Transcript Plant Genetic Engineering Genetic Engineering The process of manipulating and transferring instructions carried by genes from one cell to another Why do scientists.

Plant Genetic Engineering
Genetic Engineering
The process of manipulating and transferring instructions
carried by genes from one cell to another
Why do scientists want to change gene
instructions?
 to produce needed chemicals
 to carry out useful processes
 to give an organism desired characteristics
DNA IS
EVERYWHERE
Plant Genetic Engineering
Process
Cell
Plant cell
Extracted DNA
A single
gene
Transformation
Transgenic plant
Cell division
Why Plants?
Plants are also very flexible and can produce a wide
variety of proteins.
 Crop plants can synthesize a wide variety of
proteins that are free of mammalian toxins and
pathogens.
 Crop plants produce large amounts of biomass at
low cost and require limited facilities.
 Crops are therefore well suited for the production of
safe low-cost proteins

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
Transformation methods
DNA must be introduced into plant cells
Indirect
-Agrobacterium tumefaciens
Direct
Microprojectile bombardment
Electroporation
 Microinjection
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
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
Agrobacterium tumefaciens
Agrobacterium
Plant cell
Genomic DNA
Genomic DNA
Ti plasmid
(carries the gene of
interest)
Restriction
enzyme A
Restriction
enzyme A
+
Empty
plasmid
Gene of
interest
Ti plasmid with the gene of interest
Agrobacterium tumefaciens
Ti plasmid with the new gene
cell’s
DNA
+
Agrobacterium
Transformation
Plant cell
The new
gene
Transgenic plant
Cell division
T-DNA
binary
vector
A. tumefaciens
Factor determining the success
Species
 Genotypes
 Explant
Agrobacterium strains
 Plasmid


Direct gene transfer
Introducing gene directly to the target cell
1. Electroporation
2. Microinjection
3. Particle Bombardment
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
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
“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


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 (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
Selection Strategies
Positive
 Negative
 Visual

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
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
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