BIOTECHNOLOGY: Engineering plants for the future

1. 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

2. 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

3. 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

4. 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

5. Insert the isolated gene in vector: Plasmid DNA by cutting both with EcoRI • Sticky ends, DNA ligase

6. 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

7. 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

8. 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

9. 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

10. 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.

11. 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

12. 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

13. - 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

14. 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

15. A. Unsafe for consumers “Frankenfoods”

16. 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”

17. Ethical arguments against GM foods GM foods are wrong no matter how great the benefits may be.

18. GM foods are wrong because it’s wrong to: 1. Play God 2. Invent world changing technology • Cross species boundaries (Cold gene from fish But mules, hybrid wheat) • Reproduce by nonsexual means (Plant Cuttings) • Disrupt integrity, beauty, balance of nature (Monarch Butterfly) 6. Harm sentient beings

19. 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

20. Are valid concerns • Demand scientific and political attention

21. Many Support: Regulatory oversight on case-by-case basis • Many Do not support: a ban on all GM crops

22. 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

23. Enhanced nutrition Vitamin A Rice Iron Enhanced Rice Amino Acid Balance

24. 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

25. Disease resistance • Potatoes • Squash • Tomatoes • Corn • Rice • Canola • Soybeans • Grapes • Cantaloupes • Cucumbers

26. 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

27. Represent valid concerns • Demand scientific and political vigilance

28. Necessary: Regulatory oversight on case-by-case basis • Not necessary: A ban of GM foods

29. Following reagents except this one are required in a typical PCR reaction: • Primer • dNTP • DNA template • Polymerase enzyme • Protein stabilizer

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

31. Glyphosate kills plants because it • It prevents Electron Transport chain in photosynthesis • It prevents respiration • It prevents synthesis of secondary metabolites • It prevents synthesis of aromatic amino acids

32. GMO identification by PCR uses the following DNA sequences as target • except • 35S promoter • NOS terminator • House keeping gene • Inserted gene

33. 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

34. Some people in the society consider GMO food as wrong because they think it is • Playing God • Crossing cross-species boundaries • Disrupting the balance of nature • All of the above

35. A typical PCR method has the following steps except • DNA denaturation (Double strand separation) • Annealing (Primer binding to target) • Re-annealing (Separated strands join back) • Extension (Each strand is copied)

36. In our lab, DNA extraction from GMO/non-GMO samples requires negatively • Charged beads to • 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)

37. PCR can be used to distinguish between • Organic and non-organic food • Biological parent and non-biological parent • Tilapia and red snapper • Criminal and innocent • All of the above