Transcript Plant Genetic Engineering

Plant Genetic Engineering
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
- Chemical method
- Electrical method
- Physical methods
Chemical Method
1. Use of PEG (Polyethylene glycol (PEG)-mediated )
2. Protoplasts are incubated with a solution of DNA
and PEG
Electrical method
Electroporation (electropermeabilization)
Physical Methods
1. Particle bombardment
2. Microinjection
3. Silicon Carbide whiskers
Agrobacterium tumefaciens
Plant parasite that causes Crown Gall Disease
Encodes a large (~250kbp) plasmid called
Tumor-inducing (Ti) plasmid
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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
1. Then integrated into plant genome
2. 2 x 23bp direct repeats play an important role in
the excision and integration process
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
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Part of plasmid is inserted into plant DNA
Wound = entry point  10-14 days later,
tumor forms
Agrobacterium tumefaciens

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
• Opine synthesis genes (e.g. octopine or
nopaline)
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Carbon and nitrogen source for A. tumefaciens
growth
Insertion genes
• Virulence (vir) genes
• Allow excision and integration into plant genome
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 and genetic engineering:
Engineering the Ti plasmid
Co-integrative and binary vectors
LB
RB
Co-integrative
Binary vector
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%
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
Rupture disk
Pressure gauge
Disk with DNA-coated partic
Stop plate
Vacuum line
Gas line
Vacuum chamber
Sample goes here
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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.

Model from BioRad:
Biorad's Helios Gene
Gun
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%
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 nontransformed.
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.
Screening (selection)
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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
Selection Strategies
Positive
 Negative
 Visual
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Positive selection
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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
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
Transformation series of events
Callus formation
Transform
individual cells
Remove from sterile conditions
Auxins
Cytokinins
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 nondestructive assay so the same
cells can be monitored all the way through
GFP
protoplast
colony derived
from protoplast
mass of callus
regenerated plant
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
T-DNA
binary
vector
A. tumefaciens