1. There are two main methods of gene transfer - direct and indirect gene transfer. Indirect transfer uses Agrobacterium-mediated transformation while direct transfer uses physical or chemical methods. 2. Agrobacterium-mediated transformation uses Agrobacterium tumefaciens to transfer T-DNA containing the gene of interest into the plant genome. The process involves co-cultivation of plant explants with Agrobacterium followed by selection and regeneration of transgenic plants. 3. Direct physical methods include biolistic transformation, microinjection, electroporation, and macroinjection. Direct chemical methods include PEG-mediated, calcium phosphate co-precipitation, and liposome-mediated transformation

1. Methods of Gene Transfer
Prepared By,
Prof. T. A. Pagar
Dept of PHFBT,
K. K Wagh College of Agricultural Biotechnology, Nashik.
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2. Transformation
• Gene transfer is the uptake of foreign DNA or
transgene by plant cells.
• It is the subsequent stable integration & expression of
a foreign DNA into the genome.
• The method used for gene transfer is known as
transformation.
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3. Methods of Transformation
• There are mainly 2 methods of gene transfer:
1. Indirect or Agrobacterium-mediated gene transfer
Gene transfer is done by using the bacteria
Agrobacterium tumificiens.
2. Direct gene transfer
By using various techniques (listed on next page) the
gene is directly transferred into the host.
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Methods of Genetic Transformation
Indirect Method of
Gene Transfer
Direct Method of Gene
Transfer
Agrobacterium
mediated gene
transfer
Physical
Electroporation
Biolistic
Macroinjection
Microinjection
Liposome
mediated
Chemical
PEG mediated
DMSO polycation
DEAE Dextran
Calcium phosphate
co-precipitation

5. Agrobacterium mediated gene transfer
• Agrobacterium is soil borne, gram negative, rod shaped,
motile found in rhizosphere.
• Causative agents of “Crown gall” in dicotyledons.
• 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
• Contain a vir region ~ 40 kb at least 8-11 vir genes
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7. T-DNA
• Size 12 – 24 kb
• Left and right border sequence (24-bp) which will be
transferred into genome of host plant
• Oncogenes e.g. Auxin, cytokinin, opines
• The T-DNA contains eight potential genes - these are
eukaryotic in nature (eukaryotic promoters,
monocistronic, eukaryotic polyadenylation signals,
eukaryotic translation mechanisms)
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8. Process of T-DNA transfer and integration
 Identify a suitable explants:
• Suitable plant tissue is removed and sterilized.
 Co-cultivate with the Agrobacterium:
• Small pieces of leaf tissue placed into a culture of
Agrobacterium for about 30 mins.
• The explants then placed on MS medium without
selective agent.
• Incubate explants with Agrobacterium for 2 days to
allow transfer of the T-DNA.
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9.  Kill the Agrobacterium with a suitable antibiotic:
• The explants are removed from the medium and washed in
cefotaxime.
 Select for transformed plant cells:
• The explant are transferred to a selective (kanamycin)
medium with cefotaxime.
• Auxin, Cytokinin are used to encourage the regeneration
of by organogenesis.
 Regeneration of whole plant:
• The shoot can be rooted by placing them on solid medium
containing a high auxin to cytokinin ratio.
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10. Electroporation
• 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.
• It can be used to deliver DNA into plant cells and
protoplasts.
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11. • There are two systems of electroporation
1. Low voltage – Long pulses
▫ 300-400 V cm-1 for 10-50 ms
▫ Produce high rates of transient transformation
2. High voltage – Short pulses
▫ 1000-1500 V cm-1 for 10μs
▫ Produce high rates of stable transformation
 Transformation frequency can be improve
 A prior heat shock treatment to protoplast
 Presence of low conc. PEG (8%)
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12. Advantages:
• Both intact cells and tissue can be transformed.
• The efficiency of transformation depends upon the plant
materials
Disadvantages
• ~40 to 50% incubated cells receive DNA
• ~50% of the transformed cells can survive
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13. Microinjection
A holding pipette holds the protoplast while an injection
pipette injects the macromolecule.
 For manipulation of protoplasts without damage,
protoplasts are cultured about 1 to 5 days before the
injection.
The injection through the partially regenerated cell wall
facilitates to target particular compartments of the cell.
Particularly useful for transformation of plant
protoplasts with exogenous genes.
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15. Macroinjection
• Macroinjection is the method tried for
artificial DNA transfer to cereals plants
that show inability to regenerate and
develop into whole plants from cultured
cells.
• Needles used for injecting DNA are with
the diameter greater than cell diameter.
• DNA injected with conventional syringe
into region of plant which will develop
into floral tillers
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16. Advantages
• This technique does not require protoplast.
• Instrument is simple and cheap.
• Methods may prove useful for gene transfer into cereals
which do not regenerate from cultured cell easily.
• Technically simple.
Limitations
1. Less specific.
2. Less efficient.
3. Frequency of transformation is very low.
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17. Biolistic Method
• Firstly used by Klein et al (1987) & Sanford et al (1987).
• Also called as,
• Ballistic method / Gene gun method / Particle bombardment
/ Particle gun method / Microprojectile
• Gene gun is developed to enable penetration of the genetic
material containing a gene of interest in the cell wall .
• 1-2µm tungsten or gold particles are used, coated with the
DNA.
• Acceleration is given to enter the micro-projectiles into the
plant cells.
• Acceleration achieved by using a device varies in design &
function.
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18. Devices
1. Pressurized helium gas
or
2. The electrostatic energy
released by a droplet of
water exposed to high
voltage.
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19. Advantages
• This method can be use to transform all plant species.
• No binary vector is required.
• Transformation protocol is relatively simple.
Disadvantages
• 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.
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20. Liposome mediated gene transfer
• Liposomes are spheres of lipids used to transport
molecules into the cells.
• These are artificial vesicles that can act as delivery
agents for exogenous materials including transgenes.
• They are considered as sphere of lipid bilayers
surrounding the molecule to be transported and
promote transport after fusing with the cell
membrane.
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21. • Cationic lipids are those having a positive charge are used for
the transfer of nucleic acid.
• Liposomes are able to interact with the negatively charged cell
membrane more readily than uncharged liposomes
• Due to fusion between cationic liposome and cell surface
results in the delivery of DNA directly across the plasma
membrane.
• Cationic liposomes can be produced from a number of cationic
lipids, e.g. DOTAP and DOTMA.
• These are commercially available lipids that are sold as an in
vitro transfecting agent, as lipofectin.
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22. Advantages
• High degree of reproducibility.
• Long term stability.
• Low toxicity.
• Protection of nucleic acid from degradation.
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24. PEG mediated gene transfer
• Polyethylene glycol (PEG), in the presence of divalent
cations (using Ca2+), destabilizes the plasma membrane
of protoplasts and renders it permeable to naked DNA.
In this way, the DNA enters nucleus of the protoplasts
and gets integrated with the genome.
• Culture of protoplasts is taken into a tube and to this
tube 40% PEG 4000 (w/v) dissolved in mannitol and
calcium nitrate is added slowly.
• Then incubated for few min.
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25. Advantages
• A large number of protoplasts can be simultaneously
transformed.
• Can successfully use for a wide range of plant species.
Limitations
• The DNA is susceptible for degradation and
rearrangement.
• Random integration of foreign DNA into genome may
result in undesirable traits.
• Regeneration of plants from transformed protoplasts is a
difficult task.
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26. Calcium Phosphate co-precipitation
• The DNA is allowed to mix with calcium chloride solution and
isotonic phosphate buffer to form DNA-calcium phosphate
precipitate.
• When the actively dividing cells in culture are exposed to this
precipitate for several hours, the cells get transformed.
• The success of this method is dependent on the high
concentration of DNA and the protection of the complex
precipitate.
• Addition of dimethyl sulfoxide (DMSO) increases the
efficiency of transformation.
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27. DNA Imbibition By Cells/Tissues:
• Some workers have seriously tried to transform cells by
incubating cell suspensions, tissues, embryos and even
seeds with DNA. The belief is that the DNA gets imbibed,
and the cells get transformed. DNA imbibition approach
has met with little or no success.
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28. DMSO polycation
• It involves use of a polycation, polybrene, to increase the
absorption the absorption of DNA to the surface followed by a
brief treatment by 25-30% DMSO to increase the membrane
permeability and enhance the uptake.
• The major advantage of polybrene is that it is less toxic than
other polycations and a high transformation efficiency
requires very small quantities of plasmid DNA to be used.
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29. DEAE Dextran
• The desirable DNA can be complexed with a high
molecular weight polymer diethyl amino ethyl (DEAE)
dextran and transferred.
• The efficiency increased to 80% when DMSO shock is
given.
• The major limitation of this approach is that it does not
yield stable transformants.
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