1. What is Biotechnology?
Title page.

2. What is Biotechnology? General Definition
The application of technology to improve a biological organism
Detailed Definition
The general definition is very broad. Many individuals prefer this definition because they can claim process such as plant breeding or mutagenesis are actually biotechnology. The detailed definition points to the fact that a foreign gene needs to be inserted for a product to be considered a biotech product.
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
Genetic engineering is the collection of techniques necessary to create a transgene. These procedures include isolating the gene-of-interest from the tens of thousands of genes found in the genome of a species. Once that gene is isolated, it is modified so it functions better in an organism. That gene is then mixed with other genes to prepare it to be introduced into another organism. This whole step develops 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
Here I am pointing out the difference between the process (using a transgene) and the product (the transgenic plant).
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
Here you are pointing out the difference between the process (using a transgene) and the product (the transgenic plant).
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
GMO: notice that foreign DNA is a requirement for this definition. Plant breeding products are not GMOs, as some would like to claim.

7. Important Terms Breeding Beneficial gene added from the same species
Gene delivered by mating within the species
Source: USDA
Transformation
More definitions to know.
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
Again, here were trying to show that breeding is not biotechnology. Some would like to claim breeding is biotechnology. In that way, they can claim that we actually been using biotech products for a long time. This is a hollow argument that will backfire if pressed.

9. What are the structures in molecular genetics?
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.

10. Interspecific Cross X Wheat Rye Triticale New species, but
NOT biotechnology
products
These photos illustrate the use of plant breeding to produce a new crop of agronomic utility.

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
Notice that the mutagenic treatment changed a single base in the gene sequence. This change created a resistant plant because the gene product targeted by herbicide is not affected by the herbicide. This is a change of gene in the plant; a foreign gene was NOT involved.
Resistant
Mutant
Gene
ATTGGA

12. Transformation Cassettes
Contains
1. Gene of interest
The coding region and its controlling elements
2. Selectable marker
Distinguishes transformed/untransformed plants
All transformation cassettes contain three regions. The “gene of interest” region contains the actual gene that is being introduced into the plant. This is the gene that provides the new function to the plant. In this diagram, the region is shown in red.
Many plant tissues are treated with the transformation cassette during the transformation step. Not all of these tissues actually receive the cassette. To distinguish those that contain the gene from those that don’t, it is necessary to use a selection process. The selectable marker is a gene that provides the ability to distinguish transformed from non-transformed plants. This is shown by green.
The most common method to introduce the transformation cassette is by using the plant pathogen Agrobacterium. For this system to work it is necessary that the cassette contain insertion sequences that are used by the bacteria. These are shown by the gray.
3. Insertion sequences
Aids Agrobacterium insertion

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

14. Delivering the Gene to the Plant
Transformation cassettes are developed in the lab
They are then introduced into a plant
Two major delivery methods
Agrobacterium
Two techniques are used to deliver DNA found in the transformation cassette into plant tissues during the plant transformation process. One is a biological system based on the plant pathogen Agrobacterium tumefaciens. The second is a mechanical method where the DNA is “shot” into plant cells using a gene gun.
Regardless of the delivery method, the delivery system must use a plant tissue source that can be manipulated to produce new plants.
Tissue culture
required to generate
transgenic plants
Gene Gun

15. The Next Test Is The Field
Herbicide Resistance
Non-transgenics
The last step in plant genetic engineering is field testing. This slide shows a field that contains herbicide resistant and tolerant plants.
Transgenics

16. Final Test of the Transgenic
Consumer Acceptance
RoundUp Ready Corn
What is needed is for the public to accept these crops. Examples such as these, were a corn crop is freed of weed pressure make a compelling case for acceptance of these new agricultural products. But, it should be noted that these traits are all producer orientated.
Before
After

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 stands for Bacillus thuringiensis, a bacteria that produces a toxin that kills the insects. The gene that encodes the toxin protein was inserted into plants.
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
There a multiple forms of herbicide resistance protect the crop against a variety of herbicides. Glyophosate and imidazolinone are the primary classes that the plants are resistant against. Virus resistance is obtained by inserting the viral coat-protein gene into the plant. When this protein is produced in the plant, the viral immune system is activated, and the plant is resistant.
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
These are somewhat tough labeling regulations. Companies actually have opposed labeling, but feel this might be the best that can be achieved from Europe at this time.

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?
This is just a list of the concerns the public has expressed regarding biotechnology.

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 proteins not native to the crop.
2. Adverse effects on non-target organisms, especially pollinators and 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. Ti plasmid contains several regions of importance:
Disease caused by the action of a plasmid in Agrobacterium called the Ti plasmid (Ti = Tumor Inducing).
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 right borders 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 ( V) is given for 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.

51. 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.

52. Advantages:
Bombardment is able to penetrate intact cells thus avoiding the need to remove the cell wall.
It can work with any plant species.
It was the first reliable technique to work with soybeans and moncots such as corn and rice.

53. Problems:
You need to be able to regenerate whole plant from the single bombarded cell.
If complex tissue is used for bombardment, you can get chimeric plants containing both transformed and non-transformed tissue.
Expensive.