Plants have been engineered to produce human
antibodies against HIV and Epicyte
Pharmaceuticals has begun clinical trials with
herpes antibodies produced in plants.
5. The reasons that using plants may be more costeffective than bacteria:
a)
Scale-up involves just planting seeds.
b)
Proteins are produced in high quantity.
c)
Foreign proteins will be biologically active.
d)
Foreign proteins stored in seeds are very
stable.
e)
Contaminating pathogens are not likely to be
present.
Applications of Plant Biotechnology
6.
Edible Vaccines
a)
People in developing countries have limited access to
many vaccines.
b)
Making plants that produce vaccines may be useful for
places where refrigeration is limited.
c)
Potatoes have been studied using a portion of the E. coli
enterotoxin in mice and humans.
d)
Other candidates for edible vaccines include banana and
tomato, and alfalfa, corn, and wheat are possible
candidates for use in livestock.
e)
Edible vaccines may lead to the eradication of diseases
such as hepatitis B and polio.
Applications of Plant Biotechnology
7.
Biopolymers and Plants
a)
Plant seeds may be a potential source for plastics that
could be produced and easily extracted.
b)
A type of PHA (polyhydroxylalkanoate) polymer called
“poly (beta-hydroxybutyrate”), or PHB, is produced in
Arabidopsis, or mustard plant.
c)
PHB can be made in canola seeds by the transfer of
three genes from the bacterium Alicaligenes eutrophus,
which codes for enzymes in the PHB synthesis pathway.
d)
Monsanto produces a polymer called PHBV through
Alicaligenes fermentation, which is sold under the name
Biopol.