2. GENE TRANSFER
Indirect gene transfer/
Agrobacterium mediated
Direct gene transfer/
Vector less gene transfer
1. Chemical methods- PEG
CaPO4 precipitation
DEAE-Dextran mediated
Liposome mediated
2. Physical methods- Electroporation
Microinjection
Gene gun
3. Imbibition
3. DIRECT GENE TRANSFER
Foreign gene of interest is delivered into the host plant cell
without the help of a vector.
Introduction of DNA into plant cells with out the
involvement of a biological agent, e.g., Agrobacterium
leading to stable transformation.
Stable method of gene transfer
Different chemical and physical treatments are employed
to facilitate the entry of DNA into plant cells
4.
5. • Protoplasts are treated with a solution containing various ions, PEG
and DNA. Changes in the plasma membrane allow the DNA to
penetrate and move into the cytoplasm.
• Whether PEG is directly involved in the delivery of DNA and the
mechanism of this process are still unclear
• The integration of the target DNA into the plastid chromosome is
site-directed due to the design of vectors with sequences
homologous to a specific target area in the plastid genome.
PEG MEDIATED GENE TRANSFER
6. Plant protoplasts are suspended in a transformation medium rich
in Mg2+
Linearized plasmid containing gene/DNA is added
PEG is added, adjust the PH about 8
Give 5 min heat shock at 45°C followed by transfer to ice
PROCEDURE
7. Advantages
• Yield more than 3% transformation
• More reliable and efficient
• Frequency of transformation is high, if we use 2-7% PEG
Disadvantages
• Plant cells are most sensitive to PEG
8. LIPOFECTION
Introduction of DNA into cells via liposomes
Transformation frequencies of 4Χ10-5
Plasmid DNA of 9kb and larger DNA can be transferred
Higher transformation frequencies with PEG and
electroporation make them more active
10. Advantages:
• High efficiency
• Its ease of use
• Reproducibility
• Low toxicity
Dis Advantages
Not applicable to all cell types
11. CALCIUM PHOSPHATE PRECIPITATION
• Mixing of DNA in phosphate buffer
solution, adding this in controlled manner
to a calcium chloride solution and
allowing the mixture to incubate at room
temperature
• This step generates a precipitate that is
dispersed onto the cultured cells
• The precipitate is taken up by the cells via
endocytosis or phagocytosis
14. DEAE-DEXTRAN-MEDIATED TRANSFECTION
• DEAE-Dextran (diethylaminoethyl-dextran) is water soluble and
polycationic
• Added to the transfection solution containing the DNA
• Brings about DNA uptake b the cells through endocytosis
• Interaction with –vely charged DNA and with the components of
cell surface plays an important role.
Disadvantage:
• Not efficient in producing stable transformation
15.
16.
17. MICROINJECTION
The DNA solution is directly injected directly inside the
nucleus of a cell
It uses capillary glass micropipettes with the help of
micromanipulators of microinjection assembly
Success has been claimed in the transfer of a gene for
resistance to the antibiotic kanamycin.
18. CONTI….
The protoplasts are immobilized on glass slides/petriplates
coated with polylysine or by holding them under suction by
a micropipette
Agar used as solidifying agent
High costly equipments
Successful in Brassica napus (microspore cells), Barley
21. ELECTROPORATION
Mix the DNA with protoplasts containing suitable ionic
solution
Expose high voltage electrical pulses for very brief
periods of time (300-400 V/cm for 10-50 ms)
It induce transient pores in the plasma lemma called
Electroporation
22.
23. Advantages
• Stable transformed cells
• Easy to perform
Disadvantages
• Applicable for only sensitive plants
• Low copy number
24. FIBRE MEDIATED DNA DELIVERY
Suspension culture cells were mixed with DNA
Silicon carbide fibres of 0.6μm dia and 10-80μm length
The mixture should be vortexed
DNA-coated fibers penetrate the cell wall in the presence of
small holes created in collisions between the plant cells and
fibers.
25.
26. Advantages
• Easy and quick procedure
• Low expenses and
• Usefulness for various plant materials
Dis advantages
• low transformation efficiency
• Cause damage to cells
27. LASER INDUCED DNA DELIVERY
• The plasma membrane of a cell is then exposed to a laser
beam for a small amount of time
• The generation of a photopore allows exogenous DNA
• Laser puncture transient holes in the cell membrane through
which DNA may enter into the cell cytoplasm
• High transformation frequency
• Low stable integration
28.
29. DIRECT GENE TRANSFORMATION THROUGH IMBIBITION
• Uptake of exogenous DNA of dehydrated plant tissues
• presence of 20% DMSO, suggesting that membrane
permeability was an important factor in the process.
• Dessicated somatic embryos of alfalfa, showed transient
GUS expression at frequencies up to 70%.
• The stable transformation of rice by embryo imbibition
was also reported.
30. Advantages
Most simple of methods, as they require no specialist
equipment
preparation of target plant tissues generally simple
Dis advantages
They can be applied only to very specific organs or tissues (i.e.
newly pollinated flowers or hydrating embryos)
It is still not clear that they lead to stable, and heritable
transformation.
31. DENDRIMER MEDIATED GENE TRANSFER
• +vely charged amino groups on the surface
of the dendrimer molecule interact with the –
vley charged DNA to form DNA-dendrimer
complex
• DNA-dendrimer complex ha overall positive
charge and can bind negatively charged
surface molecules on membrane of
eukaryotic cells
• Complexes bound to the cell surface are
taken into the cell by nonspecific endocytosis
• The complexes are transferred to the
endosomes
32. REFERENCES
• Text book of Biotechnology expanding horizon, By B.D Singh
• Text book of Life Scineces fundamentals and practice part II by Pranav kumar and Usha mina
• http://www.scribd.com/doc/54262070/Direct-Gene-Transfer-Methods#scribd
• http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10-0826_transfection_tutorial_interactive.pdf
