What is Biotechnology?

1. What is Biotechnology?

2. What is Biotechnology? General Definition The application of technology to improve a biological organism Detailed Definition The application of the technology to modify the biological function of an organism by adding genes from another organisms

3. What About the Term Genetic Engineering? Genetic engineering is the basic tool set of biotechnology Genetic engineering involves: • Isolating genes • Modifying genes so they function better • Preparing genes to be inserted into a new species • Developing transgenes

4. What is a transgenic? Concept Based on the Term Transgene Transgene – the genetically engineered gene added to a species Ex. – modified EPSP synthase gene (encodes a protein that functions even when plant is treated with Roundup) Transgenic – an organism containing a transgene introduced by technological (not breeding) methods Ex. – Roundup Ready Crops

5. Biotechnology Terms You Probably Heard Transgene: the foreign gene added to a species Ex. – modified EPSP synthase gene (encodes a protein that functions even when plant treated with Roundup) Transgenic: an organism containing a transgene introduced by technological (not breeding) methods Ex. – Roundup Ready Crops

6. Biotechnology Develops GMOs - Genetically modified organisms • GMO - an organism that expresses traits that result • from the introduction of foreign DNA • Also called transgenic organism

7. Important Terms • Breeding • Beneficial gene added from the same species • Gene delivered by mating within the species Source: USDA • Transformation • Beneficial gene added from another species • Gene delivered by plant genetic engineering Source: USDA

8. Let’s Be Up Front • Breeding  Biotechnology •  Breeding only exchanges genes found in the species. • Breeding can transfer the transgene to other breeding materials • BUT it is not the same as biotechnology. • Biotechnology adds traits not available in the species •  Soybean does not have a gene to breakdown Roundup • The gene comes from bacteria

9. Molecular genetics: study of genes and how they are expressed. Chromosome: part of cell nucleus that contains heredity information and promotes protein synthesis. Gene: basic unit of heredity on a chromosome. DNA: molecule in a chromosome that codes genetic information. What are the structures in molecular genetics?

10. Interspecific Cross Wheat Rye X Triticale New species, but NOT biotechnology products

11. Mutagenesis: New Trait, No Foreign Gene • Mutagenesis changes the sequence of a gene • New, useful traits can be obtained Mutagenesis Treatment Susceptible Normal Gene ATTCGA Resistant Mutant Gene ATTGGA

12. 1. Gene of interest • The coding region and its controlling elements 2. Selectable marker • Distinguishes transformed/untransformed plants 3. Insertion sequences • Aids Agrobacterium insertion Transformation Cassettes Contains

13. Tissue must be capable of developing into normal plants • Leaf, germinating seed, immature embryos • Develop shoots • Root the shoots Transformation Steps Prepare tissue for transformation Introduce DNA • Agrobacterium or gene gun Culture plant tissue Field test the plants • Multiple sites, multiple years

14. Agrobacterium Tissue culture required to generate transgenic plants • Gene Gun Delivering the Gene to the Plant • Transformation cassettes are developed in the lab • They are then introduced into a plant • Two major delivery methods

15. Non-transgenics Transgenics The Next Test Is The Field Herbicide Resistance

16. Before After Final Test of the Transgenic Consumer Acceptance RoundUp Ready Corn

18. Crop Biotech Market Dominated By Four Countriesa 6% 3.2 mha 68% 35.7 mha 3% 1.5 mha 22% 11.8 mha Total = 99% of market a2001 growing season data.

19. Agriculture Products On the Market Insect resistant cotton • Bt toxin kills the cotton boll worm • toxin gene from a bacteria Source: USDA Insect resistant corn • Bt toxin kills the European corn borer • toxin gene from a bacteria • Rootworm GM approved (2/26/03) Normal Transgenic

20. Herbicide resistant crops • current: soybean, corn, canola • coming: sugarbeet, lettuce, strawberry, alfalfa, potato, wheat (2005) • resistance gene from bacteria Source: Monsanto Virus resistance • papaya, squash, potato • resistance gene from a virus

21. Economic Effect of Bt Cotton In China • $200/acre increase in income • $750 million increase nationally

22. EU Labeling Regulations • Foods with less than 0.9% of GM gene product • Labeling not required • Products derived from a GM crop • Labeling required • Applies even if the product does not contain the GM • gene product • Ex: Corn syrup: does not have the Bt protein, but must • be labeled • Animal feeds from GM crops • Same guidelines apply

23. What Are the Public Concerns? Economics Are we changing the economics on the farm? Environmental Are we irreversibly modifying the environment?   Globalization Is technology becoming centralized in too few hands? Social Will we develop a class of genetic outcasts? Religious Are we playing God?

25. Benefits of Plant Biotechnology • Greater production efficiencies: It can help plant breeders improve a crop’s yield • Less chemical damage • Hardier plants: it leads to produce plants that will resist diseases and unfavorable weather conditions. • Improved food quality. • New crops • Improved protection against human and animal diseases

26. Concerns associated with GM crops • Possible production of allergenic or toxic proteinsnot native to the crop. • 2. Adverse effects on non-target organisms, especially pollinatorsand biological control organisms. • 3. Loss of biodiversity. • 4. Genetic pollution (unwanted transfer of genes to other species). • 5. Development of pest resistance. • 6. Global concentration of economic power and food production. • 7. Lack of "right-to-know" (i.e., a desire for labeling transgenic foods).

27. File to support registration of new crop variety- conventional breeding

29. Biological systems for transformation

30. Agrobacterium tumefaciens • Agrobacteria are soil bacteria. • They naturally infect dicotyledonous plants. • Because host range is limited, procedure has not been used for some major crops such as corn, wheat, rice, etc. • Life cycle of Agrobacterium involves living in the soil until it encounters a plant and then infecting the plant. • Infection causes a rapid proliferation of plant cells around the infection leading to formation of a crown gall tumor

31. For Agrobacterium hizogenes, masses of roots emerge from the gall forming hairy root disease. • Once the gall is produces, it provides a haven for the bacteria to proliferate. • To obtain food, the bacteria also subjugates the plant to produce an unusual class of compounds called opines. • Opines are condensation products of the amino acid arginine and carbon compounds present in the Kreb cycle. • Most common are octopine and nopaline. Opines cannot be metabolized by host plant but are used by the bacterium for food and amino acids.

32. Disease caused by the action of a plasmid in Agrobacterium called the Ti plasmid (Ti = TumorInducing). • Ti plasmid is a large circular plasmid, 180 kbp in size. • Only one present in each bacterial cell. • Ti plasmid contains several regions of importance: • 1)  Transfer or T-DNA: Is a region of the plasmid that is transferred from the bacteria to the host plant cell during the infection process. • Once in the host, it becomes stably integrated in one of the host's chromosomes. • T-DNA is ~25-kbp long bracketed by two 25-bp direct repeats called left and right borders.

33. Ti plasmid

34. Between the borders are several genes: • Isopentyl adenine transferase (IPT): synthesizes cytokinins. • Tryptophan monooxygenase • Indoleacetamide hydrogenase: both enzymes involved in the biosynthesis of the auxin, indoleacetic acid. • Gene for synthesis of a specific type of opine, either the octopine or nopaline type. • The first three genes are involved in making the plant hormones, cytokinin and auxin. • Massive production of these hormones at the site of infection causes the surrounding plant cells to divide and create the gall tumor.

35. 2) virulence or vir region: Region that contains many genes required for the infection process. • Important ones are vir A, B, C, D1, D2, E, G, and pin F that are required for the transfer and integration of the T-DNA into host. • Vir region does not have to be physically connected to the T-DNA; region can work in trans on a separate plasmid.  • Basis for the construction of binary Ti plasmids. • 3)  Replication origin for Agrobacterium. • 4) Genes responsible for opine metabolism: Allows Agrobacterium to metabolize opines back into arginine and carbon compounds.

36. Course of events during Agrobacterium infection • Agrobacterium present in the soil detects dicot plants susceptible to infection by the secretion of polyphenols from the roots or from wound sites. • Since such wounds are a site of easy infection by bacteria, so they use the polyphenol signal to identify good targets. • Bacteria move up chemical gradient of polyphenols to find the plant.

37. Polyphenols binds to a receptor encoded by vir A gene. • Binding activates vir A which then activates the vir G protein by phosphorylation. • Both vir A and G are constitutively expressed. • Vir G protein is a transcription factor which then initiates transcription of the rest of vir genes on Ti Plasmid as well as vir genes on the Agrobacterium chromosome (CHV genes). • Specific vir gene products then cut T DNA at left and right borders (Vir D1, D2, C). • Single stranded copies of the T DNA region are synthesized, creating the T-strand.

38. T-strand is coated with single stranded DNA binding proteins (Vir E) and the ss DNA/Vir E complex is shuttled out of the bacterium and transferred to plant cell where it is integrated in the host chromosome. Process similar to bacterial conjugation. • Once integrated in the plant chromosome, T-DNA genes become active, producing the oncogenic proteins for the synthesis of auxins and cytokinins, thus forcing the cells to proliferate. The opine synthesis enzyme is also produced and the manufactured opines are used as food for bacteria.

39. Life cycle of Agrobacterium make it a perfect vehicle for the stable introduction of foreign DNA into plants. • Method involves insertion of DNA to be introduced between left and rightborders of T-DNA and then infect the plant. • Early methods used natural Ti plasmids that contained the oncogenes for hormone biosynthesis and the opine bosynthesis genes. • Created transformed plants but presence of oncogenes caused plants to remain as galls (Hardly useful as a crop).

40. Newer methods for transformation use highly modified version of the Ti-plasmid • Are disarmed ("non-obcogenic") by deletion of the oncogenes. • Ti-plasmid is divided into two plasmids, a larger one containing the vir region and a smaller one containing only the T-DNA region.  • Smaller T-DNA plasmid contains two replication origins, one for E. coli and one for Agrobacterium, and antibiotic resistance gene for selection in E. coli and Agrobacterium.  • All natural genes are removed from the between the T-DNA borders (including those for opines) and replaced with a multiple cloning site to faciliatate insertion of your gene, and a selectable marker. Some plasmids also contain reporters in the T-DNA region.

41. Transformation with Ti plasmids involves: • Preparation of Ti plasmid containing the gene to be transferred. • Incubate with plant tissue wounded in some way to facilitate entry of bacterium into the plant. • Plating leaf section on media containing: • Antibiotic to kill remaining Agrobacterium. • Balance of plant hormones to allow leaf cells to divide and form callus tissue. • Suitable toxin (e.g., kanamycin, phosphinothricin) to kill all cells that begin dividing that are not transformed and thus do not contain the NPT II gene (Process called selection).

42. Transferring individual callus onto appropriate media with right hormone balance to allow regeneration of callus cells into intact plants. • Transformed plants will be hemizygous for inserted gene. Self pollination with convert some progeny into homozygous transformed lines.

43. Problems with the use of Agrobacterium for transformation: • Same DNA between T-DNA borders can be inserted into multiple chromosomal regions of the transformed plants. Easy to get as many as 10 copies inserted during a single transformation. Makes generating of homozygous plant difficult. • Only able to transform dicotyledonous plants with sufficient efficiency. Attempts to expand the host range of bacterium has met with little success.

44. Direct transfer of DNA in plant cells • Electroporation • Electroporation involves the use of electrical discharges to make cell leaky. • Leaks then provide avenues for DNA to enter cell. • Technique cannot transport DNA across cell wall so it must be removed to generate protoplasts. • Cell wall removed by fungal enzymes that specialize in digesting cellulose, pectins and other cell wall polymers. • Once missing the cell wall, protoplast are very fragile and sensitive to osmotic shock.

45. Protoplasts are mixed with DNA to be introduced and placed in a cuvette lined with two electrodes. • Both stable and transient expression increased as DNA concentration is increased. Electric shock (200-400 V) is given for 50-100 msec. • Cuvettes are cooled to reduce heat. • Then the protoplasts are allowed to recovered and regenerate their cell wall. • When placed on hormone media containing a toxin suitable for selection only those cells that are transformed will multiply producing calli (stable transformants). • Electroporation was the first technique to transform cereals like corn.

46. Advantages: • Works for any plant species and cell type. • Provides quick and accurate data on expression using transient assays. • Can test to see if a particular gene you have created will work once stably integrated without having to wait to regenerate a transgenic plant. • Delivery of the DNA is quick and relatively inexpensive so you can do lots of tests.

47. Problems: You need to produce protoplasts first. Since for many species, you cannot regenerate easily intact fertile plants from protoplasts, this method may be not suitable for producing stably transformed plants.

48. Microprojectile bombardment • Technique developed by Sanford at Cornell and the patent was sold to DuPont. • It is a technique for the delivery of DNA in intact plant cells using DNA-coated particles accelerated to high velocities. • Such particles have enough momentum to penetrate the cell wall and become lodged inside cells.  • Following bombardment, cells repair the holes and can survive. • To penetrate the cell wall, particles must have sufficient momentum (p). Because p = mv, the faster and heavier the particle the better.

49. Small (~10 uM diameter) particles made with dense metals such as tungsten or gold are used. • Particles coated with naked DNA (usually plasmids containing the gene to be inserted) are made by mixing the bead with a solution containing the DNA and then the solution is dried. • Usually the plasmid contains both a selectable marker and the DNA of interest.  • Once particles are lodged in the cells, the DNA/RNA will dissolve. • The RNA can be directly translated, and DNA can be transcribe and translated;

50. If the particles carrying DNA become lodged in the nucleus, the released DNA can stably integrate into the host chromosomes (stable transformation). • Occurs at a very low frequency, so a strong selection is necessary. • Since the individual cells that become transformed must regenerate into a whole plant, tissue culture cells, callus, and embryonic cells are typically used.