2. • In molecularbiology, transformation is genetic
alteration of a cell
resulting from the direct uptake, incorporation
and expression of exogenous genetic material
(exogenous DNA) from its surroundings and taken up
through the cell membrane(s).
3. • Transformation occurs naturally in some
species of bacteria, but it can also be affected
by artificial means in other cells. For
transformation to happen, bacteria must be in
a state of competence, which might occur as a
time-limited response to environmental
conditions such as starvation and cell density.
4. Techniques for plant genetic
transformation
• Indirect method- Agrobacterium mediated
gene transfer
• Direct methods-
– Particle bombardment (biolistics)
– Microprojectile gun method
– Electroporation
– Silicon carbide fibres
– Polyethylene glycol (PEG)/protoplast fusion
– Liposome mediated gene transfer
5. Agrobacterium mediated gene transfer
Agrobacterium-
– Soil borne, gram negative, rod shaped, motile found in
rhizosphere
– Causative agents of “Crown gall” disease of
dicoltyledones
– Have ability to transfer bacterial genes to plant genome
by nature.
– Attracted to wound site via chemotaxis in response to
chemicals (sugar and Phenolic molecules:
acetosyringone) released from damaged plant cells
– Contains Ti plasmid which can transfer its T-DNA region
into genome of host plants
7. Ti-plasmid features
• Two strains of Ti-plasmid:
-Octopine strains- contains two T-DNA region: TL (14 kb)
and TR ( 7 kb)
-Nopaline strains- contain one T-DNA region(20 kb)
• Size is about 200 kb
• Has a central role in Crown-gall formation
• Contains one or more T-DNA region that is integrated into
the genome of host plants
• Contain a vir region ~ 40 kb at least 8~11 vir genes
• Has origin of replication
• Contains a region enabling conjugative transfer
• Has genes for the catabolism of opines
9. 07 March 2002 Dr. Michael Parkinson 9
Gene construction
• Plant specific promoter
• Plant RBS
• Useful gene
• Signal peptides*
• PolyA-tail
DNA
mRNA
Polypeptide chain
transcription
translation
Post-translational
modification
Nucleus
Cytoplasm
10. 07 March 2002 Dr. Michael Parkinson 10
Plasmid construction
• Useful gene construct
• Visible marker
• Selectable marker*
11.
12. T-DNA
• Size~ 12 to 24 kb
• Left and right border sequence(24-bp) which will be
transferred into genome of host plant
• Oncogenes e.g Auxin, cytokinin, opines
• tm1 gene for determining the tumour size
• the T-DNA contains eight potential genes - these are
eukaryotic in nature (eukaryotic promoters, monocistronic,
eukaryotic polyadenylation signals, eukaryotic translation
mechanisms)
• crown gall tumorigenesis is due to the "activation" of
unregulated phytohormone synthesis in the transformed
cells
13.
14. Forms of T-DNA that are found in
Agrobacterium
•ds circles - found only in induced bacteria, not
(apparently) in plant cells•
•ds linear T-DNA - found only in induced bacteria, not
(apparently) in plant cells•
• ss linear T-DNA - found in bacteria and plant cells•
•what is not found - Ti plasmids with evidence that T-
DNA has been precisely deleted
15. Process of T-DNA transfer and integration
1. Signal recognition by Agrobacterium:
-Agrobacterium perceive signals such as sugar and phenolic
compounds which are released from plants
2. Attachment to plants cells:
Two step processes: i) initial attachment via polysaccharide ii)
mesh of cellulose fiber is produced by bacteria.
Virulence genes (chv genes) are involved in the attachment of
bacterial cells to the plants cells.
3. Vir gene induction:
VirA senses phenolics ans subsequently phosphorylating and
thereby activating VirG. VirG then induces expression of all
the vir genes.
4. T-strand production:VirD1/virD2 complex recognises the LB
and RB. virD2 produces single-stranded nicks in DNA. Then
16. 5. Transfer of T-DNA out of bacterial cells: T-DNA/VirD2 is
exported from the bacterial cell by “T-pilus” composed of
proteins encoded by virB operon and VirD2. VirE2 and VirF are
also exported from bacterial cells.
6. Transfer of the T-DNA and Vir proteins into the plant nuclear
localization: T-DNA/VirD2 complex and other Vir proteins
cross the plasma membrane through channels formed from
VirE2. VirE2 protect T-DNA from nucleases, facilitate nuclear
localization and confer the correct conformation to the T-
DNA/virD2 complex for passage through the nuclear pore
complex (NPC). The T-DNA/VirD2/VirF2 /plant protein
complex the nucleus through nuclear pore complex. And
integrated into host chromosome.
Process of T-DNA transfer and integration (continu)
17. Practical application of Agrobacterium-
mediated plant transformation
• 1. Agrobacterium mediated transformation methods
are thought to induce less rearrangement of the
transgene.
• Lower transgene copy number that direct DNA
delivery methods.
• Successful production of transgenic plants depends
on the suitable transformation protocols.
18. Agrobacterium-mediated transformation
of Tobacco
• Several factors have to be considered in the design and implementation
of any plant transformation study:
• -1. plant tissue to be transformed. The explant should be capable of
producing whole plant and should contain high number of cells that are
competent for transformation.
• 2. The vector used to deliver the transgene into the genome of plant.
Vector should Ti-plasmid binary vector which have LB and RB of Ti-
plasmid, bacterial selectable marker gene, selectable marker gene for
selection of transformed plant. and Multiple cloning sites.
• 3. Strain of Agrobacterium used: The choice of strain for crop plants is not
critical to the success of transformation but for recalcitrant plants, choice
of strains is a major factor to successful transformation. For dicotyledons
plants: LBA4404, GV3001 etc., For cereals ( which are not infected by
naturally infected) Supervirulent strain such as EHA101, EHA105 are used
19. The basic protocol used for any Agrobacteruim
mediated transformation experiments
1. Identify a suitable explants: Suitable plant tissue is removed
and sterilized. Leaf is used for Tobacco.
2. Co-cultivate with the Agrobacterium: Leaf tissue is cut into
small pieces and placed into a culture of Agrobacterium for
about 30 mins. The explants are subsequently removed from
the bacterial culture and placed on to the MS medium that
contain no selective agent. The incubation of explants with
Agrobacterium is allowed to continue for 2 days to allow
transfer of the T-DNA transfer to the plant cells.
3. Kill the Agrobacterium with a suitable antibiotic: The
explants are removed from the medium and washed in
antibiotic (cefotaxime) solution that kill Agrobacterium cells.
20. 4. Select for transformed plant cells: The explant are
transferred to fresh solid medium supplemented
with a selective agent (kanamycin). It also contains
cefotaxime. Auxin, Cytokinin are used to
encourage the regeneration of by organogenesis.
High cytokinin to auxin ratio promotes shoot
formation from the explants.
5. Regeneration of whole plant :The shoot can be
rooted by placing them on solid medium
containing a high auxin to cytokinin ratio.
The basic protocol used for any Agrobacteruim
mediated transformation experiments (Conti-)
21. Direct gene transfer methods
• The trem “Direct gene transfer” is used to discriminate
between the methods of plant transformation that rely on
Agrobacterium (indirect method) and those that do not
(direct methods). Direct gene transfer methods all rely on the
delivery of large amount of naked DNA whilst plant is
transiently permeabilised.
Direct methods-
Particle bombardment (biolistics)
Microprojectile gun method
Electroporation
Silicon carbide fibres
Polyethylene glycol (PEG)/protoplast
fusion
Liposome mediated gene transfer
22. Advantages and disadvantages of direct
gene transfer
• Adv- Widespread use of transformation of
cereal crops that initially proved difficult to
transformation with Agrobacterium.
• Disadv- They tend to lead higher frequency of
transgene rearrangement and higher copy
number. This can lead to high frequency of
gene silencing.
23. Particle bombardment
• Why Biolistics or Biolistic bombardment?
• Is the most powerful method for introducing nucleic acids into
plants, because the helium pressure can drive microcarriers
through cell walls
• Is much easier and less time consuming than microinjecting
nucleic acids into plant cells or embryos
• Allows transformation of animal cells that have unique growth
requirements and that are not amenable to gene transfer
using any other method
• Requires less DNA and fewer cells than other methods, and
can be used for either transient or stable transformation
24. Principle
• The gold or tungsten particles are coated with the DNA that is
used to be transform the plant tissue.
• The particles are propelled at high speed into the target plant
material where the DNA is released within then cell and can
integrate into the genome.
• Two types of plant tissues are used for particle bombardment:
• a) Primary explants that are bombarded and then induced to
become embryogenic
• b) Proliferating embryonic cultures that are bombarded and
then allowed to proliferate further and subsequently
regenerate.
•
25. PDS-1000/He bombardment System
Fig: Schematic representation of the PDS-1000/He
system upon activation. The arrows indicate the
direction of helium flow
Fig: The PDS-1000/He system, shown here
with magnified view of the Hepta adaptor.
26. How the PDS-1000/He System Works
• The sample to be transformed is placed in the bombardment chamber,
which is evacuated to subatmospheric pressure
• The instrument is fired; helium flows into the gas acceleration tube and is
held until the specific pressure of the rupture disk is reached
• The disk bursts, and the ensuing helium shock wave drives the
macrocarrier disk (which carries the coated microparticles) a short
distance toward the stopping screen
• The stopping screen retains the macrocarrier, while the microparticles
pass through the screen into the bombardment chamber and penetrate
the target cells
• The launch velocity of microcarriers depends on a number of adjustable
parameters: the helium pressure (rupture disk selection, 450–2,200 psi),
the amount of vacuum, the distance from the rupture disk to the
macrocarrier, the distance from the microcarrier launch assembly to the
stopping screen, and the distance between the stopping screen and target
cells. Adjusting these parameters allows you to produce a range of
velocities to optimally transform many different cell types.
27. Polyethylene glycol (PEG) mediated
transformation method
Plant protoplast can be transformed with naked DNA by
treatment with PEG in the presence of divalent cations e. g.,
Calcium.
PEG and divalent cations destabilize the plasma membrane of the
plant protoplast and rendered it permeable to naked DNA.
DNA enters the nucleus and integrates into the host genome.
Disadvantage and advantages:
Regeneration of fertile plants from protoplasts is a problematic
for some species.
The DNA used for transformation is also susceptible to
degradation and rearrangement.
Despite the limitations, the technique have the advantages
and protoplast can isolated and transformed in number of plants
species.
28.
29. Electroporation
• It can be used to deliver DNA into plant cells and protoplasts.
• The genes of interest require plant regulatory sequence.
• Plant materials is incubated in a buffer solution containing
DNA and subjected to high-voltage electric pulse.
• The DNA then migrates through high-voltage-induced pores in
the plasma membrane and integrates into the genome.
• It can be used to transform all the major cereals particularly
rice, wheat, maize.
• Advantages and disadvantages:
• Both intact cells and tissue can be transformed.
• The efficiency of transformation depends upon the plant
materials, electroporation and tissue treatment conditions
used for transformation.
• ~40 to 50% incubated cells receive DNA
• ~50% of the transformed cells can survive
30. Silicon carbide fibres-Whiskers
• Plant materials (Cells in suspension culture, embryos and embryo-
derived callus) is introduced into a buffer containing DNA and the
silicon fibers which is then vortexed.
• The fibers (0.3-0.6 μm in diameter and 10-100μm long) penetrate
the cell wall and plasma membrane, allowing the DNA to gain
access to the inside of the cells.
• Disadvantages and advantages
• The drawbacks of this technique relate to the availability of
suitable plant material and the inherent dangers of the fibers,
which require careful handing.
• Many cereals, produce embryonic callus that is hard and compact
and not easily transformed with this technique.
• Despite the some disadvantages, this method is recently used for
successful transformation of wheat, baerly, and maize without
the need to cell suspension.
31. Microinjection
Microinjection techniques for plant protoplasts utilize a holding
pipette for immobilizing the protoplast while an injection pipette is
utilized to inject the macromolecule.
In order to manipulate the protoplasts without damage, the
protoplasts are cultured for from about 1 to 5 days before the
injection is performed to allow for partial regeneration of the cell
wall.
It was found that injection through the partially regenerated cell
wall could still be accomplished and particular compartments of the
cell could be targeted.
The methods are particularly useful for transformation of plant
protoplasts with exogenous genes.
