2. GENE TRANSFER TECHNIQUES
(TRANSFORMATION TECHNIQUES)
• Genetic Engineering - Recent approach in
Biotechnology →Transfer and expression of
foreign genes into higher plants – involves the
transfer of specifically constructed gene
assemblies through various transformation
techniques.
• Plants obtained through genetic engineering
contain a gene or genes usually from an
unrelated organism – transgenes – transgenic
plants.
3. • Genetic modification (genetic manipulation) of higher
plants by introducing DNA into their cells extend
their gene pool- create genetic variability – helpful in
enhancing their agricultural efficiency.
• Uptake of foreign DNA or transgenes by plant cells –
Transformation.
Gene transfer techniques
i) Direct gene transfer techniques –without the
involvement of a biological agent.
ii) Indirect gene transfer technique – with the
involvement of a biological agent, Agrobacterium
tumefaciens.
(Agrobacterium mediated gene transfer technique)
4. Direct Gene Transfer Techniques
• Introduction of DNA into plant cells without
the involvement of a biological agent →
transformation.
• Spontaneous uptake of DNA by plant cells is
very low – employ chemical and physical
treatments – facilitate the entry of DNA into
plant cells.
5. 1. Chemically stimulated DNA uptake
by Protoplast
Chemicals – Polyethylene glycol (PEG)
Polyvinyl alcohol (PVA)
Calcium phosphate
6. Chemically stimulated DNA uptake by
Poly Ethylene Glycol (PEG)
Mix freshly isolated protoplasts (protoplast
= cell without cell wall – prepared by using
cell wall degrading enzymes) with
DNA(linearised plasmid DNA containing the
gene construct ).
Add 15 – 20 % of PEG dissolved in a buffer
containing divalent cations (Mg ²+, Ca²+).
7. Incubate for 30 minutes - PEG causes
precipitation of ionic molecules like DNA –
stimulates their uptake by endocytosis –
transforms 0.1 to 0.4 % of the total protoplasts
treated.
Wash the protoplasts.
Treated protoplasts are plated in Petri dish for
culture and growth – regenerate cell wall →
callus colonies .
8. A selectable marker is used for the selection
of transformed protoplasts.
Regenerate into plants.
Generally used for stable transformation of
monocot protoplasts.
Used for direct gene transfer in Nicotiana
protoplasts.
9. 2. Transduction
( Zinder & Lederberg 1952)
A mechanism of genetic recombination in Bacteria
mediated by a lysogenic (Temperate) phages.
phage – serving as a vector , transferring a portion of
bacterial DNA (a few genes) from one bacterium to
another.
Temperate phages (Lysogenic phages) – do not lyse
host bacterium.
The phage DNA get integrated with the host bacterial
chromosome in a non- infectious stage – prophage .
10. • Sometimes the phage DNA may get excised
from the host bacterial chromosome
→initiates Lytic life cycle (Prophage
induction).
• Packaging error during assembly – may get
filled with bacterial chromosomal DNA or a
mixture of chromosomal and phage DNA
→aberrant or abnormal or transducing phages
– attach to other bacteria – introduce
bacterial DNA into them.
11. Types of Transduction
i) Generalized Transduction – a random
fragment of bacterial DNA is packaged in the
phage head in place of phage chromosome –
transducing phages – can transport any
bacterial gene from one bacterium to
another.
e.g. P22 in Salmonella typhimurium , P1 in
E.coli.
13. ii) Specialized Transduction -Transfer only
certain genes between bacteria.
E.g. Lambda phage carries only the gal
(required for the utilization of Galactose – a
monosaccharide- as the source of energy)
and bio ( essential for the synthesis of
Biotin) genes from one E.coli bacterium to
another.
16. 3. Electroporation
• Done with an instrument – Electroporator.
• High voltage electricity for a very brief period is
used to introduce DNA into cells.
• Exposure of cells to high voltage induce
transient pores in the plasma membrane – a
passage – foreign DNA can enter into the
protoplasts – increase transformation frequency.
• Optimal voltage and time – depends on plant
species, source of protoplasts and resistance of
the medium.
17. Methods
1. Low voltage – long pulse method: 300 to
400 V cm⁻¹ for 10 – 50 ms (Milliseconds)
2. High voltage – short pulse method:1000-
1500 V cm ⁻¹ for 10 µs (Microseconds)→ high
rates of stable transformation.
18. • Suspend plant protoplasts in a suitable ionic solution
containing linear recombinant plasmid DNA.
• Expose to chosen voltage
• Culture → cell colonies → plants
Transformation frequency can be enhanced by
Heat – shock to protoplasts just prior to
electroporation
Presence of a low concentration (about 8%) of Poly
ethylene glycol during electroporation.
• Electroporation can also be used to introduce DNA
into intact cells – cell wall weakened by a mild
enzymatic treatment – DNA entry → cell.
• Used in Tobacco, Petunia, Maize etc.
19. 4. Microinjection
Microinjection of DNA into fertilized eggs or
Embryos → transgenic animals.
• Eggs are surgically removed from the female
parent.
• in vitro fertilization.
• Microinjection of DNA into the male pronucleus
(haploid nucleus contributed by sperm, prior to
nuclear fusion) of the fertilized egg through a
very fine – tipped glass needle.
21. • The injected zygote is implanted into the
female to complete their development.
• Integration of injected DNA molecules may
occur early during embryonic development
at random sites in the genome.
22. • The animals that develop from the injected
eggs (G0 generation) – genetic mosaics- some
somatic cells carrying the transgene and
others not carrying.
• All cells of the progeny of initial transgenic
animals carry the transgene.
24. TRANSFECTION
• Uptake of DNA by a eukaryotic cell, followed
by its incorporation into the cell’s genome -
Transforming embryonic stem cells (ES cells)
growing in tissue culture with the desired
DNA
25. • Injection of DNA into large populations of cultured
cells derived from embryonic stem cells – introduce
these cells to other developing embryos – some form
adult tissues – progeny may contain its own cells and
those derived from the cultured transfected ES -cells
(Chimeras).
• If the ES cells contribute to the chimera’s germ line,
the introduced foreign DNA may be transmitted to
next generation.
27. 5. Microprojectiles(Shot gun method, Particle gun method,
Biolistic process,Particle acceleration, Microprojectile
bombardment)
Klein et.al. 1988
• Shooting DNA – coated 1- 2 µm tungsten or
gold particles into plant cells using a device -
accelerates particles by pressurized Helium or
electrostatic energy released by a droplet of
water exposed to a high voltage.
• Can deliver DNA into all tissues
28. Gene transfer using Shot gun method
Image: http://www.artsci.wustl.edu/~anthro/blurb/fg8.t.gif
30. Can be used to transform
shoot apical meristem
leaf blades
immature and mature embryos
mature pollen etc.
Used to produce stable gene transfers in
• Cotton
• Maize
• Rice
• Sugar cane
• Tobacco etc.
31. Indirect Gene Transfer Technique
Agrobacterium mediated gene transfer
Agrobacterium tumefaciens
• a gram negative, rod shaped, motile, soil
bacterium.
• Invades many dicots and some
gymnosperms- enters through fresh wound –
attach to the cell wall – transfers a part of its
Tumour inducing plasmid (Ti plasmid) known
as T-DNA (Transferred DNA) – integrates into
host chromosome – causes crown gall –
tumourous growth.
32. • Two components of Ti plasmid , the T-DNA
and vir (for virulence ) are essential for the
transformation of plant cells – vir region
contains genes required for the T-DNA
transfer process.
• Some genes in the T-DNA encode enzymes -
catalyze the synthesis of phytohormones -
auxin, IAA and cytokinin, isopentenyl
adenosine – responsible for the tumourous
growth.
33. Ti plasmid – Ideal vector for Plant
Genetic Engineering
Image: https://en.wikipedia.org/
36. Steps in Agrobacterium mediated
gene transfer
1. Isolation of foreign gene – The desired
foreign gene is isolated from the desired
strain of plants or micro organisms.
2. Construction of intermediate vector
plasmid, pBR 322- using naturally occurring
ColE1 plasmid of E. coli.
3. Isolation of Ti plasmid – from
Agrobacterium tumefaciens and T DNA
portion is separated.
37. 4. Construction of Shuttle vector – The T-DNA
is inserted in pBR 322 plasmid.
5. Construction of rDNA – T-DNA is cut with a
restriction enzyme and the prepared foreign
DNA fragment is inserted into the T-DNA
→recombinant DNA (rDNA or Chimeric DNA).
6. Transformation – rDNA is inserted into E.coli
in presence of Calcium chloride solution.
38. 7. Transformation of Agrobacterium
tumefaciens – The mixture of transformed
E.coli and Agrobacterium tumefaciens are
incubated for a few hours → Conjugation
between E. coli and Agrobacterium →
transfer of rDNA (recombinant plasmid/
chimeric DNA) to Agrobacterium – rDNA
undergo genetic recombination with its Ti
plasmid- inserted foreign gene is transferred
to the unmanipulated Ti plasmid of
Agrobacterium.
39. 8. Transformation of plant cells – The
transformed Agrobacterium are allowed to
infect the cultured plant cells whose
genomes are to be improved – This
Agrobacterium insert the foreign DNA into
plant cells – get integrated with the
homologous sequence of plant genome –
thus the plant cell is genetically transformed
to synthesize the product of the desired
gene.
