A brief account of some direct and indirect gene transfer techniques

1. GENE TRANSFER TECHNIQUES
Saji Mariam George
Associate Professor
Assumption Autonomous College
Changanacherry

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.

12. Stable gene transfer into
recipient cell chromosome

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.

14. Images:https://slideplayer.com

15. Image:https://slideplayer.com

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.

20. Microinjection
Image:https://www.rockefeller.edu/

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.

23. Image:https://web.wpi.edu/

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.

26. Image:https://www.biology-pages.info/

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

29. Image:https://groisman.physics.ucsd.edu/

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/

34. Image: http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF

35. Crown Gall
Image: OSU Plant Clinic collection,2012.
https://pnwhandbooks.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.

40. THANK YOU