For the fulfillment of the course 'Molecular basis of host pathogen interaction' offered by Kerala Agricultural University.

1. Different methods of gene transfer and
biosafety issues related to GM crops
Pl.Path 604 Molecular basis of host pathogen interaction
Kiran Mohan (2019-27-003)

2. Content
• Introduction
• Methods of gene transfer
• Biosafety issues related to gene transfer
• Conclusion

3. • Gene – Segment of nucleic acid that encodes a
functional protein or RNA and is the unit of
inheritance.
• Genetic engineering - Considerable efficiency
and skill in gene transfer into organisms and
desirable expression have been achieved.
• Transgenic animals and plants can be obtained
by introduction of exogenous DNA into targeted
animals and plants accompanied by the stable
expression.

4. Methods of gene transfer
• There are mainly two methods;
1. Vector-Mediated Gene Transfer
2. Direct or Vector-less DNA Transfer

5. Direct / Vector less gene transfer
1. Natural methods
1. Conjugation
2. Bacterial transformation
3. Transposition
4. Phage transduction
5. Retroviral transduction
2. Artificial methods
Physical methods
• Macroinjection
• Microinjection
• Protoplast fusion
• Biolistic transformation /
Microprojectile bombardment
• Pollen tube pathway
Chemical methods
• DNA transfer by calcium phosphate
method
• Polyethylene glycol mediated
• Polybrene/DMSO assisted
• DEAE-Dextran mediated
• Liposome mediated
• Silicon carbide mediated
transformation
Electrical methods
• Electroporation
Horizontal gene transfer
/ Lateral gene transfer

6. Conjugation
• Conjugation is a natural microbial
recombination process.
• During conjugation, two live bacteria (a
donor and a recipient) come together,
join by cytoplasmic bridges and transfer
single-stranded DNA (from donor to
recipient).
• Conjugation can occur among the cells
from different genera of bacteria.
Bacterial transformation
/ Bactofection
• It is a method of direct gene
transfer using bacteria into the
target cell, tissue, organ or
organism.
• The genes located on the
plasmids of the transformed
bacterial strains are delivered and
expressed into the cells.
• Strains that are invasive and
having better cell to cell spread
are more efficient.

7. Transduction
• Transduction is the process by
which DNA is transferred from
one bacterium to another by
a virus.
• Transduction does not require
physical contact between the
cell donating the DNA and the
cell receiving the DNA.
• Movement of DNA from one
location to another.
• Segments of DNA with this ability
to move are called transposable
elements or transposon.
• Transposition can be either
"replicative" or "conservative" in
nature.
• Method of gene silencing.
Transposition

8. Macroinjection
• Used in rye (Secale cereale)
plants.
• Needles used for injecting
DNA are with the diameter
greater than cell diameter.
• DNA injected with
conventional syringe into
region of plant which will
develop into floral tillers.
• Timing of injection is
important and should be
fourteen days before
meiosis.
• Dela Pena and coworkers in
1987.
• Used to study the cellular
functions of plant cells and plastid
physiology, e.g. in tobacco, alfa
alfa and Vicia faba
• Very fine needles or glass
micropipettes having the diameter
of 0.5 to 10 μm are used.
• Computerized control of holding
pipette, needle, microscope stage
and video technology has
improved the efficiency.
• Crossway and Reich in l986.
Microinjection

9. Macroinjection
Advantages
• This technique does not require
protoplast.
• Instrument will be simple and
cheap.
• Methods may prove useful for
gene transfer into cereals which
do not regenerate from
cultured cell easily.
• Technically simple.
Disadvantages
• Less specific.
• Less efficient.
• Frequency of transformation is
very low.
• Only proved on rye.
Microinjection
Advantages
• Frequency of stable integration of
DNA is far better as compare to
other methods.
• Method is effective in transforming
primary cells as well as cells in
established cultures.
• The DNA injected in this process is
subjected to less extensive
modifications.
• Mere precise integration of
recombinant gene.
• Disadvantages
• Costly & Skilled person required.
• Limited use in plants due to
characteristic of plant cell wall.

10. Protoplast fusion
• Protoplasts are the cells of which
cell walls are removed and
cytoplasmic membrane is the
outermost layer.
• Protoplast fusion is a physical
phenomenon, reported successful
b/w Brassica napus and Diplotaxis
harra.
• Normally isolated protoplast do not
fuse with each other because the
surface of isolated protoplast
carries negative charges (-10mV to -
30mV)
• Some agents required to reduce the
electronegativity of the isolated
protoplast and allow them to fuse
with each others.
Mechanical fusion
• Under the microscope and using
micromanipulator or perfusion
micropipette, protoplasts brought
into intimate contact
Chemical fusion
• Sodium nitrate, polyethylene
glycol, Ca2+ commonly used.
• Non specific, in expensive and
cause massive fusion product
having less frequency
Electofusion
• Electric field of low strength 10
Kvm-1 is used.
• Easy to control with a fusion
frequency 100 %
• Sophisticated and expensive
procedure.

12. • Developed by Sanford & co-
workers in 1987.
• The use of DNA-coated
microprojectiles (tungsten or
gold) for the delivery of genes
into intact cells and tissues.
• Successfully used for
transformation in soybean,
tobacco, maize, rice, wheat, oats
and tall fescue.
• These microprojectiles, normally
1-3pm in diameter and the
acceleration is achieved either by
an explosive charge (cordite
explosion) or by using shock
waves initiated by a high voltage
electric discharge.
Advantage
• Used to transform all plant
species.
• No binary vector is required.
• Transformation protocol is
relatively simple.
Disadvantage
• Difficulty in obtaining single
copy transgenic events.
• High cost of the equipment
and microcarriers.
• Intracellular target is random.
• Transfer DNA is not protected.
Biolistic transformation / Microprojectile bombardment

14. • It will be ideal to introduce the
transgene into the microspore
when it is uninucleate, so that
the transgene is passed on to
both the generative cells.
• Touraev and co-workers
pioneered this method in
tobacco during 1997.
• Although scientists were
pondering over pollen
transformation for a long time
without success, it can
possibly become a successful
technique in future if right
efforts are made
Advantage
• Fast and regeneration independent
method
• Avoids somaclonal variation
• Genotype independent
• Cheap method
• No need for skilled workers
• No need of in vitro conditions
• Can be routinely employed
• Availability of large number of pollen
grains
Disadvantage
• Not demonstrated in many species
• Genes have to be introduced into
pollen at correct stage
• Frequency of transformation is low
Pollen tube Transformation

16. • 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
dependent on the high
concentration of DNA and the
protection of the complex
precipitate.
• Addition of dimethyl sulfoxide
(DMSO) increase the efficency of
transformation.
Limitations
• Frequency is very low
• Integrated gene undergo
substantial modification
• Many cells do not like having
the solid precipitate adhering
to them and the surface of
their culture vessel
• Integration with host cell
chromosome is random
Calcium phosphate mediated transformation

18. Polybrene/DMSO assisted transformation
• Simple and versatile method
• Capable of producing high
number of stable transfectants
with low quantities of
exogenous DNA.
• Two step procedure
1. Adsorption
2. internalization
• Adsorption is mediated by a
polycation polymer called
polybrene
• Unifrom coating of target cells
with polybrene-DNA
complexes
• Internalization of DNA
complexes is by the exposure
to dimethyl sulfoxide (DMSO) .
• During 25-30% DMSO
treatment, cell membrane
permeability increases which
enhance the uptake.
Advantage
• Less toxic than other
polycations.
• High transformation efficency
requires very small quantities
of plasmid DNA to be used.

19. • This method is only for protoplast
• Protoplast kept in a solution
containing PEG and plasmid DNA.
• Calcium chloride is added and
sucrose and glucose acts as
osmotic buffering agent.
• PEG (15-25%) in the presence of
Ca2+ , destabilize plasma
membrane of the protoplast
renders it permeable to naked
DNA
• Which stimulates uptake of DNA
by endocytosis without any gross
damage to protoplasts.
• After transfer of DNA to
protoplast, PEG is allowed to get
removed
Advantage
• A large number of protoplast can
be simultaneously transformed
• Can successfully use for a wide
range of plant species
Limitations
• DNA is susceptible for degradation
and rearrangements.
• Random integration of foreign
DNA into genome may result
undesirable traits.
• Regeneration of plants from
transformed protoplasts is a
difficult task.
Polyethylene glycol mediated transformation

20. • DNA complexed to the high
molecular weight polymer Di
Ethyl Amino Ethyl Dextran
(DEAE) is used as a method of
gene transfer.
• If DEAE-Dextran treatment is
coupled with Dimethyl
Sulphoxide (DMSO) shock,
then upto 80% transformed
cell can express the
transformed gene.
• Treatment with chloroquinine
increase transient expression
of DNA
Advantage
• It is cheap and simple
• Can be used for transient cells
which cannot survive even
short exposure of calcium
phosphate
Disadvantage
• Stable expression is very
difficult to obtain
• After transformation, further
improvement of plant tissue
culture is not good
• Truncation, recombination,
rearrangement and silencing
may occur.
DEAE – Dextran mediated transformation

21. • Liposomes are spheres of lipids
which can be used to transport
molecules into the cells called
lipofection.
• Cationic liposomes fuse with
cell surface resulting in the
delivery of the DNA directly
across the plasma membrane.
• These are commercially
available lipids that are sold as
an in vitro-transfecting agent.
• Cationic liposomes can be
produced from a number of
cationic lipids like DOTAP and
DOTMA
Advantages
• Protection of DNA/RNA from
nuclease digestion.
• Low cell toxicity.
• Stability and storage of nucleic
acids due to encapsulation in
liposomes.
• High degree of reproducibility.
• Applicability to a wide range of
cell types.
• Simplicity.
Liposome mediated transformation

23. • Plant materials like cell
suspension culture, embryos
etc introduced into a buffer
containing DNA and the silicon
fibres which is then vortexed.
• The fibres (0.3 – 0.6 micro
meter in dia. and 10-100 micro
meter long) penetrate the cell
wall and plasma membrane,
allowing the DNA to gain access
to the inside of the cells
• Cellular penetration is likely
enhanced by frequent and
forceful intercellular collision
encountered in the vortexed
millue.
Advantage
• Improvement in yield
• Herbicide resistance
• Improvement in quality
• Insect resistance
• Resistance to abiotic stress
• Rapid and accurate technique
Disadvantage
• Inherent dangers of fibres,
require careful handling
• Many cereals produce
embryonic callus which is hard
and compact.
Silicon carbide mediated transformation

24. • This method was introduced by
Fromm and his coworkers in 1986.
• High field strength electrical
impulses used to reversibly
permeabilize the cell membranes
for the uptake of DNA.
• DNA then migrates through high-
voltage-induced pores in the
plasma membrane called
electrospores.
• Low voltage long pulses (300-400 V
cm-1 for 10-50 ms) or High voltage
short pulses (1000-1500 V cm-1 for
10 µs) used.
• Reported in Tobacco, petunia,
maize, rice, wheat and sorghum
etc.
Advantage
• Both intact cell and tissue can
be transformed.
• Less costly.
• Applied for a number of cell
types.
• Simultaneously a large number
of cell can be treated.
• High percentage of stable
transformants can be produced
Disadvantage
• About 50% of the transformed
cells can survive.
• No proof was available for
transfer of gene into walled
cells through this method.
Electroporation

26. Vector-mediated gene transfer
• Agrobacterium mediated transformation
• Virus mediated transformation

27. Agrobacterium mediated transformation
• It is rod shaped and motile, and belongs to the
bacterial family of Rhizobiaceae
• It is known as natural genetic engineer
A. tumefaciens that induces crown gall disease.
A. rhizogenes that induces hairy root disease.

28. Organization of Ti plasmid

29. T-DNA transfer and integration
1. Signal induction to Agrobacterium
2. Attachment of Agrobacterium to plant cells
3. Production of virulence proteins
4. Transfer of T-DNA out of Agrobacterium
5. Transfer of T-DNA into plant cells and integration

31. Ti plasmid derived vector system
• Ti plasmid of Agrobacterium has
the ability to genetically transform
plants.
• Insert a desired DNA sequence
(gene) into the T-DNA region (of Ti
plasmid).
• Use A. tumefaciens to deliver this
gene(s) into the genome of plant
cell.
• There are some limitations to use
Ti plasmid directly as vector.
• Ti plasmid- based vectors with
suitable modifications are used for
transgenesis.
• Two types of Ti plasmid-derived
vectors are used for genetic
transformation of plants— co-
integrate vectors and binary
vectors
Co-integrate vector
• T-DNA genes for hormone
biosynthesis are removed.
• In place of the deleted DNA, a
bacterial plasmid (pBR322) DNA
sequence is incorporated -
intermediated cloning vector.
Binary vector
• Binary system is a pair
of plasmids consisting of a binary
plasmid and a helper plasmid.
• Binary plasmid: shuttle vector
• Helper plasmid: Entire T-DNA
region including borders deleted
while vir gene is retained.

32. Steps for Agrobacteium mediated transformation

33. Advantages
• This is a natural method of
gene transfer.
• Agrobacterium can
conveniently infect any
explant (cells/tissues/organs).
• Even large fragments of DNA
can be efficiently transferred.
• Stability of transferred DNA is
reasonably good.
• Transformed plants can be
regenerated effectively.
Limitations
• There is a limitation of host
plants for Agrobacterium,
since many crop plants
(monocotyledons e.g.
cereals) are not infected by
it.
• The cells that regenerate
more efficiently are often
difficult to transform.

34. Virus mediated transformation
• Natural vectors for genetic engineering.
• They can infect the intact plants and amplify the
transferred genes through viral genome replication.
• They can introduce the desirable gene(s) into almost all
the plant cells since the viral infections are mostly
systemic.
• Plant viruses are non-integrative vectors: They spread
systemically within the host plant where the new
genetic material is expressed.
• The three groups of plant viruses used – Caulimo
viruses, Gemini viruses, RNA viruses

35. Caulimo viruses
• The caulimo viruses contain
circular double- stranded
DNA, and are spherical in
shape.
• Widely distributed and
Cauliflower mosaic virus
(CaMV) is the most important
for gene transfer.
• Limited capacity for insertion
of foreign genes.
Gemini viruses
• Twin and paired capsid
structures with one or two
single-stranded circular DNAs.
• They can infect a wide range
of crop plants.
• But it is very difficult to
introduce purified viral DNA
into the plants.
RNA plant viruses
• Two type of single stranded
RNA viruses.
1. Mono-partite viruses -
Tobacco mosaic virus
2. Multipartite viruses -
Brome mosaic virus
– good efficiency to infect
cells
– and spread to different
tissues.
– high level of gene expression
- difficulty of joining RNA
molecules in vitro.

36. Biosafety issues related to GM crops

38. Bt cotton
• Bt is a naturally-occuring
soil borne bacterium found
worldwide.
• Shigetane Ishiwattari in
1901 first isolated.
• Gram positive, facultative
aerobic, rod-like, motile
and sporulating bacterium.
• Produces crystals of
endotoxin (cry protein or
delta toxin)- toxic to insect
mainly in the larval stage .
• These crystal protein (cry
protein) are insect stomach
poisons.
• Thus they act as insecticide.
• Insect stop feeding within
two hours of bite,if enough
toxin is eaten, die with in
two or three days.
Mechanism
1. Ingestion.
2. Solublization & proteolytic
activation.
3. Binding to target site.
4. Formation of toxic lesions.

40. Biosafety issues related to GM crops
1. Bio-safety of human and animal health
2. Labelling of GM and non GM food
3. Ecological concerns
4. Public attitude
5. Socio economic and ethical consideration

41. 1.Bio-safety of human and animal
health
a. Safety of source organism and
genes
b. Effectiveness of antibiotics
c. Potential toxicity and allergenicity
d. Changes in nutritional level
2. Labelling of GM and non GM
food
a. Keeping GM and non GM crops
seperately.
3. Ecological concerns
a. Creation of weediness
b. Erosion of biodiversity
• Pollution of gene pools
4. Public attitude
• Consumer response depends
on perceptions about risks and
benefits of GM crops
• Communicate to the
customers and stake holders
• Media, individuals, scientists ,
administrators, politicians and
NGO have the responsibility.
5. Socio economic issues
• Due to increasing seed market,
developing countries may get
dependent on few suppliers
• Countries with effective and
efficent technology will obtain
and sustain advantage of
international markets

42. Direct effect on non- target organism
In may 1999, reported that pollen
from Bt resistant corn had a
negative impact on Monarch
butterfly larvae
This report raised concern about
Monarchs and other non target
organisms.
Issue of accidental cross breeding
• A recent study of Jean-
Francois Arnaud published
by Royal society reported
the cross breeding of GM
crops.
• He studied three separate
crops of commercial sugar
beet, wild sea beet and
weed beet .
• From the DNA matching he
found that , weed beet act
as a bridge between the
wild and commercial crops
via accidental seed flow.

43. Impact of Bt Brinjal in Bengladesh
• Brinjal (Solanum melongena L.),
also known as eggplant, is a
popular multiuse vegetable
cultivated in Asian countries
• The eggplant fruit and shoot
borer (FSB) is responsible for the
chronic and widespread
infestation
• In severe cases, the infestation
levels may exceed 90% and
causing yield loss of up to 86% in
Bangladesh.
• On 30 October 2013, Bangladesh
approved the official release of
four genetically modified,
varieties of insect-resistant Bt-
brinjal for seed production and
initial commercialization.
Impact of the Bt brinjal
• This survey found that 89% of
the farmers believe the
cultivation of Bt-brinjal
improved quality of brinjal
• 59% of farmers opined that
price was reduced due to Bt-
brinjal cultivation because of
higher production
• The farmers (97%) informed
that Bt-brinjal cultivation
reduced pesticide use.
• 96% of farmers consider Bt-
brinjal is safe for human health

44. Possibility of pest shift
• Northern China reported long term ecological effect of
Bacillus thuringiensis on non target pest.
• Over a period of 10 years, Bt cotton cultivation become
increased and insecticide use in this crop dropped
considerably.
• Mirid bugs (Heteroptera: Miridae) have progressively
increased the population size and acquired pest status.

45. Regulatory mechanism in India
• Biosafety regulations cover
assessment of risks and the
policies and procedures adopted
to ensure environmentally safe
applications of biotechnology.
Rules and policies
1. Rules, 1989 under Environment
Protection Act (1986).
2. Seed Policy, 2002.
Guidelines
1. Recombinant DNA guidelines,
1990
2. Guidelines for research in
transgenic crops, 1998
Agency for implementation
• Ministry of Environment, Forests
and Climate Change.
• Department of Biotechnology,
Government of India.
Competent authorities
1. Recombinant DNA Advisory
Committee (RDAC)
2. Review Committee on Genetic
Manipulation (RCGM)
3. Genetic Engineering Approval
Committee (GEAC)
4. Institutional Biosafety
Committees (IBSC)
5. State Biosafety Coordination
Committees (SBCC)
6. District Level Committees (DLC).

46. Conclusion
• Different types of methods are available for
gene transfer.
• The choice of methods of DNA transfer
depends upon the target cells and objectives
of gene manipulation.
• Agrobacterium mediated transfer is reported
to be widely adopted one.
• Biosafety issues should be addressed before
releasing GM crops.

47. Reference
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• https://bio.libretexts.org/@go/page/9298
• https://vikaspedia.in/agriculture/crop-production/advanced-
technologies/transgenic-crops-biosafety-concerns-and-
regulations-in-india
• Khan, K.H. 2010. Gene transfer technologies and their
applications: roles in human disease. Asian J. Biol. Sci. 1: 208-218.
• Khan, K.H.2009. Gene transfer technologies in plants: roles in
improving crops. Recent Res. Sci. Technol. 1(3): 116-123.
• Shelton A. M., Sarwer, S. H., Hossain, Md. J., Brookes, G and
Paranjape, V. 2020. Impact of Bt Brinjalcultivation in the market
value chain in five districts of Bangladesh. Front. Bioeng.
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