1 / 60

TOPICS IN (NANO) BIOTECHNOLOGY Dining with DNA Lecture 8

PhD Course. TOPICS IN (NANO) BIOTECHNOLOGY Dining with DNA Lecture 8. 19 th May, 2006. Genetic Engineering: Is it the next Magic Pill?. Population growth Finite resources (land and water) Access to food. Technology options to increase productivity

carol
Download Presentation

TOPICS IN (NANO) BIOTECHNOLOGY Dining with DNA Lecture 8

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. PhD Course TOPICS IN (NANO) BIOTECHNOLOGY Dining with DNA Lecture 8 19th May, 2006

  2. Genetic Engineering: Is it the next Magic Pill?

  3. Population growth Finite resources (land and water) Access to food Technology options to increase productivity Agricultural and food policy options Global Context

  4. Population Growth • Increasing by 80 million people per year • 95% of the increase in developing countries • 1.3 billion people live in absolute poverty currently

  5. Demand for Food • Projected increase in global demand between 1993 and 2020 • Cereals: 41% • Meats: 63% • Roots and Tubers: 40% • Feed vs Food • Cereals for animal feed will double • Cereals for human consumption will increase by 47%

  6. Food Availability • Per capita availability of food will increase 7% by 2020 • Global income growth projected to increase 2.7% per year • Availability does not imply access

  7. Options to Meet Demand • Increase in cultivated land: < 20% contribution • Productivity increase: Cereal yields expected to decrease to 1.5% from 2.3% • Water stress: Demand for water to increase by 2.4% per year • Technology?

  8. Technology Options • Integrated Pest Management • Improved cultural practices • Improved food preservation techniques • Identifying new genetic sources • Genetic engineering

  9. What is Genetic Engineering?

  10. Definitions • Any food that has been made from genetically altered plants or animals • Genetic alteration: Altering gene/s using recombinant technique • Gene: Small segment of DNA that codes for a specific protein • Recombinant technique (rDNA): Methods used to alter gene/s

  11. Is Genetic Engineering Different from Traditional Breeding? • No! • Traditional breeding also involves gene transfer but thousands of genes, good and bad, are moved

  12. Plant Breeding

  13. Hybridization or Cross-breeding 1000 Genes in Line A 1000 Genes in Line B 1000 Genes in Hybrid

  14. DOMESTICATION OF MODERN DAY CROPS TEOSINTE TO MAIZE

  15. Some domestics and their (never domesticated) close relatives

  16. Is Genetic Engineering Different from Traditional Breeding? • Yes! • Specific gene/s from “any” source can be introduced and is faster

  17. How are GMOs Created

  18. Some history... • In 1953 Francis Crick and James Watson published their discovery of the structure of DNA, which led to scientists being able to splice genes from one organism and insert them into the DNA of another. • In 1973 Stanley Cohen and Herbert Boyer created the first successful recombinant DNA organism. • In 1980 U.S. Supreme Court ruled that genetically altered life forms can be patented in the case of Diamond vs. Chakrabarty. This decision allowed an oil eating organism to be patented by Exxon Oil Company.

  19. GMO Timeline • 1986 – Federal “Coordinated Framework” for regulating biotech • 1993 – FDA approves rBGH • 1994 –First biotech food approved (Flavr Savr tomato) • 1996 – First GM corn seed is sold; GM crops enter the food supply

  20. 1996 – Mad cow disease linked to human brain disease 1997 – European consumers protest US shipments; Monsanto targeted 1999 – Activists get violent; Secretary Glickman is pummeled in Italy; Monsanto PR campaign backfires in the EU; Brazil, Australia and China threaten ban; Monarch butterfly scare 2000 –Starlink corn crisis Non-US GMO Timeline

  21. World Political Timeline • 2001 – Application for GM fish is submitted to FDA; EU says labeling will be mandatory, trade war lingers; Mexican maize contamination reported; Monsanto abandons New Leaf potato • 2002 –Prodigene episode • 2003 – SubSaharan African nations reject US food aid with GM corn; US sues EU in WTO • 2004 – New EU rules go in effect; Monsanto shelves GM wheat; Glofish released unregulated

  22. GMO Foods - VERY controversial! http://www.teachersdomain.org/9-12/sci/life/gen/lp_bioengfood/index.html

  23. Example of genetically modified foods? • Also called genetically modified organisms (GMO). • Involves the insertion of DNA from one organism into another OR modification of an organism’s DNA in order to achieve a desired trait. 4 5 A strawberry resistant to frost + = Arctic fish DNA strawberry

  24. A. Totipotency • Definition • Entire plant can be generated from a single, non-reproductive cell • Single cells can be separated from leaf, stem or root tissue using enzymes to digest pectin holding cells together (pectinase)

  25. A. Totipotency • Clones from cuttings in tissue culture • Asexual reproduction of plants can occur using fragments of plants • Shoots or stems or leaves = EXPLANTS • In tissue culture, cells divide from exposed cell  a callus forms • Callus = undifferentiated cluster of rapidly dividing cells • Adventitious roots often form from callus

  26. A. Totipotency • Callus tissue regeneration • Callus tissue will develop if cells are grown with proper balance of nutrients and plant hormones • Magenta boxes, sterile medium and transfer instruments • Murishigee and Skoog medium (MS medium) – Artificial medium (agarose, nutrients and hormones) • Under influence of increased cytokinin, shoots will differentiate • Transferred to increased auxins, roots will establish • Eventually transferred to soil  entire plant with reproductive structures (ovules, pollen) • Calluses can be split into many smaller pieces before hormones are added to increase # of plants

  27. B. DNA inserted into plants  Transgenic plant • Characteristics of transgenic plants • All cells in the plant are derived from one cell • All cells express the desired genetic information • Why make transgenic plants? • Genes from distantly related plant families can be introduced without need for breeding (some families of plants are incompatible) • To improve crop hardiness and characteristics of final plant product • Protein content • Ripening rate • Drought resistance…..

  28. B. DNA inserted into plants  Transgenic plant • Procedures for generating transgenic plants • Microinjection • DNA constructs injected using fine glass pipettes in combination with phase contrast microscopy • Electroporation of protoplasts • Electric pulses of high field strength • Reversibly permeabilize cell membranes • Electric discharge gun – Gold beads • Firing DNA-coated pellets using a modified .22 caliber gun

  29. B. DNA inserted into plants  Transgenic plant • “Whiskers” of silicon carbide – holes punched, DNA introduced • Agrobacterium tumefaciens • Viral vectors • Cauliflower mosaic virus vectors • Gemini virus vectors • Liposome-mediated transformation of protoplasts • Artificial lipid vesicles = Liposomes • Chemically-stimulated DNA uptake by protoplasts • Polyethylene glycol + CaCl2

  30. B. DNA inserted into plants  Transgenic plant • Protoplast fusion can also be used to fuse two different plant types together  New Plant Varieties (hybrid plantlet) • Fused cell acquires some of the characteristic of both genetic backgrounds and can be regenerated into a plant with some traits from both parental plants • Fusigenic agents (polyethylene glycol) or electroporation used to fuse membranes • Useful if species are sexually incompatible or cross with difficulty

  31. B. DNA inserted into plants  Transgenic plant • US commercially important plants that can be grown from single somatic (non-seed) cells • Asparagus • Cabbage • Citrus fruits • Carrots • Alfalfa • Millet • Tomatoes • Potatoes • Tobacco • More than 30 different crop plants developed with rDNA techniques are being tested in field studies

  32. C. Agrobacterium tumefaciens • Characteristics • Plant parasite that causes Crown gall disease • Encodes a large (~250kbp) plasmid called Tumor-inducing (Ti) plasmid • Portion of the Ti plasmid is transferred between bacterial cells and plant cells  T-DNA (Tumor DNA) • T-DNA integrates stably into plant genome • T-DNA ss DNA fragment is converted to dsDNA fragment by plant cell • Then integrated into plant genome

  33. C. Agrobacterium tumefaciens

  34. C. Agrobacterium tumefaciens • How is T-DNA modified to allow genes of interest to be inserted? • In vitro modification of Ti plasmid • T-DNA tumor causing genes are deleted and replaced with desirable genes (under proper regulatory control) • Insertion genes are retained (vir genes) • Selectable marker gene added to track plant cells successfully rendered transgenic [antibiotic resistance gene  geneticin (G418) or hygromycin] • Ti plasmid is reintroduced into A. tumefaciens • A. tumefaciens is co-cultured with plant leaf disks under hormone conditions favoring callus development (undifferentiated) • Antibacterial agents (e.g. chloramphenicol) added to kill A. tumefaciens • G418 or hygromycin added to kill non-transgenic plant cells • Surviving cells = transgenic plant cells

  35. C. Agrobacterium tumefaciens • Techniques to transform plant cells by A. tumefaciens • Wounding and direct inoculation • Inoculation of explants in vitro • Transformation of leaf-disks • Co-cultivation of Agrobacterium with protoplasts

  36. C. Agrobacterium tumefaciens

  37. II. Examples of Crop Improvement Measures

  38. A. Nitrogen fixation • To enable plants to fix atmospheric N2 so that it can be converted into NH3, NO3-, and NO2- providing a nitrogen source for nucleic acid and amino acid synthesis • Thereby eliminating need to fertilize crops with nitrogen • Exploit N2 fixation metabolic machinery of bacteria and fungi • Some live freely in soil and water • Some live in symbiosis • Rhizobium spp. live in symbiosis with leguminous species of plants in root nodules (e.g. soy, peas, beans, alfalfa, clover)

  39. B. Frost Resistance • Ice-minus bacteria • Ice nucleation on plant surfaces caused by bacteria that aid in protein-water coalescence  forming ice crystals @ 0oC (320F) • Ice-minus Pseudomonas syringae • Modified by removing genes responsible for crystal formation • Sprayed onto plants • Displaces wild type strains • Protected to 23oF • Dew freezes beyond this point • Extends growth season • First deliberate release experiment – Steven Lindow – 1987- sprayed potatoes • Frost Ban • Different strain of bacteria – Julie Lindemann led different project – 1987 • Strawberries in California

  40. C. Resistance to biological agents • Anti-Insect Strategy – Insecticides • From Bacillus thuringensis • Toxic crystals found during sporulation • Alkaline protein degrades gut wall of lepidopteran larvae • Corn borer caterpillars • Cotton bollworm caterpillars • Tobacco hornworm caterpillars • Gypsy moth larvae • Sprayed onto plants – but will wash off

  41. C. Resistance to biological agents • Monsanto Chemical Company – 1991 Trials • BT  into cotton plants using A. tumefaciens vector • Cottton bollworms  protection in 6 loctions, 5 different states, consistent results • First crops – 1996 • Corn • Cotton • Seed potatoes • Soybean • Others

  42. C. Resistance to biological agents • Cloned BT toxin gene into a different bacterium that lives harmlessly in corn plants • Pressure applied to introduce modified bacterium into seeds • Corn stalks protected from corn borers • BT in poplar and white spruce  caterpillar resistance • BT-resistant strains are beginning to emerge in some caterpillars

  43. C. Resistance to biological agents • Anti-Bacterial Strategies • Resistance to Xanthomonas oryzae (rice wilting) • Conferred by cloning resistance genes from wild rice strains • Anti-Worm Strategies (Animal pest) • Nematode resistance gene from wild beet plants • To protect sugar beet

  44. Resistance to herbicides • Glyphosate resistance • Glyphosate = “Roundup”, “Tumbleweed” = Systemic herbicide • Glyphosate inhibits EPSP synthase (S-enolpyruvlshikimate-3 phosphate – involved in chloroplast amino acid synthesis) • Escherichia coli EPSP synthase = mutant form  less sensitive to glyphosate • Cloned via Ti plasmid into soybeans, tobacco, petunias • Increased crop yields of crops treated with herbicides

  45. Resistance to herbicides • Bromoxynil • = bromine-based herbicide • Bromoxynil resistant cotton • Concern over movement of resistance genes into weeds  making compounds useless

  46. Examples

  47. Examples

  48. Specific Examples

  49. Specific Examples

More Related