Methods of plant transformation

and direct gene transfer

Khushpreet Singh
B tech Biotech ‘09
Thapar University Patiala INDIA
 Plant Transformation
 The genetic manipulation of plants has been an ongoing
science since prehistoric times, when early farmers along the
Euphrates began carefully selecting and maintaining seed from
their best crops to plant for the next season. Early Americans
also bred plants, and modern corn is a result of thousands of
years of genetic manipulation.
 With the advent of recombinant DNA technology in the 1970s,
the genetic manipulation of plants entered a new age. Genes
and traits previously unavailable through traditional breeding
became available through DNA recombination, and with greater
specificity than ever before.
 Genes from sexually incompatible plants, or from animals,
bacteria or insects can now be introduced into plants. Products
of modern plant genetic engineering are already on the market
in various regions of the U.S. Examples include a slow-softening
tomato and cotton plants resistant to herbicides and insects.
With many more products in the pipeline, the genetic
engineering of plants will have a profound impact on the future
Keys
• DNA must be moved into nucleus
• DNA must be integrated into the genome
• DNA must be stable
• Tissue culture techniques are needed for
– Preparation of cells amenable to receive
DNA
– Selecting transgenic cells and tissues
– Regenerating transgenic plants
– Transgene(s) inherited in Mendelian fashion
Formula
Tissue culture + DNA delivery
and
 Plant Transformation using
Agarobacterium tumefaciens
 Modern plant genetic engineering involves the transfer of the
desired genes into the plant genome, and then regeneration of a
whole plant from the transformed tissue.
 Currently, the most widely used method for transferring genes into
plants is Agrobacterium-mediated transformation.
 Agrobacterium is a naturally occurring pathogenic bacteria in the
soil that has the ability to transfer its DNA into a plant's genome.
 Agrobacterium infection and gene transfer normally occurs at the
site of a wound in the plant, and causes a characteristic growth
referred to as a crown gall tumor.
 Scientists have taken advantage of this naturally occurring transfer
mechanism, and have designed DNA vectors from the tumor-
inducing plasmid DNA found in the bacteria that are capable of
carrying desired genes into the plant.
 The engineered or constructed genes are inserted into the
Agrobacterium vectors and enter the plant by the bacteria's own
internal transfer mechanisms.
 Transformation is typically done on a small excised portion of a
plant known as an explant. This small piece of transformed plant
tissue is then regenerated into a mature plant through tissue
culture techniques.
 Agrobacterium tumefaciens
Agrobacterium
tumefaciens is the
causal agent of
Crown Gall disease
(the formation of
tumours) in over 140
species of dicot. It is
a rod shaped,
Gram negative soil
bacterium.
Symptoms are
caused by the
insertion of a small
segment of DNA
(known as the T-DNA,
Causative agent of crown gall
disease
Transformation by A.
tumefaciens
If a plant is wounded or damaged, A.
tumefaciens can infect the at the wound
site.
Crown gall formation depends on the
presence of a plasmid in A. tumefaciens
known as Ti plasmid.
Part of this plasmid is actually transferred
from the bacterium into the plant cell, where
it integrates into the host genome.
The T-DNA is involved in the biosynthesis of-
Plant hormones (auxins and cytokinins).
Novel plant metabolites (opines and
agropines).
The Ti plasmid
Ti plasmid has a central role in crown gall
formation and it is the portion of Ti plasmid
that is integrated into the host genome.
This Ti plasmid is responsible for the
tumorous phenotype.
Ti plasmid features-
They contain one or more T-DNA regions.
They contain a vir region.
They contain an origin of replication.
They contain a region enabling conjugative
transfer.
They contain genes for the catabolism of
opines(a class of amino acid conjugates).
The T-DNA
 The T-DNA region of any Ti plasmid is defined by the
presence of the right and the left border sequences.
 These border sequences are 24 bp imperfect
repeats.
 Any DNA between the borders will be transferred into
the genome of the plant.
 The oncogenes:-
 Two genes auxA and auxB encode proteins involved in
the production of auxins, similarly gene cyt for cytokinin
production which are the prime determinants of the
tumour phenotype.
 Other genes :-
 The tml gene which is involved in determining tumor
size is found in some species, is also found in the T-
DNA.
 Genes responsible for the T-DNA transfer are also
situated on the Ti plasmid.
Key steps from natural
Agrobacterium to “useful
• Some vir genes deleted--disarmed
– Opines not going to be produced
– Deleting tumorogenesis function
• Choosing strains that transfer DNA in lab
• Clone in genes of interest, antibiotic
resistance genes, etc.
• Binary system-- two plasmids are better
than one Ti plasmid
 The process of T-DNA transfer
and integration
Agrobacteriumcontains a tumour-inducing (Ti)
plasmid, which includes virulence (vir) genes and a
transferred-DNA(T-DNA) region.
Genes of interest can be inserted into the T-DNA.
Wounded plant cells produce phenolic defence
compounds, which can trigger the expression of the
Agrobacterium virgenes. The encoded virulence
(Vir) proteins process the T-DNA
region from the Ti-plasmid, producing a 'T-strand'.
After the bacterium attaches to a plant cell, the T-
strand and several types of Vir proteins are
transferred to the plant through a transport channel.
Inside the plant cell, the Vir proteins interact with the
T-strand, forming a T-complex.
 The process of T-DNA transfer
and integration
 Step 1. Signal recognition by Agrobacterium.
 Step 2. attachment to plant cells.
 Two step process, attachment via polysaccharide and
subsequently by a mesh of cellulose fibers produced by bacterium.
 Step3. induction of vir genes.
 Vir A senses phenolics, autophosphorylates and subsequently
phosphorylates Vir G which induces the induction of all the vir
genes.many sugars enhance the vir genes induction.
 Step 4. T-strand production.
 the left and right borders are recognized by VirD1-VirD2 complex
and Vir D2 becomes covalently attached to 5’ end and displaces
single stranded T-DNA strand .
 Step 5. transfer of T-DNA out of the bacterial cell.
 Done by a T-pilus(membrane channel secretory system) composed
of proteins encoded by virB operon and Vir D4. Vir E2 and Vir F are
exported from bacterial cell.
 Step 6. transfer of T-DNA and Vir proteins into the
plant cell and nuclear localization.
 TDNA-VirD2 complex and other vir proteins cross the plant plasma
Desirable features of any plasmid
vector
Be of a small size. Small plasmids are easy
to handle invitro as they are less liable to
damage by shearing.
There is also less chance of the vector
having other sites for restriction
enzymesmaking the design and
integrationof a MCS simple.
Confer a selectable phenotype on the hist
cell so that they can be selected for like
antibiotic resistant gene.
Contain single site for a large number of
restriction enzymes to enable the
DIRECT GENE
BIOLISTIC GUN
ELECTROPORATION
TRANSFORMATION USING SILICON CARBIDE
FIBERS
PEG MEDIATED TRANSFER
Direct gene transfer
Advantage:
 This method can be use to transform all plant
species.
 No binary vector is required.
 Transformation protocol is relatively simple.

Disadvantage:
 Difficulty in obtaining single copy transgenic events.
 High cost of the equipment and microcarriers.
 Intracellular target is random (cytoplasm, nucleus,
vacuole,
plastid, etc.).
 Transfer DNA is not protected.
 Gene Delivery System
Biolistic-mediated transformation
Also known as:
 Particle Bombardment
 Biolistics
 Microprojectile bombardment
 Particle acceleration
 Particle inflow gun
 Gene gun
 Using a gene gun directly shoots a
piece of DNA into the recipient plant
tissue.

 Tungsten or gold beads are coated in

the gene of interest and fired
through a stopping screen,
accelerated by Helium, into the plant
tissue. The particles pass through
the plant cells, leaving the DNA
Overview of the process
Mechanism
Equipment
DNA-coated microcarriers are
loaded on microcarrier.
Micro-carriers are shot
towards
target tissues during helium
gas
decompression.
A stopping screen placed
allowing
the coated microprojectiles
to
pass through and reach the
Particle bombardment of Vicia faba
cotyledons
Biolistic Transformation
parameters
A number of parameters has been
identified and need to be considered
carefully in experiments involving
particle bombardment
Parameter categories:
- Physical parameters
- Biological parameters
- Environmental parameters
 Physical parameters
 Nature, chemical and physical properties of the metal particles used as
a macrocarrier for the foreign DNA
 Particles should be high enough mass in order to possess adequate
momentum to penetrate into appropriate tissue.

 Suitable metal particles include gold, tungsten, palladium,

rhodium,platinum and iridium.

 Metals should be chemically inert to prevent adverse reaction with DNA

and cell components.

 Nature, preparation and binding of DNA onto the particles

 The nature of DNA (single vs double stranded, circular vs linerized DNA).
Optimal: double stranded circular DNA molecules (e.g. plasmid)
 In the process of coating the metal particls with DNA certain additives
such as spermididne and CaCl 2 appear to be useful.
 Target tissue
 It is important to target the appropriate cells that are competent for both
transformation and regeneration.
 Depth of penetration is one of the most important variables in order to
achieve particle delivery to particular cells.
Biological parameters

-Biological parameters
Temperature, photoperiod and humidity
These parameters have a direct effect on the
physiology of tissues.
Such factors will influence receptiveness of
target tissue to foreign DNA delivery and also
affect its susceptibility to damage and injury
that may adversely affect the outcome of
transformation process.
Some explants may require a “healing” period
after bombardment under special regiments of
light, temperature, and humidity.
 PROTOPLAST
ELECTROPORATION
 Protoplasts are cells
stripped of their cell
walls and maintained in
culture
 Electroporation, or
electropermeabilizati
on, is a significant
increase in the
electrical conductivity
and permeability of the
cell plasma membrane
caused by an externally
applied electrical field.
 The electroporation of
cells can be used to
 The vectors used
can be simple
plasmids.
 Material is
incubated in buffer
solution containing
DNA & subjected to
high voltage
electrical pulse.
 DNA migrates
through high
voltage induced
pores in plasma
membrane and
integrates into the
genome.
 Plant material used
for electroporation
require specific
treatment such as
pre and post
The vectors used can be simple plasmids.
Material is incubated in buffer solution
containing DNA & subjected to high voltage
electrical pulse.
DNA migrates through high voltage induced
pores in plasma membrane and integrates
into the genome.
Plant material used for electroporation
require specific treatment such as pre and
post electroporation incubation in buffers of
high osmotic pressure.
Advantages of protoplast
Electroporation

Amount of DNA deliverd to each cell is low

which has a benefit of producing
transformants with a low transgene copy
number.

All the cells are in the same physiological

state after transformation unlike the
situation with particle bombardment where
damage can occur from transformation.
Transformation by Silicon carbide
fibres : WHISKERS™
Transformation by Silicon carbide
fibres : WHISKERS™(contd.)

This technique requires no special

equipment.
Plant material is introduces into a buffer
containing DNA and silicon carbide fibres,
which is then vortexed.
The fibre penetrates the cell wall and
plasma membrane allowing DNA to gain
access to the inside of the cell.
Drawbacks
Related to availability of suitable plant
materials.
Fibres require careful handling.
This procedure has been utilized for friable
callus for maize and is limited only to few
genotypes of maize and oats.
Many cereals ,the natural targets for the
procedure produce embryogenic callus that
is hard and compact and is not easily
transformed at present.
Microinjection
• The microinjection
method uses a fine
needle to inject a
solution of DNA into
a cell.
 Transformation with
Protoplasts
Microinjection
 Protoplasts attached to
slide by embedding in
agarose using a
holding pipet
 DNA injected using
injection pipet
PEG mediated transformation
Polyethylene Glycol
Transformation of naked
DNA done by treatment
with PEG in presence of
divalent cations.
PEG and divalent
cationsdestabilize the
plasma membrane of
plant protoplast and
render it permeable to Figure: GUS staining of
naked DNA. PEG-transformed
protoplasts derived
DNA then enters the from roots of
Arabidopsis ecotype
Columbia after
Advantages
The PEG-mediated transformation is
simple and efficient, allowing a simultaneous
processing of many samples.
Yields a transformed cell population with
high survival and division rates
Method utilizes inexpensive supplies and
equipments,
Helps to overcome a hurdle of host range
limitations of Agrobacterium-mediated
transformation.
The PEG-mediated DNA transfer can be
readily adapted to a wide range of plant
Disadvantages
Plant protoplasts are not easy to work with,
and the regeneration of fertile plants from
protoplasts is problematic for some species.
The DNA used is also susceptible to
degradation and rearrangement
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