Gene transfer methods

1. Genetic Transformation Methods
Dr. Saraswathi, M.S.c, Ph.D
Assistant Professor,
Dept. of Life Sciences,
Kristu Jayanti College, Autonomous, Bengaluru

2. Genetic transformation
• Genetic transformation is a powerful tool and a significant
strategy for studying plant functional genomics.
• Gene exploration, new insights into gene regulation and the
analysis of genetically regulated characteristics.
• Genetic engineering allows the insertion of alien genes into
crop plants and the accelerated creation of new genetically
modified organisms.
• Gene transformation and genetic engineering lead to the
overall production of crops.

3. Importance of gene transfer technologies to plants
i Provide resistance against viruses
ii. Acquire insecticidal resistance
iii. To strengthen the plant to grow against bacterial diseases
iv. Develop the plants to grow in draught
v. Engineering plants for nutritional quality
vi. Make the plants to grow in various seasons
vii. Herbicide resistant plant can be made
viii. Resistance against fungal pathogens
ix. Engineering of plants for abiotic stress tolerance
x. Delayed ripening can be done

4. • The process of transfer, integration and expression of transgene in the
host cells is known as genetic transformation.
• A foreign gene (transgene) encoding the trait must be incorporated into
plant cells, along with a "cassette" of extra genetic material to add a
desirable trait to a crop.
• Various genetic transfer techniques are grouped into two main categories.
1) Vector mediated gene transfer (Indirect method)
2) Vector less gene transfer (Direct method)
Gene transfer technologies in plants

6. Plant transformation technique using Agrobacterium

7. In general, most of the Agrobacterium-mediated plant transformations
have the following basic protocol:
1. Development of Agrobacterium carrying the co-integrate or
binary vector with the desired gene
2. Identification of a suitable explant e.g. cells, protoplasts,
tissues, calluses, organs
3. Co-culture of explants with Agrobacterium
4. Killing of Agrobacterium with a suitable antibiotic without
harming the plant tissue
5. Selection of transformed plant cells
6. Regeneration of whole plants

8. i. It is a natural means of gene transfer
ii. Agrobacterium is capable of infecting plant cells and tissue and
organs
iii. Agrobacterium is capable of transfer of large fragments of
DNA very efficiently
iv. Integration of T DNA is a relative precise process
v. The stability of gene transferred in excellent
vi. Transformed plants can be regenerated effectively
Advantages

9. Limitations
i. Host specificity: There is a limitation of host plants for
• Agrobacterium, since many crop plants (monocotyledons e.g.
cereals) are not infected by it.
• In recent years, virulent strains of Agrobacterium that can infect a
wide range of plants have been developed
ii. Inability to transfer multiple genes: The cells that regenerate
• more efficiently are often difficult to transform, e.g. embryonic
• cells lie in deep layers which are not easy targets for Agrobacterium
iii. Somaclonal variation
iv. Slow regeneration

14. Particle Bombardment/Microprojectile/Biolistic/Gene Gun
• Particle bombardment is a technique used to introduce
foreign DNA into plant cells Particle (or micro projectile)
• Bombardment is the most effective method for gene
transfer, and creation of transgenic plants.
• This method is versatile due to the fact that it can be
successfully used for the DNA transfer in mammalian cells
and microorganisms.

16. • The process of transformation employs foreign DNA
coated with minute 0.2-0.7 μm gold (or) are tungsten
particles to deliver into target plant cells.
• The coated particles are loaded into a particle gun and
accelerated to high speed-pressurized helium gas.
• By electro static energy released by a droplet of water
exposed to a high voltage.
• In order to protect plant tissues from being damaged by
bombardment, cultures are maintained on high
osmoticum media or subjected to limited plasmolysis.

18. Factors affecting bombardment
i Nature of micro particles: Inert metals such as tungsten,
gold and platinum are used as micro particles to carry DNA.
These particles with relatively higher mass will have a better
chance to move fast when bombarded and penetrate the
tissues.
ii) Nature of tissues/cells: The target cells that are capable
of undergoing division are suitable for transformation.
iii) Amount of DNA: The transformation may be low when
too little DNA is used. On the other hand, too much DNA
may result is high copy number and rearrangement of
transgenes.

19. Plant material used in bombardment:
1. Primary explants which can be subjected to
bombardment that are subsequently induced to
become embryo genic and regenerate
2. Proliferating embryonic tissues that can be
bombarded in cultures and then allowed to
proliferate and regenerate

20. Plant transformation process using particle bombardment

21. Advantages of particle bombardment
• Gene transfer can be efficiently done in organized tissues
• Different species of plants can be used to develop transgenic
plants.
Limitations of particle bombardment
• Instability of transgene expression due to gene silencing
• The target tissue may often get damaged due to lack of control
of bombardment velocity
• Sometimes, undesirable chimeric plants may be regenerated

22. Electroporation
• Electroporation is the incorporation of DNA into the cell
by exposing them to high voltage electrical pulses for a
very short period of time to cause temporary pores in
the plasma lemma.
• Plant cell electroporation generally uses protoplast,
while thick plant cell walls restrict the movement of
macromolecule.
• The plant material is incubated in a buffer solution
containing the desired foreign/target DNA, and
subjected to high voltage electrical impulses.

23. Electroporation

24. • After entry into the cell, the Foreign DNA gets incorporated
with the host genome.
• Resulting the genetic transformation the protoplasts are then
cultured to regenerate in to whole plants.
• This method can be used in those crop species in which
regeneration from protoplast is possible.
• In the early years, only protoplasts were used for gene transfer
by electroporation.
• Now a day, intact cells, callus cultures and immature embryos
can be used with suitable pre-and post-electroporation
treatments.

25. • Electroporation has been successfully used for the production
of transgenic plants of many cereals e.g. rice, wheat, maize.
(1) Isolate protoplasts from leaf tissues.
(2) Inject DNA-coated particles into the protoplasts using particle
gun.
(3) Regenerate into whole plants.
(4) Acclimate the transgenic plants in a greenhouse

26. Advantages of electroporation
• This technique is simple, convenient and rapid, besides being
cost effective
• The transformed cells are at the same physiological state after
electroporation.
• Efficiency of transformation can be improved by optimising the
electrical field strength.

27. Limitations of electroporation
• Under normal conditions, the amount of DNA
delivered into plant cells is very low
• Efficiency of electroporation is highly variable
depending on the plant material and the treatment
conditions
• Regeneration of plants is not very easy,
particularly when protoplasts are used

28. Chemical gene transfer methods
• Polyethylene glycol (PEG), in the presence of divalent
cations (using Ca2+), destabilizes the plasma membrane
of protoplasts and renders it permeable to naked DNA.
• The DNA enters nucleus of the protoplasts and gets
integrated with the genome.
• The procedure involves the isolation of protoplasts and
their suspension, addition of plasmid DNA, followed by a
slow addition of 40% PEG-4000 (w/v) dissolved in
mannitol and calcium nitrate solution.
• As this mixture is incubated, protoplasts get transformed.

29. • The naked plant protoplasts are mixed with molecules of
linearized plasmid DNA containing the foreign gene.
• The two are mixed in a transformation medium rich in Mg2+
ions in place of Ca2+ ions, following which 20% polyethylene
glycol (PEG) solution is added.
• After the treatment, the PEG concentration is reduced and
Ca2+ concentration is enhanced.
• It promotes the frequency of transformation.

30. Advantages of PEG-mediated transformation
i) A large number of protoplasts can be simultaneously transformed
ii) This technique can be successfully used for a wide range of plant species
Limitations of PEG-mediated transformation
i) The DNA is susceptible for degradation and rearrangement
ii) Random integration of foreign DNA into genome may result in undesirable
traits
iii) Regeneration of plants from transformed protoplasts is a difficult task.
. Sometimes the foreign DNA is degraded in the cytoplasm before reaching the
nucleus

31. DEAE dextran-mediated transfer
• 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 trans-formants.

32. 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.
• 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.

35. Applications of Transgenic Plants
The six applications are:
(1) Resistance to Biotic Stresses
(2) Resistance to Abiotic Stresses
(3) Improvement of Crop Yield and Quality
(4) Transgenic Plants with Improved Nutrition
(5) Commercial Transgenic Crop Plants and
(6) Transgenic Plants as Bioreactors.

36. i. Resistance to biotic stresses i.e. resistance to diseases caused
by insects, viruses, fungi and bacteria.
ii. Resistance to abiotic stresses-herbicides, temperature (heat,
chilling, freezing), drought, salinity, ozone, intense light.
iii. Improvement of crop yield, and quality e.g. storage, longer
shelf life of fruits and flowers.
iv. Transgenic plants with improved nutrition.
v. Transgenic plants as bioreactors for the manufacture of
commercial products e.g. proteins, vaccines, and biodegradable
plastics.

38. • Almost all the stresses, either directly or indirectly, lead to
the production of reactive oxygen species (ROS) that create
oxidative stress to plants.
• This damages the cellular constituents of plants which is
associated with a reduction in plant yield.
• Bacillus thuringiensis (Bt) toxin:

39. Resistance to diseases (insect, microorganisms).
ii. Improved nitrogen fixing ability.
iii. Higher yielding capacity.
iv. Resistance to drought and soil salinity.
v. Better nutritional properties.
vi. Improved storage qualities.
vii. Production of pharmaceutically important compounds.

40. Delay in fruit ripening has many advantages:
i. It extends the shelf-life, keeping the quality of the fruit
intact.
ii. Long distance transport becomes easy without
damage to fruit.
iii. Slow ripening improves the flavour.

41. Transgenic Plants with Improved Nutrition:
• Amino Acids of Seed Storage Proteins:
• Golden Rice —The Provitamin A Enriched Rice:
• Commercial Transgenic Crop Plants: