Department of Botany

Content

• Introduction
• Electroporation
• Microinjection
• Particle Bombardment
• Comparative Aspect
• Reference
Vectorless DNA transfer or Direct Gene
transfer

1) It is the process of gene transfer into the host cell without using
a vector.
2)This is possible by four methods namely :-
1) Chemical mediation transfer
2) Electroporation
3) Microinjection
4) Particle bambardment
• foreign gene of interest is delievered into the host plant cell
without vector.
 Electroporation

1) The process in which transient holes are

produced in the plasma membrane of the target cell
to incorporate foreign DNA is called electroporation.
2) Creation, by means of an electrical current, of
transient pores in the plasmalemma to allow
exogenous material, especially DNA, orgenes to
enter the cell from the external medium.
Principle:- 1) Makes protoplast temporarily permeable to DNA.
• Procedure :-
1. Plant cell protoplasts are suspended in an ionic solution
containing the vector DNA in a small chamber that has electrodes
at opposites ends.
2. A pulse of high voltage is applied to the electrodes which
produces transients pores (Ca. 30mm) in the plasma
membrane,allowing the DNA to diffuse into the cell.
3. After a short while, the membrame reseals.
If treated well,
The cells can regenerate cell wall.

Divide to form callus.

Regenerate complete plant.

• . With a 1 cm gap between the electrodes
and protoplasts of 40 44 um diameter,1-1.5
kV cm-² of field strength for 10 us is
required for effiecient in introduction of
DNA
MICROINJECTION

• The technique of introducing foreign DNA into a target

cell by injecting the DNA directly into to nucleus with
the help of micro-needle is called as microinjection.
• Principle :- Insertion of genes or DNA into cells or
embryo using a very thin (<10 µm ) glass needle.
• Apparatus :- Fine tipped pipette (0.5-10 µm)
• Procedure :-
1) Direct insertion of plant protoplasts or cells using fine
tipped pipette.
2) Cells or protoplasts are immobilized on a solid support
such as agarose or held with a micro pipette under
suction.
3) Protoplasts regenerates cell wall started dividing with
the new gene.
PARTICLE BOMBARDMENT

• The process of DNA transfer in which foreign DNA is coated

on Tungsten or Gold particles bombarded directly on cell or
tissue is called as particle bombardment.
• Principle :- Desired gene is bombarded on cell or tissue with
the help of Helium pressure gun.
• Procedure :-
1) 1.2 µm Gold or Tungsten particles coated with DNA are
shot into plant cells using a Helium pressure gun device.
• This method overcome many of the biological
barrier associated with other methods of
transformation such as host range specificity of the
the agrobacterium and regeneration of complete
plant from protoplasts.
• This method initially named as ‘bio-listics’ by
Sanford (1987).
COMPARATIVE ASPECTS

Particle
Electroporation Microinjection Bombardment
Protoplasts Needed Either Not Needed
Marker Gene Needed Not Needed Needed
Apparatus Electric setup Micro-suction pipette Gene Gun
Transistent pores Gene directly DNA coated Gold
Working made in protoplast injected into the particles bombarded
with the help of nucleus of on cell or tissue using
electric current . protoplasts/cell. Gene Gun.
Lycopersicon
Transformed escalentum , Carrot, Brassica napus, Crop plants like
plants Soybean, Maize Barley Wheat, Rice, Maize
(Tissue Poration)
REFERENCES

• Biotechnology by U. Satyanarayana
• Plant Cell And Tissue Culture by Karl- Harmann
Neumann
THANK
YOU