TRANSGENIC TECHNOLOGY
Traits that plant breeders
would like in plants
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High primary
productivity
High crop yield
High nutritional
quality
Adaptation to intercropping
Nitrogen Fixation
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Drought resistance
Pest resistance
Adaptation to
mechanised farming
Insensitivity to
photo-period
Elimination of toxic
compounds
Plant transformation
getting DNA into a cell
getting it stably integrated
getting a plant back from the cell
Requirement
1. a suitable transformation method
2. a means of screening for transformants
3. an efficient regeneration system
4. genes/constructs
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Vectors
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Promoter/terminator
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reporter genes
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selectable marker genes
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‘genes of interest’
Transformation methods
DNA must be introduced into plant cells
Indirect
- Agrobacterium tumefaciens
Direct
- Microprojectile bombardment
- Electroporation
- Polyethylene glycol (PEG)
- Glass-beads
- Silicon carbide whiskers
Method depends on plant type, cost, application
Agrobacterium-mediated
transformation
Transformation by the help of agrobacterium
Agrobacterium is a ‘natural genetic engineer’
i.e. it transfers some of its DNA to plants
Agrobacterium
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A natural genetic engineer
2 species
• A.tumefaciens (produces a
gall)
• A. rhizogenes (produces
roots)
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Oncogenes (for auxin and
cytokinin synthesis) +
Opines
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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.
Agrobacterium tumefaciens
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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
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
Agrobacterium 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  10-14 days later, tumor forms
What is naturally encoded in T-DNA?
•Enzymes for auxin and cytokinin synthesis
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Causing hormone imbalance  tumor formation/undifferentiated callus
Mutants in enzymes have been characterized
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•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
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Ti plasmid of A. tumefaciens
Ti plasmid of A. tumefaciens
1. Auxin, cytokinin, opine
synthetic genes
transferred to plant
2. Plant makes all 3
compounds
3. Auxins and cytokines
cause gall formation
4. Opines provide unique
carbon/nitrogen source
only A. tumefaciens can
use!
Agrobacterium tumefaciens
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How is T-DNA modified to allow genes of
interest to be inserted?
• In vitro modification of Ti plasmid
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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
Agrobacterium and genetic engineering:
Engineering the Ti plasmid
Co-integrative and binary vectors
LB
RB
Co-integrative
Binary vector
Expose wounded plant cells to transformed
agro strain
Electroporate TDNA vector into
Agrobacterium
and select for tetr
Induce plant regeneration and select
for Kanr cell growth
Factor determining the success
Species
 Genotypes
 Explant
Agrobacterium strains
 Plasmid
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Direct gene transfer
Introducing gene directly to the target cell
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
Microprojectile bombardment
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.
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Model from BioRad:
Biorad's Helios Gene
Gun
“Gene Gun” Technique
DNA coated
golden particles
Gene gun
Cell’s DNA
Plant cell
A plant cell with
the new gene
Transgenic plant
Cell division
Electroporation
Explants: cells and protoplasts
Most direct way to introduce foreign DNA into the nucleus
Achieved by electromechanically operated devices
Labour intensive and slow
Transformation frequency is very high, typically up to ca. 30%
Power supply
Electroporation
Technique
Plant cell
Duracell
Protoplast
DNA containing
the gene of interest
DNA inside the
plant cell
The plant cell with
the new gene
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%
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
Competent cells
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
Screening technique
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
Screening
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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 screening agent like antibiotic resistance.
The NptII gene encoding Neomycin phospho-transferase II
phosphorylates kanamycin group antibiotics and is
commonly used.
Screening
Investigating based on a large number of organism for
the presence of a particular property as in screening
for a mutation for antibiotic resistance
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Screen at the level of the intact plant
Screen 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
Selection methods
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The most common procedure was mass-selection which in turn was
subdivided into negative and positive
Negative selection
The most primitive and least widely used method which can lead to
improvement only in exceptional cases
It implies culling out of all poorly developed and less productive
individuals in a population whose productivity is to be genetically
improved
The remaining best individuals are propagated as much as necessary
Positive selection
Only individuals with characters satisfying the breeders are selected from
population to be used as parents of the next generation
Seed from selected individuals are mixed, then progenies are grown
together
Selection Strategies
Positive
 Negative
 Visual
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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
Negative selection
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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.
Visual selection
Only useful for colored or fluorescent compounds
Plate out at about 50,000 cells per plate.
Pick off colored / fluorescent compounds
Possible to screen about 1,000,000 cells in a days work.
Positive and Visual Selection
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
Regeneration
Plant tissue culture uses growth regulators and nutrients to
regenerate plants in vitro
Regeneration of shoots from leaf protoplasts in Arabidopsis thaliana
Somatic embryogenesis in peanut
Organogenesis
Gene construct
BamHI
P SAG12 ipt
nptII
LB T 35S
P 35S
gus-intron
T nos T 35S
P 35S
RB
Cloning Vectors
Plasmids that can be modified to carry new genes
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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
A typical plasmid vector with a
polylinker
Chimeric Plasmids
Named for mythological beasts with body parts from
several creatures
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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
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
Promoter
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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
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)
Reporter gene
easy to visualise or assay
- ß-glucuronidase (GUS)
(E.coli)
-green fluorescent protein (GFP)
(jellyfish)
- luciferase
(firefly)
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.
GUS
Bombardment of GUS gene
- transient expression
Stable expression of
GUS in moss
Phloem-limited expression of GUS
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 rootspecific expression.
Inducible expression
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 non-destructive assay so the same cells can be
monitored all the way through
GFP
protoplast
colony derived from
protoplast
regenerated plant
mass of callus
Selectable Marker Gene
let you kill cells that haven’t taken up DNA- usually genes that confer
resistance to a phytotoxic substance
Most common:
1. antibiotic resistance
kanamycin, hygromycin
2. herbicide resistance
phosphinothricin (bialapos); glyphosate
Only those cells that have taken up
the DNA can grow on media
containing the selection agent
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:
1. Non plant genes
2. Plant genes
pathogen-derived genes
Exogenous genes
(non-plant genes)
bacterial genes
any other organism
Endogenous genes
(Plant genes)
Enzymes in biochemical pathway
Natural resistance genes