Gene transfer methods @ujjwasirohi

gene transfer methods
transformation methods
ELECTROPORATION,PEG, MICROINJECTION, MACROINJECTION, LIPOSOME MEDIATED, BIOLISTIC GUN, AGROBACTERIUM MEDIATED

1. @ujjwalsirohi
PhD scholar

2. Transformation
Gene transfer is the uptake of foreign DNA or
transgeneby plant cells.
It is the subsequent stable integration &
expression of a foreign DNA into the genome.

3. Methods of Transformation
There are mainly 2 methods of gene transfer:
Indirect (Agrobacterium-mediated) gene transfer
Gene transfer is done by using the bacteria Agrobacterium
tumificiens.
Direct gene transfer
the gene is directly transferred into the host by using various
techniques.

5. 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.

7. 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%)

8. 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

9. Microinjection
Direct injection of DNA into plant protoplast or cell
using fine tipped (0.5-10um diameter)pipette.
Protoplasts are immobilisedon the agaroseor held
with a micropipette under suction.
DNA is injected into the cytoplasm or nucleus.
Frequency of transformation:
Nucleus(14%)
Cytoplasm(6%)
Successful transformation achieved in tobacco, alfalfa,
Brassicasp.
Transformation frequency ranging from 14-60%

10. disadvantages
Extreme slow process.
Require expensive setup.

12. Macroinjection
Macroinjectionis 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. (>10-100um).
First study (1987), DNA was injected into developing
rye(cereal grain), & a low frequency of 0.07%was
recorded.

13. 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.(0.07%)

14. BiolisticMethod
Firstly used by Kleinet al (1987) & Sanfordet 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.
1-2μm tungsten or gold particles (micro-
projectiles)are used, coated with the DNA.
Acceleration is given to enter the micro-projectiles into
the plant cells.

15. 1000 psi

17. Advantages
This method can be use to transform all plant species.
Transformation protocol is relatively simple.
Disadvantages
High cost of the equipment and microcarriers.
Intracellular target is random (cytoplasm, nucleus,
vacuole, plastid, etc.).
Transfer DNA is not protected.

18. Liposome mediated gene transfer
Liposomesare 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.

20. Cationic lipids are those having a positive charge are
used for the transfer of nucleic acid.
Liposomesare 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.

21. Advantages
High degree of reproducibility.
Long term stability.
Protection of nucleic acid from degradation.

23. 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 mannitoland
calcium nitrate is added slowly.
Then incubated for few min.

24. Process
Protoplast suspended in medium (Mg and Ca ions)
Heat shock treatment (5min, 45 ˚C)
PEG added (20-28% conc.)
Incubation (calcium conc. enhenced)
cultured

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.
Random integration of foreign DNA into genome may
result in undesirable traits.
Regeneration of plants from transformed protoplasts is
a difficult task.

26. Agrobacteriummediated gene
transfer
Agrobacteriumis soil borne, gram negative, rod shaped,
motile found in rhizosphere.
Causative agents of “Crown gall” .
Size odplasmid is about 200 kb.
Contain a virregion ~ 35-40 kb at least 8-11 virgenes.

28. T-DNA
Size 12 –24 kb
Left and right border sequence (24-bp) which will be
transferred into genome of host plant
The T-DNA contains eight potential genes.

31. 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
Agrobacteriumfor about 30 mins.
The explants then placed on MS medium without selective
agent.
Incubate explants with Agrobacteriumfor 2 days to allow
transfer of the T-DNA.

32. Kill the Agrobacteriumwith a suitable antibiotic:
• The explants are removed from the medium and washed in
cefotaxime.
Select for transformed plant cells:
The explantare transferred to a selective (kanamycin)
medium with cefotaxime.
Auxin, Cytokininare 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 auxinto cytokininratio.

33. Thank you