Direct or vector less dna transfer

1. Transgenic Plants
Direct or Vector-less DNA Transfer
Amandeep Singh
Assistant Professor,
Department of Biotechnology,
GSSDGS Khalsa College,
Patiala.

2. Physical Gene Transfer Methods
• Electroporation
Electroporation basically involves the use of
high field strength electrical impulses to
reversibly permeabilize the cell membranes
for the uptake of DNA.
Advantages:
• Simple, Convenient
• Transformed cells at same physiological state
• Efficiency can be optimized
Disadvantages:
• Amount of DNA delivered is low
• Variable efficiency
• Low regeneration

3. Microprojectile Injection/ Particle Bombardment/Gene Gun/
Biolistic Gun method/ Bioblaster
PDS-1000/He
Particle Delivery System

4. Biolistic Gun Method

5. The Success of Bombardment
First commercial genetically
modified (GM) crops such as
Maize containing Bt-toxin
gene were developed by this
approach.

6. Factors affecting bombardment
• Nature of microparticles: Inert materials (Tungsten, Gold, Platinum) are used
as microparticles to carry DNA. These particles with relatively high mass will
have a better chance to move fast when bombarded and penetrate the tissues.
• Nature of tissues/cells: Cells that are capable of undergoing divisions are
suitable for transformation. e.g. embryonic tissues.
• Amount of DNA: DNA used should be balanced. Chemical aminosiloxane used
to coat the microparticles with low quantities of DNA.
• Environmental factors: Temperature, humidity, photoperiod influence
physiology of plant material and thus gene transfer.
Advantages:
• Efficient
• Different species of plants can be used to develop transgenic plants.
Disadvantages:
• Gene silencing (because of high copy number)
• Target tissue can be damaged by high velocity of bombardment

7. Microinjection
• Direct physical method involving
the mechanical insertion of
desirable DNA into target cell.
• Transfer of gene through a
micropipette (0.5=10.00 µm tip)
into the cytoplasm/nucleus of a
plant cell or protoplast.
• It is done by keeping the recipient
cells immobilized in agarose
embedded & held by a suction
holding pipette.

8. Liposome mediated Transformation
Advantages:
• DNA is protected from environmental factors
• DNA is stable and can be stored for sometime prior to transfer
• Applicable to a wide range of plant cells
• Good productivity
Liposomes: Artificially created lipid vesicles containing a phospholipid membrane.
Liposomes carries the gene to be inserted and are fused with plant cells using
Polythylene glycol (PEG).
Plant cell Attachment Fusion Release
PEG

9. Silicon Carbide Fiber mediated
Transformation
Silicon Carbide Fiber: 0.3-0.6 micrometer (in diameter)
10-100 micrometer (in length)
DNA coated silicon carbide + Plant material (suspension culture, calluses)
Vortexed
DNA coated SCF enter plant tissue
SCF with trade name Whiskers are available in the market.

10. Chemical Gene Transfer Methods
• Polyethylene Glycol
• DEAE (Dextran mediated transfer)
• Calcium phosphate Co-transfer method
DNA
Calcium chloride + Phosphate buffer
DNA-calcium phosphate precipitate
Transformation
Efficiency increased by the addition of dimethyl sulfoxide (DMSO)
Cells to be transformed