Download Goals of Genetic Enginnering - ASAB-NUST

Applications of plant Genetic
Engineering
KOMAL JAMIM SHEHZADI
Asim Farooq
Basma Sarfraz
Goals of Genetic Enginnering
 Improvement of agronomically important crops.
 Production of oils and lubricants.
 Modification of lipid composition of seed crops.
 Mnufacturing of industrially important chemicals.
 And pharmeceutically active compounds.
 Development of stress tolerant plants.
Genetically Engineered traits (Big Six)
Six genetically traits which are
Herbicide resistance
Insect resistance
Viruses resistance
Altered oil contents
Delayed fruit ripening
pollen control
Herbicides resistance
• Weeds reduces the yields.
• Compete for nutrients,sunlight,and water etc.
• Herbicides reduces the impact of weeds.
Mode of action of herbicides
Herbicides disrupts disrupt one or more metabolic
pathway or other vital processes to kill the weeds.
Disadvantage of using Herbicides
• Weeds still reduce crop productivity by approximately 12%.
• Some traditional crops are more sensitive to herbicides.
Solution
•
•
•
•
Modification of crops with foreign genes.
Reduction of number of application of herbicides.
Using only single chemical.
Using biodegradable or less toxic herbicides.
Critics
• If chemical approach to weed control continues there
will be
Incresed amount of chemicals in our environment.
Incresed dependency on toxic chemicals
Weeds may become resistance to herbicides.
Types of GE herbicides resistance
• There are two types
1)modification of target (protein) so that it no
longer binds to herbicides.
2) production of new protein that inactivates
the herbicides.
Herbicides and resistance modified
crops
• Crops plants have been engineered to be resistance to four
herbicides.
Herbicide: Resistance modified crops
Glyphosate: soyabeans,corn,canola,cotton
and sugarbeets
Glufosinate: soyabeans,canola,cotton,corn
sugarbeets rice.
Bromoxynil: cotton
sulfonlurea: cotton and flax
Glyphosate resistance
• Most commonly used herbicide.Known as Roundup
Mode of Action
Inhibits EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) in
the shikimate pathways in both plants and bacteria.
Gene encoding EPSPS in a glyphosate resistance E.coli strain was
isolated.
Under control of plant promoter transferred into plant cells.
e.g soyabeans,corn,canola and cotton.
Glufosinate resistance
• Market name Basta,and Liberty.
Mode of Action
Active ingredient (phosphinothricin) in glufosinate mimics
the structure of glutamine.
This blocks the activity of glutamine synthase(required for
nitrogen metabolism).
Gene is isolated from the bacterium streptomyces,whose
protein products inactivates phosphinothricine in the
herbicide.
Bromoxynil resistance
• Nitrile herbicide marketed as Buctril,and bronate.
Mode of Action
Inactivates photosynthesis in plants.
Bromoxnil resistance plants are producedby
transferring the gene encoding the enzyme bromoxnil
nitrilase (BXN).
Gene is obtained from soil bacterium klebsiella
pneumoniae.
Mode of Action
Sulfonlurea
• Environmentally friendly herbicide.Discovered by Dupont
crop protection in 1975.
Mode of Action
Blocks an enzyme acetolactase required for the synthesis of
amino acid(valine,lucine,and isolucine).
Herbicide resistance is developed by modifying the enzyme by
gene mutation and then transferred into plants.
e.g cotton.
Non Resistant
Resistant
Insect Resistance
 Biopesticides
 Bt-based insect resistance; successfully engineered Bt gene into
different plant varieties
 Protease inhibitors
 PI’s are present in Storage Tissues contributing to the total of
10% of protein content (Huma and Khalid, DOB, UOK, Aug, 2007)
 First observed in larvae growing on soya bean products
 Transgenic plants produced; cowpea trypsin inhibitor gene,
potato protease inhibitor, α-amylase inhibitor
 One gene killing more insects in different plants
Continued….
 Resistance of insects against Bt toxin and PIs
 Two methods of PI resistance
High level expression of soybean-trypsin-inhibitor gene in
transgenic tobacco plants failed to confer resistance against
Helicoverpa armigera
Delaying insect resistance to transgenic crops
High dosage refuge strategy can be used to somehow delay the
process of insect resistance (Bruce E.T. Department of
Entomology, University Of Arizona)
Virus Resistance
 Every year, tons of plants and plant products are lost due to viral
infections
 Different chemicals supplied to kill viral vectors
 Viral DNA multiplies within the cell, thousands of copies are
produced, virus infects next cell(s)/Plant(s)
 Two genetic techniques are involved:
 Coat protein-mediated resistance
 RNA mediated virus resistance
Coat Protein-Mediated Resistance
 Plant is genetically engineered with a particular viral coat protein
gene
 Roger N. Beachy from Donald Danforth Plant Science centre, St.
Louis, USA carried out studies on TMV an reported that
transgenically expressed CP interferes with disassembly of TMV
particles in the inoculated transgenic cell. Using the known
coordinates of the three–dimensional structure of TMV, mutant
forms of CP were developed that had stronger inter–subunit
interactions, and confer increased levels of CP–MR compared with
wild–type CP
Continued…
 Numerous transgenic plant varieties have been produced for
resistance against Cucumber mosaic virus, alfalfa mosaic virus,
tobacco rattle virus etc.
 Resistance genes for a many other disease causing agents like
fungi and bacteria have also been produced
 Genes from tomato have been isolated against P. syringae and C.
fulvum. A flax gene develops resistance against fungal rust
disease
 Transgenic varieties of Yellow Squash and Papaya
RNA-Mediated Virus Resistance
 Directs the used of genetically altered RNA to stop viral
multiplication within the cell
 49 plants were generated that express the 5' untranslated region
of the tobacco etch potyvirus (TEV) genome ligated to a mutated
version of the TEV coat protein gene sequence that rendered it
untranslatable. Eight different transgenic plant lines were
analyzed for transgene expression and for resistance to TEV
(Dougherty et al, DOM, Oregon State University)
Altered Oil Content
 Various types of oils are produced in plants which differ due to
difference in the fatty acid content etc
 Plant oils are largely used commercially as well as domestically
 Genes with altered properties of improved oil traits are cloned
into soybean. These genes are selected through the particular
gene promoters.
 Reduced palmitic acid content, 80% oleic acid and 30% stearic
acid content
 Considerable change in fatty acid content
Delayed Fruit Ripening
 Basically a hormone regulated process
 Genetically engineered tomatoes were marketed, the only delayed
fruit of the times
 Technique not much developed
 The ACC synthase gene obtained from fruits was engineered in
antisense form in the papaya tissue through the particle gun. The
gene translated to protein which resulted in delayed fruit ripening
(Evelyn Mae, IPB, University of Philippines)
Flavr Savr Tomato
 First genetically modified food
 Polygalacturonase gene was blocked
 Resulted in delayed ripening of tomato
 Antisense gene technology was used
 FDA approved in May 1994
 Produced only for 1 year
Pollen Control
 Due to improved qualities, hybrid crops are used in agriculture
 Hybridization must be in a controlled manner
 Male flower part is removed before pollen release
 Production of male sterile plants by inserting a gene from Bacillus
amyloliquefaciens , producing barnase
 Barstar gene technique is also used
 Barstar pollen fertilizes male sterile barnase plant and a fertile hybrid
is produced
 Corn and chicory have been genetically engineered through this
technique
• The initial objective was to improve crop
protection.
1. through the introduction of resistance
against plant diseases.
2. through increased tolerance towards
herbicides.
• Few incidents:
• In 1989, GE supplement L-tryptophan… outbreak of
Eosinophilia Myalgia Syndrome (EMS)… 5000 people
suffered.
• In 1999, GA potatoes… poisonous to mammals… when
fed to rats… caused highly weight reduction of many
organs, impairment of immunological responsiveness
and signs suggestive of viral infection.
• Cornell University scientists reported recently that 44%
of monarch butterfly larvae died within
four days when fed milkweed that had
been dusted with pollen from GA corn.
• The safety assessment of GM foods generally
investigates:
(a) direct health effects (toxicity).
(b) tendencies to provoke allergic reaction (allergenicity).
(c) specific components thought to have nutritional or
toxic properties.
(d) the stability of the inserted gene.
(e) nutritional effects associated with genetic
modification.
(f) any unintended effects which could result
from the gene insertion.
• The first genetically modified soybeans were planted in
the US in 1996, that was Roundup Ready (RR) soybean,
was developed by the biotech giant Monsanto.
• In 2007, 216 million tones of soybeans were produced
worldwide. The world’s leading soybean producers are
the US(33%), Brazil (27%), Argentina (21%), and China
(7%).
• Each year, the EU imports approximately 40 million
tones of raw soy products, primarily destined for use as
cattle, swine, and chicken feed.
• In addition, soybeans are used to produce numerous
food ingredients , oils and additives. Lecithin, for
example, is used as an emulsifier in
chocolate, ice cream, margarine, and
baked goods.
• Even a small change in the amount of oil a
soybean produces could affect industry profits
marginally, the main aim was creating higher oil
content without disrupting protein content.
• Enhancement of soybean oil content was
achieved by the introduction of a seed-specific
transgenic for a DGAT2-type enzyme from the oilaccumulating fungus Umbelopsis ramanniana.
• In these studies, the oil content was increased
from approximately 20% of the seed weight to
approximately 21.5%.
• The high-oleic soybean, developed by DuPont
is one of the first in a wave of bioengineered
cash crops that are being altered for
nutritional purposes by removing primary
antigen( protein P34) from soybean.
• Currently, Monsanto has also introduced two
varieties of biotech soybeans.
1. By promising to eliminate trans fats.
2.And produce oil with omega-3 fatty acid for
use in yogurt, granola bars and spreads.
• For the first time since 1996, acres of Roundup
Ready genetically modified soybeans could
drop as more farmers decide to plant nonGMO.
• “Farmers upset with Monsanto”.
• Hamsters Experiment showed devastating
results- made upset.
• GM foods have the potential to solve many of
the world's hunger and malnutrition
problems.
• It helps protect and preserve the environment
by increasing yield and reducing reliance upon
chemical pesticides and herbicides.
• Yet there are many challenges ahead for
governments, especially in the areas of safety
testing, regulation, international policy and
food labeling.
• Genetically engineered corn was first sold in 1996,
and since then, scientists have introduced a variety
of new genes.
• In 2002, 32% of field in US was genetically
engineered planted within 12.4 million hectares.
• Most common is Bt insect resistant corn, e.g. from
European corn borer and corn root worm.
• Other include resistance to different herbicides.
Disease-resistant corn crops may have lower levels
of mycotoxins, potentially
carcinogenic compounds.
• The study found that three strains of molded
crops -- MON 810 and MON 863, which are
resistant to pests, and NK 603, which is
fortified to withstand weed killer -significantly disrupted the blood chemistry of
rats who ate them.
• A genetically engineered rapeseed that is tolerant
to herbicide was first introduced to Canada in
1995.
• In 2008 genetically modified (GM) herbicide
tolerant canola is being grown commercially in
Australia for the first time.
• Today 80% of the acres sown are genetically
modified canola.
• In 2003, Australia's gene technology regulator
approved the release of canola altered to make it
resistant to glufosinate ammonium, a herbicide.
• Rice that has been…
1. Genetically modified.
2. Contain beta carotene.
3. Produce vitamin a once consumed.
•
•
•
•
Produced by combining genetic material from
Daffodils
Peas
Japonica rice.
• Developed by two scientists in 1999.
• Dr. Ingo Potrykus of the Institute for Plant
Sciences at the Swiss Federal Institute of
technology in Zurich, Switzerland.
• Dr. Peter Beyer of the center for Applied
Bioscience at the University of Freiburg in
Germany.
• The addition of 2 genes in the rice genome will
complete the biosynthetic pathway:
1. Phytoene synthase (psy) – derived from daffodils.
2. Lycopene cyclase (crt1) – from soil bacteria
Erwinia uredovora.
• Produces enzymes and catalysts for the
biosynthesis of carotenoids (β-carotene) in the
endosperm.
• Presence of pro-vitamin A gives rice grains a
yellowish-orange color, thus,
the name ‘Golden Rice’.
:
: Fish oil, Liver Milk, Eggs Butter,
Orange/Yellow fruits and vegetables.
: Growth Healthy skin & cells, Good
night vision.
Blindness ,weakened immune
system, increased susceptibility to infection and
cancer, anemia, deterioration of the eye tissue,
cardiovascular disease and can increase the risk
of maternal mortality.
Between 100 and 140 million children are
vitamin A deficient each year. About 250,000 to
500,000 become blind, (1/2 dying within 12
months of losing sight), 600,000 women die from
childbirth-related causes each year.
• Vitamin A in “Golden” rice is not sufficient for
the daily recommended amount.
• Even if it was enough, people require zinc,
proteins, fats and other elements (which they
also often lack) in their diets to convert the
beta-carotene to vitamin A.
• White rice is associated with culture and
tradition ; golden rice may not be accepted .
• May solve VAD in third world countries.
• Can improve amount of Vitamin A.
• May influence the acceptance of GMOs if
successful.
• Golden Rice may not be the complete solution
to VAD, but it is a start and a contribution to
the problem.
• GMOs have actually reduced the use
of herbicides and pesticides.
• Funded by the Rockefeller Foundation, the
Swiss Federal Institute of Technology, and
Syngenta, a crop protection company.
• Golden Rice Humanitarian Board-responsible
for the global development, introduction and
free distribution of Golden Rice to target
countries.
• It is the use of genetically engineered crops to
produce compounds with therapeutic value ,
including antibodies, blood products, cytokines,
growth factors, hormones, recombinant enzymes
and human and veterinary vaccines.
• Several PDP products for the treatment of
human diseases are approaching
commercialization, including
1. Recombinant gastric lipase for the treatment
of cystic fibrosis.
2. Antibodies for the prevention of dental caries
etc.
• Potential products include the development of
antigens for vaccines that might be massproduced in plants such as corn and used to
fight such diseases as cancer and diabetes.
• Today in London, UK regulators announced the
approval of Europe’s first clinical trial of an antiHIV product produced in genetically modified
tobacco plants.
• Plant-produced antibody designed to stop
transmission of HIV when applied directly to the
vaginal cavity.
• The active ingredient is an antibody called P2G12
– it recognizes proteins on the surface of HIV to
block infection. More specifically, it’s a
monoclonal antibody made from immune cells
for one specific role.
1.
2.
3.
4.
5.
6.
7.
Commercial scale-up involves simply planting seed rather than using
costlier fermenters.
Plants do not carry pathogens that might be dangerous to human
health.
On the level of pharmacologically active proteins, there are no
proteins in plants that are similar to human proteins.
Like animals, plants are complex, multicellular organisms and
therefore their process of protein synthesis is more similar to that of
animals than those of bacteria or yeast.
Purification of the desired product from plants is often easier than
from bacteria, which can be labour and cost intensive.
Transgenic plants can be grown on an agricultural scale requiring only
water, minerals and sunlight.
Plants offer advantages over live animals and animal cell cultures in
terms of safety, cost, time involved, and storage and distribution
issues.
Expressions Cost of
System
maintaining
Protein
yield
Therapeutic
risk
Type of
storage
Production
cost
-2.0°C
Gene size
(protein)
Restriction
Unknown
Yeast
inexpensive
High
Unknown
Bacteria
inexpensive
Medium
yes
-2.0°C
Unknown
Medium
Plant
viruses
inexpensive
Very high
Unknown
-2.0°C
Limited
Low
Transgenic
Plants
inexpensive
High
Unknown
RT*
Not limited
Low
Animal Cell expensive
Cultures
Medium
to high
yes
N2**
Limited
High
Transgenic
Animals
High
yes
N/A
Limited
High
expensive
Medium
• Today Molecular Farming is considered "big
business". According to the Canadian Food
Inspection Agency, in a recent report, says
that U.S. demand alone for biotech
pharmaceuticals is expanding at 13 percent
annually and to reach a market value of $28.6
billion in 2004. Molecular Farming is expected
to be worth $100 billion globally by 2020.
A vaccine that is based on the genetically engineered
expression of an antigenic protein by an edible plant.
Following consumption, the protein is recognized by
the immune system.
• The first journal publication describing edible vaccine
was published in 1992.
• The studies was conducted by Mason, Lam and Arntzen
at Texas A&M University.
• A tobacco plant was successfully transformed and
Hepatitis B surface antigens was expressed.
• It hold great promise as a cost-effective, easy-toadminister, easy-to-store, fail-safe and socioculturally
readily acceptable vaccine delivery system, especially
for the poor developing countries.
• It involves introduction of selected desired genes into
plants and then inducing these altered plants to
manufacture the encoded proteins.
• Initially thought to be useful only for preventing
infectious diseases, it has also found application in
prevention of autoimmune diseases, birth control,
cancer therapy etc.
• Edible plant-derived vaccine may lead to a future of
safer and more effective immunization.
• Resistance to genetically modified foods may affect the
future of edible vaccines.
- Human Papillomavirus.
- Rinderpest Virus.
- Cysticercosis.
- Rotavirus.
- Helicobacter pylori.
- E .coli, HBV.
- Malaria virus.
• Humanitarian- save millions of lives.
• Cut down costs
- vaccines are given orally, no purification of
protein is needed.
- elimination of medical professionals.
• Renewable resource.
• Palatability.
• The concept is intriguing, in practice it could prove
quite unsafe and risky.
• Even the creator of the edible vaccine, Charles Arntzen,
now concedes that the idea should be abandoned.
• Research in this area now focuses on non-food crops
where the vaccine is subsequently purified and
packaged as a pill or capsule. It permits wider
distribution and easier storage of the product.
• As a result, developing countries would be capable of
distributing much-needed vaccines that are impractical
in a liquid form, which requires refrigeration or must
be administered via injection.
• BIOPOLYMERS look exactly like a
conventional plastic and have similar
characteristics. The difference is just
that they are made from vegetable
raw materials.
• Biopolymers are produced from
annual crops and plants like wheat,
corn, potato and sugar cane and other
new alternatives now being developed
for the production of food packaging
materials like film sheet and foam
containers. i.e. PLA (Polylactic Acid).
• Some examples:
The input materials for the production of these
polymers may be either renewable (based on
agricultural plant or animal products) or synthetic.
• There are four main types of green polymer based
respectively on:
• 1. Starch
• 2. Sugar
• 3.Cellulose
• 4.Synthetic materials
• Plants are able to produce
using 3 genes
from bacterium
into
canola for synthesis of PHB in seeds.
is produced in
experimental plant
.
• Every biopolymer has its own material-specific
properties, e.g. barrier properties such as oxygen
permeability.
• The barrier properties are relevant to the choice
of biopolymers for the packaging of particular
products.
• Bioplastics have very promising prospects for use
in pesticide soil pins, for packaging in-flight
catering products and for packaging dairy
products.
• The Biota Water bottle looks and feels like
plastic, but it's actually made from corn.
• The new, high-tech material is just as effective
as plastic and it's biodegradable.
• If you throw it in a landfill or compost pile, the
bottle dissolves in as little as 80 days,
according to Biota.