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Applications of
Transgenic technology
Transgenic technology
Breeding method
Crop Improvement
The big six traits
 Herbicide Resistance
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Insect Resistance
 Virus Resistance
 Altered Oil Content
 Delayed Fruit Ripening
 Pollen Control
Herbicide Resistance
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.
Critics claim that genetically engineered plants will lead to more
chemical use, and possible development of weeds resistant to the
chemicals.
Herbicide Resistance
a)
Glyphosate Resistance
i. Glyphosate = “Roundup”, “Tumbleweed” = Systemic herbicide
ii. Marketed under the name Roundup, glyphosate inhibits the enzyme EPSPS
(S-enolpyruvlshikimate-3 phosphate – involved in chloroplast amino acid
synthesis), makes aromatic amino acids.
iii. 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.
Herbicide Resistance
c) 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.
d) 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.
Roundup Ready™ Soybeans
A problem in agriculture is the reduced growth of crops imposed by the
presence of unwanted weeds. Herbicides such as RoundupTM and Liberty
LinkTM are able to kill a wide range of weeds and have the advantage of
breaking down easily. Development of herbicide resistant crops allows the
elimination of surrounding weeds without harm to the crops.
Insect resistance
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Anti-Insect Strategy - Insecticides
a) Toxic crystal protein from Bacillus thuringensis
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Toxic crystals found during sporulation
Alkaline protein degrades gut wall of lepidopteran larvae
• Corn borer catepillars
• Cotton bollworm catepillars
• Tobacco hornworm catepillars
• Gypsy moth larvae
Sprayed onto plants – but will wash off
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.
Insect resistance
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.
Bt Corn
Various insect resistant crops have been produced. Most of
these make use of the Cry gene in the bacteria Bacillus
thuringiensis (Bt); this gene directs the production of a protein
that causes paralysis and death to many insects.
Corn hybrid with a Bt gene
Corn hybrid susceptible to European
corn borer
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.
Virus resistance
e) 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.
f) Yellow Squash and Zucchini
Seeds are available that are resistant to watermelon mottle virus,
zucchini yellow mosaic virus, and cucumber mosaic virus.
g) 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.
h) Papaya
Varieties resistant to papaya ring spot virus have been developed.
Virus Resistant Crops
Papaya infected with the papaya
ringspot virus
Virus resistance gene
introduced
The Freedom II squash has a
modified coat protein that confer
resistance to zucchini yellows
mosaic virus and watermelon
mosaic virus II.
Scientists are now trying to develop
crops with as many as five virus
resistance genes
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.
Laurate canola oil
Canola plant modified with thioesterase gene obtained
from California bay laurel tree
Enzyme produces lauric acid (up to 40% in oil from
genetically modified (GM) canola seeds)
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Low saturated fat content
Heat tolerant
• Does not break down
• Excellent for high temperature cooking processes
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
d) Tomatoes have been engineered to produce less ethylene so
they can develop more taste before ripening, and shipment to
markets.
Delayed Fruit Ripening
d) What happened to the Flavr Savr tomato?
i.
ii.
iii.
iv.
v.
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.
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.
First genetically modified organism to be approved by the FDA, in 1994.
Tomatoes were delicate, did not grow well in Florida, and cost much more than
regular tomatoes.
Calgene was sold to Monsanto after Monsanto filed a patent-infringement lawsuit
against Calgene, and the Flavr Savr tomato left the market.
The Flavr Savr Tomato
(First transgenic Plant Product)
DNA
Summary of Antisense
mechanism:
How enzyme is
made?
PRODUCED
When A Cloned Antisense DNA
Is Added To The Original DNA:
First biotech plant product – Flav’r Sav’r tomato
“Rot-Resistant Tomato”
Anti-sense gene  complementary to polygalacturonase (PG)
PG = pectinase  accelerates plant decay/rotting
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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 (barnase
gene). This gene is dominant gene for male sterility
d) Genetic laser approach
Genetic laser approach
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Targetting the expression of a gene encoding a cytotoxin by placing it
under the control of an ather specific promoter (Promoter of TA29
gene)
Expression of gene encoding ribonuclease (chemical synthesized
RNAse-T1 from Aspergillus oryzae and natural gene barnase from
Bacillus amyloliquefaciens)
RNAse production leads to precocious degeneration of tapetum cells,
the arrest of microspore development and male sterility. It is a dominant
nuclear encoded or genetic male sterile (GMS), although the majority of
endogenous GMS is recessive
Success in oilseed rape, maize and several vegetative species
Used antisense or cosuppression of endogenous gene that are
essential for pollen formation or function
Reproducing a specific phenotype-premature callose wall dissolution
around the microsporogenous cells
Reproducing mitocondrial dysfunction, a general phenotype observed
in many CMS
Fertility restoration
 Restorer gene (RF) must be devised that can suppress the
action of the male sterility gene (Barstar)
1. a specific inhibitor of barnase
2. derived from B. amyloliquefaciens
3. Served to protect the bacterium from its own RNAse activity by
forming a diffusion-dependent, extremely one to one complex which is
devoid of residual RNase activity
 The use of similar promoter to ensure that it would be
activated in tapetal cells at the same time and to maximize
the chance that barstar molecule would accumulate in
amounts at least equal to barnase
 Inhibiting the male sterility gene by antisense. But in the
cases where the male sterility gene is itself antisense,
designing a restorer counterpart is more problematic
Production of 100% male sterile population
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When using a dominant GMS gene, a means to produce 100%
male sterile population is required in order to produce a practical
pollination control system
Linkage to a selectable marker
Use of a dominant selectable marker gene (bar) that confers
tolerance to glufosinate herbicide
Treatment at an early stage with glufosinate during female parent
increase and hybrid seed production phases eliminates 50%
sensitive plants
Pollen lethality
add a second locus to female parent lines consisting of an RF gene
linked to a pollen lethality gene (expressing with a pollen specific
promoter)
Plant Biotechnology Revolution:
Genetically Engineered Foods.
 Foods that contain an added gene sequence
 Foods that have a deleted gene sequence
 Animal products from animals fed GM feed
 Products produced by GM organisms
Plant Biotechnology Revolution:
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 broad-spectrum
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.
Plant Biotechnology Revolution:
Genetically Engineered Foods
4. Corn
a) Bt insect resistance is the most common use of engineered corn, but herbicide resistance
is also a desired trait.
b) 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
Other engineered plants include papaya, rice, tomato, sugar beet, and red heart
chicory.
Plant Biotechnology Revolution:
Nutritionally Enhanced Plants
Golden Rice
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. 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.
4. Other enhanced crops include iron-enriched rice and tomatoes with
three times the normal amount of beta-carotene
Plant Biotechnology Revolution:
Nutritionally Enhanced Plants
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
f) Currently, no cases of allergic reactions have been reported, and the EPA ruled in
2001 that StarLink was not safe for humans.
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Plant Biotechnology Revolution:
Nutritionally Enhanced Plants
7.
Cause for Concern? Genetically Engineered Foods and Public
Concerns.
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.
Plant Biotechnology Revolution:
Nutritionally Enhanced Plants
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.
Golden Rice
Normal rice
Transgenic technology produced a type of rice that accumulates
beta-carotene in rice grains. Once inside the body, beta-carotene is
converted to vitamin A.
“Normal” rice
“Golden” rice
Plant Biotechnology Revolution:
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 massproduce 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. Drugs can also be produced in rice,
corn, and tobacco plants.
Plant Biotechnology Revolution:
Molecular Farming
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 cost-effective 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 (no
animal contaminations).
Plant Biotechnology Revolution:
Molecular Farming
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.
Pharmaceutical Production in Plants
Genetically modified plants have been used as “bioreactors” to produce
therapeutic proteins for more than a decade. A recent contribution by
transgenic plants is the generation of edible vaccines.
Edible vaccines are vaccines produced in plants that can be administered
directly through the ingestion of plant materials containing the vaccine. Eating
the plant would then confer immunity against diseases.
Edible vaccines produced by transgenic plants are
attractive for many reasons. The cost associated
with the production of the vaccine is low, especially
since the vaccine can be ingested directly, and
vaccine production can be rapidly up scaled should
the need arises. Edible vaccine is likely to reach
more individuals in developing countries.
The first human clinical trial took place in 1997.
Vaccine against the toxin from the bacteria E.coli
was produced in potato. Ingestion of this transgenic
potato resulted in satisfactory vaccinations and no
adverse effects.
Edible
Vaccines
One focus of current vaccine effort is on hepatitis B, a virus responsible for causing
chromic liver disease. Transgenic tobacco and potatoes were engineered to
express hepatitis B virus vaccine. During the past two years, vaccines against a
E.coli toxin, the respiratory syncytial virus, measles virus, and the Norwalk virus
have been successfully expressed in plants and delivered orally. These studies
have supported the potential of edible vaccines as preventive agents of many
diseases.
There is hope to produce edible vaccines in bananas, which are grown extensively
throughout the developing world.
Plant Biotechnology Revolution:
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-betahydroxybutyrate”, 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.
Areas of ongoing debate
Environment
 Human Health
 Food security
Socio-economic concerns
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Environment
Anti-GM
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Loss of biodiversity
Cross-pollination
Emergence of superweeds
and superbugs
Potential increase in use
of herbicides
*Opinions are generalized, and not all opponents or
proponents may hold all of these views.
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Pro-GM
Need to increase yields to
feed growing population
Possibility of reducing
need for pesticides,
fertilizers
Grow more food on same
amount of land
Human Health
Anti-GM
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Fear of unknown allergens
Spread of anti-biotic
resistance
Inadequate regulation of
new products
Pro-GM
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Greater regulations than
other foods
Potential benefits to
nutrition
• golden rice
• enhanced protein content
in corn
• soybean oil with less
saturated fat
Food Security
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Anti-GM
Need redistribution, not
just more
Farmers will not be able to
afford expensive seed
Developing countries
should not have to eat the
food others reject
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Pro-GM
Modified seeds will allow
farmers to grow more to
feed their family and to
sell, reducing the need for
food aid
Public-private cooperation
can transfer technology
Socio-economic concerns
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Corporations benefit, not
those in need
Products needed in
developing countries are
not being developed
because the market is not
profitable
It is wrong to patent life
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Patents needed because
new strains are intellectual
property
Publicly funded research
can benefit the public good