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-Can use protoplast or leaf -Electroporation

-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 inyoung 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

Credit: Dr. Comstock, North Carolina State University

• GMOs are wrong because risks outweigh benefits• GMOs are wrong , no matter how great the benefits

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 God2. Invent world changing technology3. 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:

– 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 Biotechnologyhttp://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

• Representvalid concerns

• Demand scientific and political vigilance

• Necessary: Regulatory oversight on case-by-case basis

• Not necessary: A ban of GM foods

Following reagents except this one are required in a typical PCR reaction:

1. Primer2. dNTP3. DNA template4. Polymerase enzyme5. Protein stabilizer

During making transgenic plants, only a small population of plants get Transformed. How do the plant molecular biologists select a transgenic plants?

1. Select for trait of interest2. Select antibiotic resistant plants after transformation (1st generation) 3. Select antibiotic resistant plants after seed production (2nd generation)4. Select antibiotic susceptible plants after transformation (1st generation)5. Select antibiotic susceptible plants after seed production (2nd generation)

Glyphosate kills plants because it

1. It prevents Electron Transport chain in photosynthesis2. It prevents respiration3. It prevents synthesis of secondary metabolites4. It prevents synthesis of aromatic amino acids

GMO identification by PCR uses the following DNA sequences as target except

1.35S promoter2.NOS terminator3.House keeping gene4.Inserted gene

Identify the wrong method of transforming plants with foreign DNA1. Electroporation2. Biolistic methods for direct entry (Gene Gun)3. Sodium Chloride based channel opening4. Agrobacterium infection

Some people in the society consider GMO food as wrong because they think it is

1. Playing God2. Crossing cross-species boundaries3. Disrupting the balance of nature4. All of the above

A typical PCR method has the following steps except

1.DNA denaturation (Double strand separation)2.Annealing (Primer binding to target)3.Re-annealing (Separated strands join back)4.Extension (Each strand is copied)

In our lab, DNA extraction from GMO/non-GMO samples requires negativelyCharged beads to

1.Exclude the proteins from the sample2.Exclude magnesium (divalent cation) to protect DNA in the sample3.Exclude RNA from the sample4.Help PCR methods amplify magnesium regulated enzymes5.Help reduce protein contamination (PCR inhibitor)

PCR can be used to distinguish between

1.Organic and non-organic food2.Biological parent and non-biological parent3.Tilapia and red snapper4.Criminal and innocent5.All of the above