1. TRANSGENIC TECHNOLOGY

2. Traits that plant breeders would like in plants
High primary productivity
High crop yield
High nutritional quality
Adaptation to inter-cropping
Nitrogen Fixation
Drought resistance
Pest resistance
Adaptation to mechanised farming
Insensitivity to photo-period
Elimination of toxic compounds

3. Plant transformation getting DNA into a cell
getting it stably integrated
getting a plant back from the cell

4. Requirement a suitable transformation method
a means of screening for transformants
an efficient regeneration system
genes/constructs
Vectors
Promoter/terminator
reporter genes
selectable marker genes
‘genes of interest’

5. Transformation methods
DNA must be introduced into plant cells
Indirect - Agrobacterium tumefaciens
Direct - Chemical method
- Electrical method
- Physical methods
Chemical Method
Use of PEG (Polyethylene glycol (PEG)-mediated )
Protoplasts are incubated with a solution of DNA and PEG

6. Physical Methods Electrical method
Electroporation (electropermeabilization)
Cells or protoplast are subjected to short electrical pulse
Physical Methods
Particle bombardment
Microinjection
Silicon Carbide whiskers

7. Agrobacterium-mediated transformation
A natural genetic engineer
2 species
A.tumefaciens (produces a gall)
A. rhizogenes (produces roots)
Oncogenes (for auxin and cytokinin synthesis) + Opines
In the presence of exudates (e.g. acetosyringone) from wounded plants, Virulence (Vir) genes are activated and cause the t-DNA to be transferred to plants. Everything between the left and right border is transferred.

8. BACTERIAL GALL DISEASES
Galls:
overgrowth or proliferation of tissue, primarily due to increased cell division (hyperplasia) and increased cell size (hypertrophy).
Bacterial Galls:
induced by bacteria in 3 different genera.
Agrobacterium
Pseudomonas
Clavibacter
Genes for plant hormone production found on bacterial plasmids!

9. Crown Gall Disease: Agrobacterium tumefaciens
Gram -
Dicots
Worldwide

10. Disease Cycle

11. 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)

12. Agrobacterium tumefaciens
T-DNA integrates stably into plant genome
Single stranded T-DNA fragment is converted to dsDNA fragment by plant cell
Then integrated into plant genome
2 x 23bp direct repeats play an important role in the excision and integration process

13. Agrobacterium tumefaciens
Tumor formation = hyperplasia
Hormone imbalance
Caused by A. tumefaciens
Lives in intercellular spaces of the plant
Plasmid contains genes responsible for the disease
Part of plasmid is inserted into plant DNA
Wound = entry point  days later, tumor forms

14. Agrobacterium tumefaciens
What is naturally encoded in T-DNA?
Enzymes for auxin and cytokinin synthesis
Causing hormone imbalance  tumor formation/undifferentiated callus
Mutants in enzymes have been characterized
Opine synthesis genes (e.g. octopine or nopaline)
Carbon and nitrogen source for A. tumefaciens growth
Insertion genes
Virulence (vir) genes
Allow excision and integration into plant genome

15. Ti plasmid of A. tumefaciens

17. Auxin, cytokinin, opine synthetic genes transferred to plant
Plant makes all 3 compounds
Auxins and cytokines cause gall formation
Opines provide unique carbon/nitrogen source only A. tumefaciens can use!

18. 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

19. Agrobacterium and genetic engineering: Engineering the Ti plasmid

20. Co-integrative and binary vectorsLB RB
Co-integrative vectors require the genes that are transferred from bacteria to go into the plasmid DNA by homologous recombination. For binary vectors, the plasmid containing the t-DNA is able to replicate in E.coli and can be mobilised into Agrobacterium by a triparental mating with a helper strain of E.coli. This greatly simplifies the process of plasmid construction.
Co-integrative
Binary vector

21. Agrobacterium-mediated transformation
Agrobacterium tumefaciens
cause ‘Crown gall’ disease
Agrobacterium is a ‘natural genetic engineer’
i.e. it transfers some of its DNA to plants

22. Agrobacterium Mediated Transfer
Expose wounded plant cells to transformed agro strain
Electroporate T-DNA vector into Agrobacterium and select for tetr
Agrobacterium
Mediated Transfer
Induce plant regeneration and select for Kanr cell growth

25. Electroporation Explants: cells and protoplasts
Most direct way to introduce foreign DNA into the nucleus
Achieved by electromechanically operated devices that control the insertion of fine glass needles into the nuclei of individuals cells, culture induced embryo, protoplast
Labour intensive and slow
Transformation frequency is very high, typically up to ca. 30%

26. Microprojectile bombardment
uses a ‘gene gun’
DNA is coated onto gold (or tungsten) particles (inert)
gold is propelled by helium into plant cells
if DNA goes into the nucleus it can be integrated into the plant chromosomes
cells can be regenerated to whole plants

27. In the "biolistic" (a cross between biology and ballistics )or "gene gun" method, microscopic gold beads are coated with the gene of interest and shot into the plant cell with a pulse of helium.
Once inside the cell, the gene comes off the bead and integrates into the cell's genome.

29. Model from BioRad: Biorad's Helios Gene Gun

30. Microinjection
Most direct way to introduce foreign DNA into the nucleus
Achieved by electromechanically operated devices that control the insertion of fine glass needles into the nuclei of individuals cells, culture induced embryo, protoplast
Labour intensive and slow
Transformation frequency is very high, typically up to ca. 30%

31. Silicon Carbide Whiskers
Silicon carbide forms long, needle like crystals
Cells are vortex mixed in the present of whiskers and DNA
DNA can be introduced in the cells following penetration by the whiskers

32. Competent cells to maximize chance of both happening
Not all cells take up DNA & not all cells can regenerate
Need an efficient regeneration system and transformation system i.e. lots of cells take up DNA and lots of cells regenerate into a plant
to maximize chance of both happening
regenerable cells
Transformed cells
Cells containing new DNA that are able to regenerate into a new plant

33. Screening technique How do we know which plants have taken up the DNA?
There are many thousands of cells in a leaf disc or callus clump - only a proportion of these will have taken up the DNA
therefore can get hundreds of plants back - maybe only 1% will be transformed
How do we know which plants have taken up the DNA?
Could test each plant - slow, costly
Or use reporter genes & selectable marker genes

34. Screening
Transformation frequency is low (Max 3% of all cells) and unless there is a selective advantage for transformed cells, these will be overgrown by non-transformed.
Usual to use a positive selective agent like antibiotic resistance. The NptII gene encoding Neomycin phospho-transferase II phosphorylates kanamycin group antibiotics and is commonly used.

35. Screening (selection)
Select at the level of the intact plant
Select in culture
single cell is selection unit
possible to plate up to 1,000,000 cells on a Petri-dish.
Progressive selection over a number of phases

36. Selection Strategies
Positive
Negative
Visual

37. Positive selection
Add into medium a toxic compound e.g. antibiotic, herbicide
Only those cells able to grow in the presence of the selective agent give colonies
Plate out and pick off growing colonies.
Possible to select one colony from millions of plated cells in a days work.
Need a strong selection pressure - get escapes

38. Negative selection
Add in an agent that kills dividing cells e.g. chlorate / BUdR.
Plate out leave for a suitable time, wash out agent then put on growth medium.
All cells growing on selective agent will die leaving only non-growing cells to now grow.
Useful for selecting auxotrophs.

39. Visual selection Only useful for coloured or fluorescent compounds
Plate out at about 50,000 cells per plate.
Pick off coloured / fluorescent compounds
Possible to screen about 1,000,000 cells in a days work.

40. Positive and Visual Selection

41. Regeneration System How do we get plants back from cells?
We use tissue culture techniques to regenerate whole plants from single cells
getting a plant back from a single cell is important so that every cell has the new DNA

42. Regeneration
Plant tissue culture uses growth regulators and nutrients to regenerate plants in vitro
Regeneration of shoots from leaf protoplasts in Arabidopsis thaliana

43. Somatic embryogenesis in peanut

44. Organogenesis

45. Gene construct

46. Cloning
Clone: a collection of molecules or cells, all identical to an original molecule or cell
To "clone a gene" is to make many copies of it - for example, in a population of bacteria
Gene can be an exact copy of a natural gene
Gene can be an altered version of a natural gene
Recombinant DNA technology makes it possible

47. Naturally occurring extrachromosomal DNA
Plasmids
Naturally occurring extrachromosomal DNA
Plasmids are circular dsDNA
Plasmids can be cleaved by restriction enzymes, leaving sticky ends
Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid

48. Restriction Enzyme
Molecular scissors; isolated from bacteria where they are used as Bacterial defense against viruses.
Molecular scalpels to cut DNA in a precise and predictable manner
Members of the class of nucleases

49. Nuclease
Breaking the phosphodiester bonds that link adjacent nucleotides in DNA and RNA molecules
Endonuclease
Cleave nucleic acids at internal position
Exonuclease
Progressively digest from the ends of the nucleic acid molecules

50. Endonuclease Type Characteristics I
Have both restriction and modification activity
Cut at sites 1000 nucleotides or more away from recognition site
ATP is required II
It has only restriction site activity
Its cut is predictable and consistent manner at a site within or adjacent to restriction site
It require only magnesium ion as cofactor
III
Cut at sites closed to recognition site

51. Restriction Enzyme
There are already more than 1200 type II enzymes isolated from prokaryotic organism
They recognize more than 130 different nucleotide sequence
They scan a DNA molecule, stopping only when it recognizes a specific sequence of nucleotides that are composed of symetrical, palindromic sequence
Palindromic sequence:
The sequence read forward on one DNA strand is identical to the sequence read in the opposite direction on the complementary strand
To Avoid confusion, restriction endonucleases are named according to the following nomenclature

52. Nomenclature
The first letter is the initial letter of the genus name of the organism from which the enzyme is isolated
The second and third letters are usually the initial letters of the organisms species name. It is written in italic
A fourth letter, if any, indicates a particular strain organism
Originally, roman numerals were meant to indicate the order in which enzymes, isolated from the same organisms and strain, are eluted from a chromatography column. More often, the roman numerals indicate the order of discovery

53. Nomenclature EcoRI E : Genus Escherichia co: Species coli
R : Strain RY13
I : First endonuclease isolated
BamHI
B : Genus Bacillus
am: species amyloliquefaciens
H : Strain H
I : First endonuclease isolated
HindIII
H : Genus Haemophilus
in : species influenzae
d : strain Rd
III : Third endonuclease isolated

54. Specificity Enzyme Source Sequence End BamHI
Bacillus amyloliquefaciens H
GGATCC
Sticky
BglII
Bacillus globigii
AGATCT
EcoRI
Escherichia coli RY13
GAATTC
EcoRII
Escherichia coli R245
CCTGG
HaeIII
Haemophilus aegyptius
GGCC
Blunt
HindII
Haemophilus influenzae Rd
GTPyPuAC
HindIII
AAGCTT
HpaII
Haemophilus parainfluenzae
CCGG
NotI
Nocardia otitidis-caviarum
GCGGCCGC
PstI
Providencia stuartii 164
CTGCAG

55. Restriction enzymes
Restriction enzymes can be grouped by:
number of nucleotides recognized (4, 6,8 base-cutters most common)
kind of ends produced (5’ or 3’ overhang (sticky), blunt)
degenerate or specific sequences
whether cleavage occurs within the recognition sequence
Become familiar with the back of your molecular biology catalog!

56. A restriction enzyme (EcoRI)
1. 6-base cutter
2. Specific palindromic sequence (5’GAATTC)
3. Cuts within the recognition sequence (type II enzyme)
4. produces a 5’ overhang (sticky end)

57. Restriction enzymes

58. Plasmids that can be modified to carry new genes
Cloning Vectors
Plasmids that can be modified to carry new genes
Plasmids useful as cloning vectors must have
a replicator (origin of replication)
a selectable marker (antibiotic resistance gene)
a cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers

61. A typical plasmid vector with a polylinker

62. Named for mythological beasts with body parts from several creatures
Chimeric Plasmids
Named for mythological beasts with body parts from several creatures
After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted
Ends of the plasmid/fragment are closed to form a "recombinant plasmid"
Plasmid can replicate when placed in a suitable bacterial host

65. Often one desires to insert foreign DNA in a particular orientation
Directional Cloning
Often one desires to insert foreign DNA in a particular orientation
This can be done by making two cleavages with two different restriction enzymes
Construct foreign DNA with same two restriction enzymes
Foreign DNA can only be inserted in one direction

67. Promoter A nucleotide sequence within an operon
Lying in front of the structural gene or genes
Serves as a recognition site and point of attachment for the RNA polymerase
It is starting point for transcription of the structural genes
It contains many elements which are involved in producing specific pattern and level of expression
It can be derived from pathogen, virus, plants themselves

68. Types of Promoter
Promoter always expressed in most tissue (constitutive)
-. 35 s promoter from CaMV Virus
-. Nos, Ocs and Mas Promoter from bacteria
-. Actin promoter from monocot
-. Ubiquitin promoter from monocot
-. Adh1 promoter from monocot
-. pEMU promoter from monocot
Tissue specific promoter
-. Haesa promoter
-. Agl12 promoter
Inducible promoter
-. Aux promoter
Artificial promoter
-. Mac promoter (Mas and 35 s promoter)

69. easy to visualise or assay
Reporter gene
easy to visualise or assay
- ß-glucuronidase (GUS) (E.coli)
green fluorescent protein (GFP) (jellyfish)
luciferase (firefly)

70. GUS
Cells that are transformed with GUS will form a blue precipitate when tissue is soaked in the GUS substrate and incubated at 37oC
this is a destructive assay (cells die)
The UidA gene encoding activity is commonly used. Gives a blue colour from a colourless substrate (X-glu) for a qualitative assay. Also causes fluorescence from Methyl Umbelliferyl Glucuronide (MUG) for a quantitative assay.

71. GUS Bombardment of GUS gene - transient expression
Stable expression of GUS in moss
Phloem-limited expression of GUS

72. HAESA gene encodes a receptor protein kinase that controls floral organ abscission. (A) transgenic plant expressing a HAESA::GUS fusion. It is expressed in the floral abscission zone at the base of an Arabidopsis flower.
Transgenic plants that harbor the AGL12::GUS fusions show root-specific expression.

73. Inducible expression

74. GFP (Green Fluorescent Protein)
Fluoresces green under UV illumination
Problems with a cryptic intron now resolved.
Has been used for selection on its own.
GFP glows bright green when irradiated by blue or UV light
This is a nondestructive assay so the same cells can be monitored all the way through

75. GFP mass of callus colony derived from protoplast protoplast
regenerated plant

76. Selectable Marker Gene
let you kill cells that haven’t taken up DNA- usually genes that confer resistance to a phytotoxic substance
Most common:
antibiotic resistance
kanamycin, hygromycin
2. herbicide resistance
phosphinothricin (bialapos); glyphosate

77. Only those cells that have taken up the DNA can grow on media containing the selection agent

78. Gene of interest
Sequence of DNA which will be inserted to the host cell and its product will be studied or beneficial for mankind
Origin of gene interest:
Non plant genes
Plant genes

79. pathogen-derived genes
bacterial genes
any other organism
Exogenous genes (non-plant genes)
Enzymes in biochemical pathway
Natural resistance genes
Endogenous genes (Plant genes)

80. The desired DNA produced by the above methods can then be introduced into a host cell by a number of methods.
The desired DNA can be inserted into cloning vectors such as plasmids and viral genomes. The desired DNA can be spliced into isolated plasmids using restriction endonuclease enzymes.
The desired DNA can be spliced into the genome of viruses capable of inserting their genome into the chromosomes of the host cell, such as modified retroviruses or temperate bacteriophages. Infection of the host cell then allows for the insertion of the viral genome (now containing the "desired" DNA) into the host cell's DNA.

81. The desired DNA can be introduced into plant cells by protoplast fusion. With protoplast fusion, the plant cell wall is enzymatically removed to create protoplasts. Polyethylene glycol is then used to enable the protoplasts to fuse together.
The desired DNA can be introduced into cells by microinjection and electroporation. In the case of microinjection, micropipettes are used to inject the DNA into the cell's cytoplasm. With electroporation, high voltage electrical impulses are used to destabilize the cytoplasmic membrane and permit entry of DNA.
The desired DNA can be introduced into plant cells by Agrobacterium or using gene guns. With agrobacterium, the plant cell is cocultivated with agrobacterium for a second. Agrobacterium will integrate the gene of interest to plant chromosome. Gene guns use helium bursts to propel microscopic gold or tungsten particles coated with DNA through plant cell walls.

83. T-DNA
binary
vector
A. tumefaciens