Transcript Herbicide resistance - Howard University > Plant Biotechnology

BIOTECHNOLOGY: Engineering plants for the future
- beer, wine, cheese through micro-organisms.
- for food quality, yield, resistance to pathogens, produce metabolites, secondary
products
- new varieties as plant breeders
-Involves manipulation of biochemistry, physiology, and development
- Advent of DNA technology revolutionized
-Components of recombinant DNA technolgy
-Tissue and protoplast culture
-Use of agrobacterium as vectors for genetic engineering
-selected examples
ENGINEERING PLANTS WITH THEIR OWN GENES
-Ethylene induced fruits lack the texture and flavor of vine ripened appearance
-Pectin degradation by Polygalacturonase (PG) cause softening
-Antisense technology: sense RNA binds with antisense RNA
-Takes twice as long as normal tomato
-Was not commercially successful
Steps in producing Transgenic
-A source of foreign DNA containing the desired gene
-A vector that carries the gene
-Means to introduce the vector into host plant
Methods for isolating/cloning genes
-Restriction enzymes from bacteria
cut double stranded DNA at specific
sequences
-EcoRI cut at CTTAAG
-Different enzymes cut at different
location, so can use combination of
enzyme to isolate fragment of DNA
-Or make cDNA from mRNA using
reverse transcriptase enzyme
-Each piece of cDNA represent a gene
-Insert the isolated gene in vector: Plasmid DNA by cutting both with EcoRI
- Sticky ends, DNA ligase
-T-DNA in Ti plasmid induce galls by auxin and cytokinin and opines for bacteria
-Disarmed by cutting vir genes replace auxin/cytokinin/opines genes with YFG
-Antibiotic genes
-Electroporation
-Can use protoplast or leaf
-Biolistic methods for direct entry
-Protoplast fusion
-Micropropagation-virus free
PLANT PROTECTION
• Competition from weeds: nutrient, space
• Attack by viruses, fungi, and predatory insects
• Extensive use of herbicide, fungicides, insecticides
• Carryover of agricultural chemicals along with food product, effect on ecosystems
• Transgenic provides opportunities with decreased reliance on dangerous chemicals
• Weeds cause $12 b crop loss annually
• Mixture of herbicides to kill pre- and post-emergence weeds
• Multiple spraying, heavy chemical load on crops and soil
• Herbicide-resistant crops are solution!
• Roundup resistant soybean.
•Glyphosate inhibits Enzyme EPSP Synthase (aromatic amino acids)
• EPSP pathway is only found in plants and in microorganisms
• Humans need aromatic amino acids in their diet
• Herbicide resistant can be achieved by over-expression of tolerant enzymes
• Find a gene that encodes an enzyme variant that is resistant to the herbicide
• Find a strong promoters
Cells expressing large amounts of enzyme will tolerate higher doses of enzyme inhibitors
Strategy: Petunia cells in culture screened for glyphosate resistance
-Most cells died, few survived
-clone variant gene (EPSP synthase inhibitor tolerant)
-express in crops with strong promoter (35S) by agrobacterium vector
-More than half of the soybeans planted in North America are glyphosate resistant
Other examples: Glufosinate (Liberty) resistant.
-Herbicide resistant weeds: undesirable side effects from selection pressure
Bacillus thuringiensis (Bt) resistance
- Economic loss from pathogens $100b
- Insecticides are not specific, may kill the pollinators; now hire commercial pollinators
- Bt toxin ingested by insects, degraded, binds to receptors in gut and interference
with normal digestion
- Humans do not have the receptor
- Resistant insects?? Plant along with non-transgenic plants.
Disease Resistance
• Express chitinase to degrade chitin: a fungal cell wall constituents
• Vaccination of plants with cDNA from TMV coat protein: TMV resistance
• C4 genes in C3 plants, net carbon gain, reduce rubisco oxygenase activity
• Improvement of nitrogen fixation ability: Less reliance on N2 fertilizers
GOLDEN RICE
-White rice no beta carotene:
precursor of vitamin A
-Vitamin A deficiency: Blindness in
young children
-Also iron rich
psy (phytoene synthase) from daffodil
(Narcissus pseudonarcissus)
crtl from the soil bacterium Erwinia
uredovora
- Erucic acid to zero in canola oil.
- Increase unsaturated fatty acids in oil
- Tobacco plants to produce drugs: use male sterile plants so no seeds
no transfer of genes.
- Oral delivery of vaccines by edible plant tissue: norwalk virus coat protein
in potato. Fed mice produced antibody against Norwalk virus.
- Plants as alternate source of renewable fuels: ethanol by fermentation with yeast
- Sunflower oil in place of diesel oil.
- More reduced and more energy content , burn cleaner
- Potential disadvantages: Displace crop plants, arable lands
Ethical arguments against
genetically modified foods
• GMOs are wrong because risks outweigh benefits
• GMOs are wrong , no matter how great the benefits
Credit: Dr. Comstock, North Carolina State University
A. Unsafe for consumers
“Frankenfoods”
Unsafe for environments “superweeds”
–Herbicide resistance - canola gene flows
into weedy relatives
–Bt toxin kills monarch butterfly larvae
Unfair to small farmers
“Rich get richer, poor get poorer”
Ethical arguments against GM foods
GM foods are wrong no
matter how great the
benefits may be.
GM foods are wrong because it’s wrong to:
1. Play God
2. Invent world changing technology
3. Cross species boundaries (Cold gene from fish
But mules, hybrid wheat)
4. Reproduce by nonsexual means (Plant
Cuttings)
5. Disrupt integrity, beauty, balance of nature
(Monarch Butterfly)
6. Harm sentient beings
• Unsafe for consumers?
Food allergens, toxins
• Unsafe for environment?
– Unintended effects on nontarget organisms
– Gene flow, development of resistant weeds
• Unfair to small farmers?
Rich get richer, poor get poorer
• Are valid concerns
• Demand scientific and political attention
• Many Support: Regulatory oversight
on case-by-case basis
• Many Do not support: a ban on all GM
crops
Ethical arguments FOR GM foods
Potential to improve:
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Diets in developing countries
Efficiency of food production
Safety and purity of food
Agricultural sustainability
Diversity of agro-ecosystems
Enhanced nutrition
Vitamin A Rice
Iron Enhanced Rice
Amino Acid Balance
Insect resistance
•Bt corn
–Insect resistance
from Bacillus
thuringiensis
–Non-toxic to humans
–Target insect: corn
borer
–40% U.S. Corn crop
Bt
–Potential to reduce
insecticide use
Disease resistance
•Potatoes
•Squash
•Tomatoes
•Corn
•Rice
•Canola
•Soybeans
•Grapes
•Cantaloupes
•Cucumbers
Regulatory Oversight in Biotechnology
http://www.aphis.usda.gov/biotechnology/index.html
• US Dept. of Agriculture
– Plant pests
– Plants
– Veterinary biologics
• US Food and Drug Administration
– food, feed
– food additives
– veterinary drugs, human drugs and medical
devices
• US Environmental Protection Agency
– microbial/plant pesticides
– new uses of existing pesticides
– novel microorganisms
• Represent
valid concerns
• Demand scientific and political
vigilance
• Necessary: Regulatory oversight on caseby-case basis
• Not necessary: A ban of GM foods
Following reagents except this one are required in a typical PCR reaction:
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Primer
dNTP
DNA template
Polymerase enzyme
Protein stabilizer
During making transgenic plants, only a small population of plants get
Transformed. How do the plant molecular biologists select a transgenic plants?
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Select for trait of interest
Select antibiotic resistant plants after transformation (1st generation)
Select antibiotic resistant plants after seed production (2nd generation)
Select antibiotic susceptible plants after transformation (1st generation)
Select antibiotic susceptible plants after seed production (2nd generation)
Glyphosate kills plants because it
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It prevents Electron Transport chain in photosynthesis
It prevents respiration
It prevents synthesis of secondary metabolites
It prevents synthesis of aromatic amino acids
GMO identification by PCR uses the following DNA sequences as target
except
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35S promoter
NOS terminator
House keeping gene
Inserted gene
Identify the wrong method of transforming plants with foreign DNA
1. Electroporation
2. Biolistic methods for direct entry (Gene Gun)
3. Sodium Chloride based channel opening
4. Agrobacterium infection
Some people in the society consider GMO food as
wrong because they think it is
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Playing God
Crossing cross-species boundaries
Disrupting the balance of nature
All of the above
A typical PCR method has the following steps except
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DNA denaturation (Double strand separation)
Annealing (Primer binding to target)
Re-annealing (Separated strands join back)
Extension (Each strand is copied)
In our lab, DNA extraction from GMO/non-GMO samples requires negatively
Charged beads to
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Exclude the proteins from the sample
Exclude magnesium (divalent cation) to protect DNA in the sample
Exclude RNA from the sample
Help PCR methods amplify magnesium regulated enzymes
Help reduce protein contamination (PCR inhibitor)
PCR can be used to distinguish between
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Organic and non-organic food
Biological parent and non-biological parent
Tilapia and red snapper
Criminal and innocent
All of the above