The document discusses various gene transfer techniques, including direct and indirect methods. Direct methods involve physical techniques like electroporation, particle bombardment, and microinjection which directly transfer DNA into cells. Chemical methods also directly transfer DNA using liposomes, DEAE dextran, or calcium phosphate precipitation. Indirect methods involve vector-mediated transfer, including Agrobacterium-mediated transfer where bacteria insert DNA into plant cells, and virus-mediated transfer using viruses like tobacco mosaic virus or cauliflower mosaic virus.

1. Gene transfer techniques
By,
Abhinava J V
BIRAC Innovation Fellow
University of Agricultural Sciences, Dharwad

2. Direct DNA transfer
• vector less
Indirect DNA transfer
• Vector-mediated

3. Direct DNA transfer
Physical gene transfer methods
Chemical gene transfer methods

4. Physical gene transfer methods
 High voltage electrical impulses are used to create the pores in the plasma
membrane.
 Through which DNA enters and gets integrated into the host cell genome.
1. Electroporation:

5. There are other names for this technique- particle gun,
gene gun, bio blaster.
DNA is coated with the tungsten or gold particles called
as Micro carriers (micro projectiles)
These are carried by macro carriers (macro projectiles).
These macro-carriers are inserted into the apparatus and
pushed downward by rupturing the disc.
The stopping plate does not permit the movement of
macro carrier while the micro carriers (with DNA) are
propelled at a high speed into the plant material.
2. Particle Bombardment:

8. The technique of microinjection involves the
transfer of the gene through a micropipette (0.5-
10.0 pm tip) into the cytoplasm/nucleus of a plant
cell or protoplast.
The recipient cells are kept immobilized in agarose
embedding, and held by a suction holding pipette.
3. Microinjection:

10. Chemical Gene Transfer Methods:
Liposomes are artificially created lipid vesicles
containing a phospholipid membrane.
They are successfully used in mammalian cells for the
delivery of proteins, drugs etc.
Liposomes carrying genes can be employed to fuse
protoplasts and transfer the genes.
The efficiency of transformation increases when the
process is carried out in conjunction with polyethylene
glycol (PEG).
 1. Liposome-Mediated Transformation:

12.  The desirable DNA can be complexed with a high molecular
weight polymer diethyl amino ethyl (DEAE) dextran and
transferred.
 2. DEAE Dextran-Mediated transfer:
 3. Calcium Phosphate Co- Precipitation-Mediated Transfer:
 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.

13. Indirect DNA transfer
Agrobacterium-Mediated Gene Transfer
Viruses-Mediated Gene Transfer

14. Agrobacterium-Mediated Gene Transfer
 Agrobacterium tumefaciens is a soil-borne, Gram-negative
bacterium.
 It is rod shaped and motile, and belongs to the bacterial family of
Rhizobiaceae.
 A. tumefaciens contain a 200-kb tumor inducing (Ti) plasmid
 Bacteria was capable to transfer a portion of the plasmid DNA into
the plant host (T-DNA).
 Infects plants through breaks or wounds.
 Expression of genes in wild-type T-DNA results in dramatic
physiological changes to the plant cell.
 T-DNA Synthesis the of plant growth hormones (auxins and
cytokinins) which cause to neoplastic growth (tumor formation)

16. Agrobacterium tumefaciens chromosomal genes:
chvA, chvB, pscA required for initial binding of the
bacterium to the plant cell and code for
polysaccharide on bacterial cell surface.
Virulence region (vir) carried on pTi, but not in the
transferred region (T-DNA). Genes code for proteins
that prepare the T-DNA and the bacterium for
transfer.
At least nine vir-gene operons have been identified.
These include vir A, vir G, vir B1, vir C1, vir D1, D2 and
D4, and vir E1, and E2.

18. Virus-Mediated Gene Transfer:
 Mono-partite viruses: e.g. tobacco mosaic virus (TMV).
 Multipartite viruses:, e.g. brome mosaic virus (BMV)
 1. DNA Based:
 Cauliflower mosaic virus (CaMV):
 Gemini Viruses as Vectors:
 1. RNA Based:

19. Thank You