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. The cassette includes a sequence of DNA called a "promoter", which determines where and when the foreign gene is expressed in the host, and a "marker gene" which allows breeders to determine by screening or selection which plants contain the inserted gene. For example, marker genes may make plants resistant to antibiotics not used routinely (e.g., agrimycin, kanamycin) or tolerant of some herbicides.

1. 1
ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY
S. V. AGRICULTURAL COLLEGE, TIRUPATI
COURSE NO : PP 603
COURSE TITLE : Molecular Approaches for improving physiological mechanisms
through traits introgression
TOPIC : Lecture 23: Agrobacterium and other methods of plant
transformation including gene gun, inplanta etc.
Submitted to:
Dr. A. R. Nirmal Kumar
Assistant Professor
Dept. of Crop Physiology
Submitted by:
P.Tejasree
TAD/2023-10
PhD (Ag) 1st
Year
Dept. of GPBR

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INTRODUCTION:
Conventional plant breeding uses crossing, mutagenesis, and somatic hybridization for
genome modification to improve crop traits by introducing new beneficial alleles from crossable
species. However, because of crossing barriers and linkage drag, conventional plant breeding
methods are time-consuming and require several generations of breeding and selection. To feed
the several billion people living on this planet, the main aim of breeders is to increase
agricultural production. Hence, new technologies need to be developed to accelerate breeding
through improving genotyping and phenotyping methods.
Genetic transformation is a powerful tool and a significant strategy for studying plant
functional genomics, i.e. gene exploration, new insights into gene regulation, and the analysis of
genetically regulated characteristics. Furthermore, the work of isolated genes utilizing map-based
cloning of mutant alleles has been verified through functional complementation via genetic
transformation. In addition, genetic engineering allows the insertion of alien genes into crop
plants and the accelerated creation of new genetically modified organisms.
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 drought
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
Gene transfer technologies in plants
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.
The cassette includes a sequence of DNA called a "promoter", which determines where and
when the foreign gene is expressed in the host, and a "marker gene" which allows breeders to
determine by screening or selection which plants contain the inserted gene. For example, marker
genes may make plants resistant to antibiotics not used routinely (e.g., agrimycin, kanamycin) or
tolerant of some herbicides.

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Various genetic transfer techniques are grouped into two main categories.
1) Vector mediated gene transfer (Indirect method)
2) Vectorless gene transfer (Direct method)
Vector mediated gene transfer (indirect method)
Vector-mediated gene transfer is carried out either by Agrobacteriummediated
transformation or by use of plant viruses as vectors. In this approach the transgene is combined

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with a vector which takes it to the target cells for integration. The term plant gene vector applies
to potential vectors both for transfer of genetic information between plants and the transfer of
genetic information from other organisms (bacteria fungi and animals) to plants. The vector
mediated transfer is strongly linked to regeneration capabilities of the host plant. The plant gene
vectors being exploited for transfer of genes are plasmids of Agrobacterium viruses and
transposable elements
Transformation Using Agrobacterium
The Agrobacterium system was historically the first successful plant transformation
system, marking the breakthrough in plant Genetic engineering in 1983. The Agrobacterium is
naturally occurring gram negative soil bacterium with two common species A Tumifacience and
A. rhizogenes there are known as natural gene engineers for their ability to transform plants. A
tumifacience induces tubers called crown galls, whereas a rhizogenes causes hairy root diseases.
Large plasmids in these bacteria are called tumour inducing (Ti plasmid) and root inducing (Ri
plasmid) respectively. The major credit for the development of plant transformation techniques
goes to the natural unique capability of A. tumefaciens.
The Ti plasmid has two major segments of interest in transformation that is T DNA and
virus region. The T DNA region of the Ti plasmid is the part which is transferred to plant cell
and incorporated into nuclear genome of cells. The transfer of T DNA is mediated by genes in
the another region of Ti plasmid called virs genes (virulence genes). Modified Ti plasmid are
constructed that lack of undesirable Ti genes but contain a foreign gene (resistant to a disease)
and a closely linked selectable marker gene (E.g.: - for antibiotic resistance). Within the T DNA
region any gene put in T DNA region of plasmid cysts transferred to the plant genome. The T
DNA is generally integrated in low copy number per cell. Transfer of gene through to wounded
plant organs A. tumifacience has limited range of host. It can infest about 60% gymnosperms and
Angiosperm. Hence Agrobacterium mediated transformation is the method of choice in
dicotyledonous plant species, where plant regeneration system are well established, However,
Monocotyledons could not be successfully utilized for Agrobacterium mediated gene transfer.
T-DNA Insertion by Agrobacterium
a) Attachment of Agrobacterium to Plant Cells - When plant tissues are wounded, they
exude organic acids, amino acids, saccharides, and other small molecules that can invoke
chemotaxis in Agrobacterium and boost the secretion of acetylated acidic
polysaccharides. Subsequently, Agrobacterium cells adhere onto the surface of plant
cells. The attachment process in which cellulose fi bers are synthesized and secreted is
regulated by attR and cel genes in the Agrobacterium genome, resulting in solid adhesion
of the bacteria to the surface of plant cells. The attachment process of Agrobacterium is
known to be related indirectly with chvA, chvB, and pscA genes in the bacterial genome,

5. 5
as well as with arabinogalactan proteins, cellulose synthase like proteins, and cell wall
proteins in host plants.
b) Activation of Virulence ( vir ) Genes - To recognize plant cells, Agrobacterium uses a
two-way signaling system, which consists of the VirA protein and the VirG:VirA protein
pair that directly perceive phenolic compounds like acetosyringone secreted from
wounded plant cells. These compounds induce autophosphorylation of a VirA domain.
The phosphate group of the VirA is then transferred to VirG, which binds to the
enhancement elements of vir genes in the Ti-plasmid and regulates their transcription
c) T-DNA Processing and T-Strand Formation - VirD2 is a nuclear localization signal
binding protein that is covalently bound to the 5′ end of the T-strand. VirD2 recognizes
the left and right borders of T-DNA sequences, makes a nick between the third and fourth
bases of antisense T-DNA border sequences, and forms covalent bonds at the 5′ end of
single-stranded DNA to form the T-strand. The VirD1 protein can change the structure of
T-DNA, which alleviates the tension and helps to stimulate T-strand formation by VirD2.
d) Transport of T-strand and Vir Proteins and T-complex Formation - VirD2 protein
transfers the T-strand into plant cells through a VirB channel, which consists of VirB and
VirD4 proteins. The VirB channel is a filamentous pilus, which connects the
Agrobacterium and host plant cell that functions as a transporter complex through cell
membranes. VirE2, which is transferred into the cytoplasm of the infected plant cell,
combines with the single stranded T-DNA, to form a T-strand/ protein polymer called the
T-complex. The T-complex protects the T-strand from the deoxyribonucleases that exist
in the plant cytoplasm and is an ideal structure to transport the large T-strand to the
nucleus of a plant cell.
e) T-complex Transfer to a Nucleus of a Plant Cell - The T-complex is larger than the
nuclear pores in the nuclear membrane of plant cells and is transferred to the plant
nucleus by active transport. VirE2, which surrounds the T-complex, and plant derived
importin-α proteins, which specifically recognize nuclear localization sequences (NLS) in
the VirD2 protein play important roles in the active transport. In Arabidopsis thaliana ,
VirD2 has been shown to conjugate specifically with NLS of AtKAPα, a member of the
karyopherin-α family, and is then transferred to the plant nucleus. VirE2 is essential for
T-DNA transport into the plant cell nucleus. VirE2 does not combine with AtKAPα, but
with the plant-derived proteins, VIP1 (VirE2 interacting protein) and VIP2, then its
transfer to the nucleus is mediated by karyopherin-α. The over-expression of the VIP1
gene particularly increases the import of T-DNA to the nucleus, and as a result the
transformation efficiency is correspondingly enhanced.
f) Insertion of T-DNA into Plant Genomes - VirE2 is not involved in insertion of T-DNA,
but it is needed to protect the T-DNA from plant deoxyribonucleases. VirE2 secures the
integrity of the T-DNA during its transportation from the cytoplasm to the nucleus, and
regulates its integration into the plant chromosome. Before the T-DNA is inserted into the
plant chromosome, the VirE2 surrounding the T-strand has to be removed. VirF is a defi

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ning factor of host-specifi city in Agrobacterium . It functions as an F-box protein and
shows target protein specifi city in the proteolysis related Skp1p–cullin–F-box protein
(SCF) complex. When transferred into pcells, VirF was found to be involved in the
proteolysis of VirE2 and VIP1 in the nucleus. VirD2 is known to be related to the precise
insertion of T-DNA into plant genomes. Because T-DNA insertion into plant genomes is
an illegitimate recombination, the host DNA repair and recombination-related genes are
expected to influence the insertion of the T-DNA.
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

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Advantages:
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
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

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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
In planta transformation
The in planta method of transformation is a new and efficient method of Agrobacterium-
mediated transformation that skips the need for the tissue culture-based regeneration of
transgenics. In this method, Agrobacterium with the required transgene is allowed to infect the
meristematic tissue of the plant directly, eliminating the intervening tissue culture steps. This is a
cost-effective, fast, and very efficient method compared to tissue culture-based transformation
and can open new gates for recalcitrant species. The first successful in planta transformation was
reported in Arabidopsis thaliana, with improved transformation when the plant was inoculated at
the flowering stage called the floral dip method
In planta transformation: a general scenario
In planta refers to the direct transformation of the plant without involving any tissue
culture step. In planta transformation is more efficient while also being less cumbersome and
time-consuming than the callus regeneration method. This can be useful for those plants, which
lack tissue culture and regeneration systems. In different crops, different types of explants were
used such as mature seeds, embryos, inflorescence, embryogenic apical meristem, spikelet, and
roots.
Comparative methodology of callus-based and in planta-based transformation methods
suggesting the approximate investment of days in these methods

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Methods for in planta transformation
In planta transformation involves the direct transfer of T-DNA to the plant genome, and
this can be achieved in various ways such as the floral dip method, the floral drop method, and
mechanical injury to the seed meristem . These methods are described in the following.
1. Vacuum infiltration method
The first attempt at the in planta transformation of A. thaliana through vacuum
infiltration was done in 1993 by Nicole Bechtold and coworkers. The whole Arabidopsis
plant was uprooted and immersed in an Agrobacterium suspension in a vacuum chamber,
and infiltration was allowed. After vacuum infiltration, the plant was potted and allowed
to grow back normally until seeding. Seeds were germinated on a selection medium for
the selection of transformed plants. This method was reported to give a 0.35%
transformation efficiency. It was an easy, less time-consuming, and efficient method that
became popular quickly. This method of vacuum infiltration was also applied to other
plant species such as Petunia hybrida , B. rapa L. ssp. chinensis , sugarcane and
Raphanus sativu L. longipinnatus.
a) Floral dip method –
In the floral dip method, the complete inflorescence of the plant is dipped in a
solution of the appropriate strain of Agrobacterium tumifaciens, then seeds are collected
from these “T0” plants and allowed to germinate in a selection for the identification of
transgenics. This method was first practiced in A. thaliana by Bechtold et al. (1998. Apart
from Arabidopsis, many other plant species have been transformed using the floral dip
technique such as B. napus and B. carinata, maize, radishes, wheat and S. lycopersicum.
b) Floral drop method —
In this method inflorescence was trimmed and then the Agrobacterium-containing
medium was directly dropped on these trimmed inflorescences so that individual spikelet
cups get the Agrobacterium-containing medium. These Agrobacterium-dropped spikelets
were then covered with bags and allowed to grow until seed collection.
c) Embryo transformation –
Embryo transformation involves the manipulation of the totipotent/meristematic
cells for the production of transgenic plants. In cotton, the apical meristem (AM) tissues
of the embryo are wounded at the seed germination time and this wounded part is used as
the explant to infect by Agrobacterium tumefaciens. Here, the seeds were first imbibed
and then germinated at 4°C for 2 weeks. These young seedlings were infiltrated with A.
tumefaciens suspension.

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Comparison of the callus method and the in planta method for rice transformation
Advantages:
• Avoids the tissue culture method
• Easy and Convenient
• Addresses the biosafety issue
• Reduction in genetic variability in the transformant
• Thousands of transformants were produced in a few year.
Disadvantages:
• Difficult to produce genotype consistently
• Low expression of transgenes
• Low transformation frequency
VECTORLESS GENE TRANSFER (DIRECT METHOD)
When the foreign DNA is directly inserted into the plant genome, the word direct or
vector less DNA transmission is used. Direct DNA transfer methods rely on naked DNA being
delivered into the plant cells. This contrasts with the transfer of agrobacterium or vector-
mediated DNA that can be considered indirect methods. The majority of the methods for direct
transfer of DNA are simple and effective. And in addition to this process has been used to
develop other transgenic plants. The introduction of DNA into plant cells without biological
agents such as Agrobacterium being involved and leading to stable transformation is called direct
gene transfer.

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TYPES OF DIRECT DNA TRANSFER
The direct DNA transfer can be broadly divided into three categories.
1. Physical gene transfer methods:
1.1 Electroporation
1.2 Particle bombardment
1.3 Micro injection
1.4 Liposome-Mediated Transformation
1.5 Silicon Carbide Fibre-Mediated Transformation
2. Chemical gene transfer methods:
2.1 Poly-ethylene glycol (PEG)-mediated
2.2 Diethyl amino ethyl (DEAE) dextran-mediated
2.3 Calcium phosphate precipitation
3. DNA imbibition by cells/tissues/organs
4. Pollen transformation
5. Delivery via growing pollen tubes
6. Laser induced transformation
7. Etc.
1. Physical gene transfer methods
1.1 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. The electric current leads to the
formation of small temporary holes in the membrane of the protoplasts through which the DNA
can pass. 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.

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Electroporation has been successfully used for the production of transgenic plants of
many cereals e.g. rice, wheat, maize
Advantages of electroporation
i. This technique is simple, convenient and rapid, besides being costeffective
ii. The transformed cells are at the same physiological state after electroporation
iii. Efficiency of transformation can be improved by optimising the electrical field
strength, and addition of spermidine
Limitations of electroporation
i. Under normal conditions, the amount of DNA delivered into plant cells is very low
ii. Efficiency of electroporation is highly variable depending on the plant material and
the treatment conditions
iii. Regeneration of plants is not very easy, particularly when protoplasts are used
1.2 Particle bombardment/ microprojectile/ biolistic/ gene gun/ particle acceleration
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. The micro projectile
bombardment method was initially named as biolistics by its inventor Sanford . Biolistics is a
combination of biological and ballistics. The process of transformation employs foreign DNA

13. 13
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-
 By using pressurized helium gas
 By electro static energy released by a droplet of water exposed to a high voltage
The target could be plant cell suspensions, callus cultures, or tissues. The projectiles penetrate
the plant cell walls and membranes. As the micro projectiles enter the cells, transgenes are
released from the particle surface for subsequent incorporation into the plant’s chromosomal
DNA.
Plant material used in bombardment: Two types of plant tissue are commonly used for
particle 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
In order to protect plant tissues from being damaged by bombardment, cultures are maintained
on high osmoticum media or subjected to limited plasmolysis.
Transgene integration in bombardment: It is believed (based on the gene transfer in rice by
biolistics) that the gene transfer in particle bombardment is a two stage process.
1. In the pre-integration phase, the vector DNA molecules are spliced together. This
results in fragments carrying multiple gene copies
2. Integrative phase is characterized by the insertion of gene copies into the host plant
genome
The integrative phase facilitates further transgene integration which may occur at the same point
or a point close to it. The net result is that particle bombardment is frequently associated with
high copy number at a single locus. This type of single locus may be beneficial for regeneration
of plants.
The success of bombardment
The particle bombardment technique was first introduced in 1987. It has been
successfully used for the transformation of many cereals, e.g. rice, wheat, maize. In fact, the first
commercial genetically modified (CM) crops such as maize containing Bt-toxin gene were
developed by this approach.

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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. Therefore, the quantity of DNA used should be balanced.
iv) Environmental parameters: Many environmental variables are known to influence
particle bombardment. These factors (temperature, humidity, photoperiod etc.) influence
the physiology of the plant material, and consequently the gene transfer. It is also
observed that some explants, after bombardment may require special regimes of light,
humidity, temperature etc.
The technology of particle bombardment has been improved in recent years, particularly
with regard to the use of equipment. A commercially produced particle bombardment apparatus
namely PDS-1000/HC is widely used these days.
Advantages of particle bombardment
i) Gene transfer can be efficiently done in organized tissues
ii) Different species of plants can be used to develop transgenic plants

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Limitations of particle bombardment
i) The major complication is the production of high transgene copy number. This may
result in instability of transgene expression due to gene silencing
ii) The target tissue may often get damaged due to lack of control of bombardment velocity
iii) Sometimes, undesirable chimeric plants may be regenerated
1.3 Microinjection
Microinjection is a direct physical method involving the mechanical insertion of the
desirable DNA into a target cell. The target cell may be the one identified from intact cells,
protoplasts, callus, embryos, meristems etc. Microinjection is used for the transfer of cellular
organelles and for the manipulation of chromosomes.
The DNA solution is injected directly inside the cell using capillary glass micropipettes
(0.5-10.0 pm tip) with the help of micromanipulators of a microinjection assembly. It is easier to
use protoplast than cells since cell wall interferes with the process of microinjection. The
protoplast are usually immobilized in agarose (or) on a glass slides coated with polylysine or by
holding them under suction by a micropipette.
As the process of microinjection is complete, the transformed cell is cultured and grown
to develop into a transgenic plant. In fact, transgenic tobacco and Brassica napus have been
developed by this approach. The major limitations of microinjection are that it is slow,
expensive, and has to be performed by trained and skilled personnel. The process of
microinjection is technically demanding and time consuming a maximum of 40-50 protoplasts
can be microinjected in one hour.

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1.4 Liposome-mediated transformation
Liposomes are artificially created lipid vesicles containing a phospholipid membrane.
They are successfully used in mammalian cells for the delivery of proteins, drugs etc. Liposomes
carrying genes can be employed to fuse with protoplasts and transfer the genes.
The efficiency of transformation increases when the process is carried out in conjunction
with polyethylene glycol (PEG). Liposome-mediated transformation involves adhesion of
liposomes to the protoplast surface, its fusion at the site of attachment and release of plasmids
inside the cell.
Advantages of liposome fusion
i) Being present in an encapsulated form of liposomes, DNA is protected from
environmental insults and damage
ii) DNA is stable and can be stored for some time in liposomes prior to transfer
iii) Applicable to a wide range of plant cells
iv) There is good reproducibility in the technique
Limitations of liposome fusion
The major problem with liposome-mediated transformation is the difficulty
associated with the regeneration of plants from transformed protoplasts. This method has
not been commonly used as it is difficult to construct the lipid vesicles.
1.5 Silicon carbide fibre-mediated transformation
The DNA is delivered into the cell cytoplasm and nucleus by silicon carbide fibres of 0.3-
0.6 µm diameter and 10-80 µm length. The fibres mediated delivery of DNA into the cytoplasm
is similar to microinjection. These fibres are capable of penetrating the cell wall and plasma
membrane, and thus can deliver DNA into the cells. The DNA coated silicon carbide fibres are
vortexed with ‘plant material (suspension culture, calluses). During the mixing, DNA adhering to
the fibres enters the cells and gets stably integrated with the host genome. The silicon carbide
fibres with the trade name Whiskers are available in the market. The method was successful with
maize and tobacco suspension cell culture.
Advantages of SCF-mediated transformation
i) Direct delivery of DNA into intact walled cells. This avoids the protoplast
isolation
ii) Procedure is simple, rapid and does not involve costly equipment
Disadvantages of SCF-mediated transformation
i) Silicon carbide fibres are carcinogenic and therefore have to be carefully handled

17. 17
ii) The embryonic plant cells are hard and compact and are resistant to SCF penetration
In recent years, some improvements have been made in SCF-mediated
transformation. This has helped in the transformation of rice, wheat, maize and barley
by using this technique
2. Chemical gene transfer methods
2.1 Polyethylene glycol (PEG)-mediated transfer
Polyethylene glycol (PEG), in the presence of divalent cations (using Ca2+), destabilizes the
plasma membrane of protoplasts and renders it permeable to naked DNA. In this way, 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
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
2.2 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.
2.3 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.

18. 18
2.4 DNA imbibition by cells tissue, embryos and seeds
When dry isolated embryos of wheat barley, rye, pea and bean are imbibed in a DNA
solution, they take up the DNA and show the expression of marker gene. Dry seeds, whose seed
coats have been removed also take up DNA when imbibed in a DNA solution. The imbibed
seeds or embryos are germinated on appropriate selective medium to isolate the transformed
embryos. It was thought that the DNA is taken up by the embryos through the cells injured
during their isolation. The DNA then moves through plasmodesmata to other cells of embryos
2.5 Pollen transformation
Involves the gene transfer by soaking the pollen grains in DNA solution prior to their use
for pollination. The method is highly attractive in view of its simplicity and general applicability
but so far there is no definite evidence for a transgene being transferred by pollen soaked in
DNA solution.
2.6 DNA delivery via growing pollen tubes
The stigmas were cut after pollination exposing the pollen tubes, the DNA was
introduced onto the cut surface that presumably diffused through the germinating pollen tube
into the ovule. This method is simple easy and very promising provided consistent result and
stable transformations are achieved the mechanism of DNA transfer into zygote through this
method is not yet established.
2.7 Laser induced transformation
It is method of introducing DNA into plant cells with a laser micro beam. Small pores in
the membrane are created by laser micro beam. The DNA from the surrounding solution may
than enter into the cell cytoplasm through the small pores. Laser-induced stress waves facilitate
targeted gene transfer. Pressure waves caused by nanosecond laser pulses can be used to deliver
macromolecules to cells and tissues. It is well established that a strong pressure wave, known as
a photomechanical or laser-induced stress wave (LISW), accompanies laser-induced plasma.

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MERITS & DEMERITS OF DIFFERENT GENE TRANSFORMATION
METHODS