1. Methods of Gene Transfer

2. Transgenic versus Cloning
Transgenic : creation of transgenic animal or plant (introduction of foreign gene into organism)
transgenic organisms produced by introduction of foreign gene into germ line (transgenic offspring!!!)
introduction of gene into somatic cells -> gene therapy
Cloning : obtaining an organism that is genetically identical to the original organism
such as Dolly the sheep
asexual propagation of plants (taking cuttings)
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3. What is a transgenic?
Transgenic
An organism containing a transgene introduced by technological (not breeding) methods
Transgene
The genetically engineered gene added to a species

4. Why transgenic plants ? Why do we need transgenic plants ?
Improvement of agricultural value of plant (resistance to herbicides, resistance to insect attack, Bacillus thuringiensis toxin)
living bioreactor, produce specific proteins
studying action of genes during development or other biological processes (knock-out plants, expression down- regulated)

5. Transgenic Plants Advantages & Disadvantages Advantages:
Plant cells are totipotent: whole plant can be regenerated from a single cell (engineered cells - engineered plants)
Plants have many offspring: rare combinations and mutations can be found
Transposons used as vectors
Disadvantages:
Large genomes (polypoid - presence of many genomes in one cell)
plants regenerating from single cells are not genetically homogenous (genetically instable)

6. Genetically modified plants
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7. 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

8. Plant Genetic Engineering Process
Cell
Plant cell
Extracted DNA
A single gene
Transgenic plant
Transformation
Cell division

9. Production of transgenic plants
Isolate and clone gene of interest
Add DNA segments to initiate or enhance gene expression
Add selectable markers
Introduce gene construct into plant cells (transformation)
Select transformed cells or tissues
Regenerate whole plants

10. Introducing the Gene Developing Transgenic Plant STEPS
Selection of transformants
Introduction of the gene
Create transformation cassette
Prepare tissue for transformation
Tissue must be capable of developing into normal plants
Leaf, germinating seed, immature embryos
Introduce DNA
Agrobacterium or gene gun
Culture plant tissue
Develop shoots & Roots
Screening of putative transformants
Field test the plants

11. Plant transformation DNA delivery systems must be
Simple
Efficient and preferably inexpensive
The method must be available for use either because it is in the public domain or because it can be licensed
System of choice depends on
the target plant
its regeneration system

12. Requirements for plant transformation
A. Cell culture and plant regeneration system
B. Cloned DNA to be introduced
1. selectable marker gene
kanamycin or G148 resistance: neomycin, phosphotransferase (NPTII), hygromycin B: hygromycin phosphotransferase (HygB)gentamicin: gentamicin acetyltransferase
streptomycin: streptomycin phosphotransferase
Bialophos: BAR
2. promoter (constituitive or inducible), coding region
C. Method of delivery of DNA into the cell
D. Proof of transformation of plant

13. Screening & Selection of transformant
Selectable marker gene
Positive selection
PMI (phospho- mannose isomerase) Plant cells without this enzyme are unable to survive in a tissue culture medium containing mannose-6-phosphate as a sole carbon source.
Removable selectable marker gene
Genes using the Cre-lox system or transposable elements
Selectable marker gene 2 1

14. Transformation Cassettes
Contains P G M TA
Promoter
Regulatory sequence/initiation site
2. Gene of interest
The coding region and its controlling elements
3. Selectable marker
Distinguishes transformed/untransformed plants
4. Insertion sequences
Aids Agrobacterium insertion

15. Commonly used promoters
Constitutive promoter
CaMV 35S : suitable for expression of foreign genes in dicots:
The maize ubiquitin promoter, also a constitutive promoter which
drives strong expression of transgenes in monocots.
Organ/ tissue specific promoters
Vicilin and phytohemaglutinin, glutenin promoters seed specific expression
a-amylase promoter for expression in the aleurone of cereal grains;
Patatin promoter for tuber specific expression in potatoes and the RuBisCo promoter for green tissue specificity

16. Marker gene screen able marker & selectable marker Selectable Markers
allow the selection of transformed cells, or tissue explants
by ability to grow in the presence of an antibiotic or a herbicide.
frequently used - kanamycin and hygromycin
Screen able markers
encode gene products whose enzyme activity can be easily assayed
allowing not only the detection of transformants
also estimation of the levels of foreign gene expression in transgenic tissue
markers such as GUS, luciferase or -galactosidase allow screening for enzyme activity by histochemical staining or fluorimetric assay of individual cells
can be used to study cell-specific as well as developmentally regulated gene expression

17. Plant Transformation Methods
Physical
Chemical
Biological
In planta
PEG
Calcium phosphate
Artificial lipids
Proteins
Dendrimers
Chemical
Physical
Agrobacterium Tumefaciens
Agrobacterium Rhizogenes
Virus-mediated
Biological & In-planta
Microinjection
Biolistics - gene gun/Particle bombardment
Electroporation
Silica/carbon fibers
Lazer mediated

18. Physical Methods of Transformation
Microinjection
Biolistics - gene gun/Particle bombardment
Electroporation
Silica/carbon fibers
Lazer mediated

19. Electroporation
1970s, 1990 versatile method – in vivo (skin and muscles)
short pulses of high voltage to carry DNA across the cell membrane
to assist the uptake of useful molecules such as a DNA vaccine into a cell
Parameters, electrical field strength [V/cm], pulse length

20. Electroporation Technique
Duracell
DNA containing
the gene of interest
Plant cell
Protoplast
Power supply
DNA inside the plant cell
The plant cell with
the new gene
Drawbacks
Limited effective range of ~1 cm between the electrodes
Surgical procedure is required to place the electrodes deep into the internal organs
High voltage applied to tissues can result in irreversible tissue damage as a result of thermal heating electron-avalanche transfection

21. This electroporator is for low-current applications such as those using small electrodes

22. Microinjection

23. Particle gun
Simplest method of direct introduction of therapeutic DNA into target cells
Looks like a pistol but works more like a shotgun with “Golden pellets”
First described as a method of gene transfer into plants
John Sanford at Cornell University in 1987
Particle bombardment -physical method of cell transformation in which high density and sub-cellular sized particles are accelerated to high velocity in order to carry DNA or RNA into living cells
MAJOR LIMITATIONS:
shallow penetration of particles
associated cell damage
the inability to deliver the DNA systemically
the tissue to incorporate the DNA must be able to regenerate and the expensive equipment .

24. Particle gun ✓ 1
For particle bombardment, tungsten or gold particles are coated with DNA and accelerated towards target plant tissues. In the early days, the force used to accelerate the particles was a .22 caliber blank. Today, most devices use compressed helium. ✓ 2
The particles punch holes in the plant cell wall and usually penetrate only 1-2 cell layers. Particle bombardment is a physical method for DNA introduction and the biological incompatibilities associated with Agrobacterium are avoided. ✓ 3
The DNA-coated particles can end up either near or in the nucleus, where the DNA comes off the particles and integrates into plant chromosomal DNA.
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26. DNA Delivery
Particle gun
Agrobacterium

27. 2. Biological Methods Agrobacterium Tumefaciens
Agrobacterium Rhizogenes
Virus-mediated

28. Agrobacterium- mediated
In the laboratory, bacteria are co- cultured or inoculated with plant tissue and the bacteria transfer part of their DNA into plant cells.
Most of the native transferred bacterial DNA is replaced with genes of interest
Agrobacterium is a soil borne gram- negative bacterium, that has a unique ability to introduce part of its DNA into plant cells.

29. Agrobacterium tumefaciens
Wild type Tk plasmid = 200 kb – too large for cloning
Intermediate shuttle plasmid is used to cut in Gene of Interest
VIR genes must be removed for genetic engineering
LB and RB are required for insertion and recombination with plant genome
Insertion into plant host is random (sort of)
First cloned gene – luciferase in tobacco plant

30. Mechanism of Agrobacterium- mediated transformation

31. Mechanism of Agrobacterium- mediated transformation

32. Mechanism of Agrobacterium- mediated transformation

33. Chemical Transformation
PEG
Calcium phosphate
Artificial lipids
Proteins
Dendrimers

34. PEG mediated
It is the oldest (direct DNA) reliable method for plant transformation. In the first report (Krens et al Nature 296:72), Agrobacterium Ti plasmid was introduced into petunia protoplasts. Formation of tumors, opine synthesis and Southern blot provided the verification, which is an extensive and complete analysis to show success of transformation.
The first report of generating transgenic plants using this method was provided by Paszkowski et al. (1984). They regenerated transformed protoplasts into plants that were kanamycin (drug) resistant.
This method has been very useful and applied to several plant species.
But it is a tedious procedure!

35. In-Planta Transformation
Non-tissue culture based
Meristem transformation ♣ ♣
Floral dip method ♣
Pollen transformation

36. Vacuum Infiltration
Plant leaf disks are placed in a suspension of bacteria and vacuum pulled
Air is release like a sponge being squeezed
Vacuum is released and solution floods tissue
Plant disk is cultured

37. Floral Dip Simple submersion of plant into bacterium suspension
No vacuum is needed
Conducted with plants grown until just flowering
Progeny seeds are harvested and germinated using selective antibiotic

38. Analysis of T0 plants
Yield characters
Physiology
Morphology
GUS expression
Gene expression
Confirmation with selectable marker, Screenable marker, Negative & Positive control

39. GFP expression in soybean tissue
Shows variability in expression pattern standard illumination on left – gfp illumination on right

40. Few Examples of Transgenic crop

41. Golden Rice Synthesis
Two Daffodil genes and one bacterial gene Erwinia uredovora were cloned into agrobacterium T DNA and inserted into rice genome to generate needed enzymes
Phytoene synthase &
Lycopene-b-cyclase
Carotene desaturase
T DNA
Germ-line transformation with agrobacterium X
Cross
T-formed rice with genes
T-formed rice with gene
Progeny rice plant with complete b carotene pathway

42. Golden Rice Golden rice contains increased levels of pro-vitamin A .
Traditional rice is white (a).
The prototype of golden rice was developed in and is a light yellow color (b). It contains 1.6 mg/g of carotenoid.
In 2005, new transgenic lines were developed that dramatically increased the amount of carotenoid synthesized, making the rice a deep golden color (c).
This latest form contains 37 mg/g of carotenoid, of which 84% is b-carotene – trial

43. World's First Blue Roses On Display In Japan Danielle Demetriou,  Daily Telegraph, October 31, 2008, See the rose at
Tokyo, Japan - World's first blue roses have been unveiled to the public for the first time at an international flower fair in Japan, following nearly two decades of scientific research.  The blue-hued blooms are genetically modified and have been implanted with a gene that simulates the synthesis of blue pigment in pansies.
Its scientists successfully pioneered implanting into the flowers the gene that produces Delphinidin, the primary plant pigment that produces a blue hue but is not found naturally in roses.
The world's first genetically modified blue roses were unveiled in the laboratory four years ago, although further research was required to make them safe to grow in nature.
The Blue Rose was developed by Suntory Flowers

44. Tearless Onion
Dr Eady
Crop & Food Research in New Zealand and his collaborators in Japan
As onions are sliced, cells are broken, alliinases - break down aa sulphoxides - generate sulphenic acids - unstable - rearrange into a volatile gas - syn-propanethial-S-oxide – diffuses by air - reaches the eye - reacts with the water to form a diluted solution of sulphuric acid - Tear glands produce tears to dilute and flush out the irritant

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

46. Thank you!