Assignment No # 01

Subject : plant Biotechnology

Topic : Methods of Gene Transformation

Submitted By : Tanzila Nayab

Registration no : BT 120222028
Submitted to : Sir Rehan Naeem

Kohat University of Science and Technology

KOHAT

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Genetic Engineering
The Production of genetically engineered plants became possible after
Bob Fraley and others succeeded to use Agrobacterium tumefaciens to
transform plant cells with recombinant DNA in the early 1980s (Vasil,
2008a). Since this breakthrough in plant biotechnology, GM crops are
now routinely developed and grown in many parts of the globe.
Engineering has been used successfully to develop novel genes of
economic importance that can be used to improve the genetics of crop
plants. Genetic engineering is the targeted addition of a foreign gene or
genes into the genome of an organism. The genes may be isolated from
one organism and transferred to another or may be genes of one species
that are modified and reinserted into the same species. The new genes,
commonly referred to as transgenes, are inserted into a plant by a
process called transformation.

Crop genetic improvement (plant breeding) is an important tool but has

limitations. First, in conventional terms, genetic improvement can only
be done between two plants that can sexually mate with each other. This

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limits the new traits that can be added to those that already exist in that
species. Second, when plants are mated, (crossed), many traits are
transferred along with the trait of interest including traits with
undesirable effects on yield potential.
Transgenesis:
Transgenesis refers to the process of introducing an exogenous or
modified gene (transgene) into a recipient organism of the same or
different species from which the gene is derived. The transgene becomes
incorporated into the genome of the host organism and can be
transmitted to the offspring.
Transgenesis can be carried out in the gonads, sperm, fertilized eggs, and
embryos through DNA microinjection, retroviruses, stem cells, and
cloning. The most effective transgenic marker at the moment is
fluorescent protein.
Transgenic plant:
Transgenic plants are plants that have been genetically modified to
possess a new trait that is not naturally present in them. This
modification is achieved by introducing genes from bacteria or other
resistant plants through various methods, such as direct DNA methods or
Agrobacterium-mediated transformation.
Transgenic plants include maize, rice, brinjal, cabbage, cauliflowers,
potato, and tomato.
Advantages of transgenic plants
This process provides advantages like improving shelf life, higher yield,
improved quality, pest resistance, tolerant to heat, cold and drought
resistance, against a variety of biotic and abiotic stresses.

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PHYSICAL GENE TRANSFER METHODS:

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ELECTROPORATION:
Electroporation is a method of transformation via direct gene transfer. In this technique
Mixture containing cells and DNA is exposed to very high voltage electrical pulses (4000 –
8000 V/cm) for very brief time periods (few milliseconds). It results in formation of transient

Pores in the plasma membrane, through which DNA seems to enter inside the cell and then
Nucleus.
A suspension of cells with plasmid DNA is taken in an electroporation cuvette placed

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between Electrodes and electrical pulses are applied. Temporary microspores are formed i cell
Membranes which allow cells to take up plasmid DNA leading to stable or transient
DNA expression. Cells which are arrested at metaphase stage of cell cycle are especially
suitable for Electroporation as these cells have absence of nuclear envelope and an
unusual permeability of the plasma membrane. Protoplasts are used for electroporation of plant
cells as thick plant cell walls restrict movement of DNA. The electroporation
method was originally developed for Protoplasts, but has given equally good results with
cells and even tissues with easy recovery of regenerated plantlets. Immature zygotic
embryos and embryogenic calli have also been Used for electroporation to produce
transgenic maize.
Transformation of protoplast is associated with low transient expression of transgenes as
Compared to organized tissues and low regeneration frequencyespeciallyIn
monocotyledonous plants. The electrical field and Chemicalsubstancesapplied to
disorganize cell walls strongly reduce the viability and capability division of protoplasts.
Electroporation as a transformation method is fast, convenient, simple, and inexpensive
and Has low cell toxicity. The disadvantage associated with this technique is difficulty
in Regenerating plants from protoplasts, if protoplast is used for electroporation.

MICROINJECTION:
The process of using a fine glass micropipette to manually inject transgene at microscopic
or Borderline macroscopic level is known as microinjection. The transgene, in the form of
Plasmids, cosmids, phage, YACs, or PCR products, can be circular or linear and need not
be Physically linked for injection.
Microinjection involves direct mechanical introduction of DNA into the nucleus or
Stable trans formants can be achieved through this method but it requires technical
expertise And is a time consuming process. Also,microinjection has achieved only limite
success in Plant transformationdue to the thick cell walls of plants and a lack of
availability of a singlecell-to-plant regenerationsystem in most plant species.In this
technique a traditional compound microscope (around 200X magnification) or Aninverted
microscope (around 200x magnification) or a dissecting stereomicroscope (around 40

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-50x)is used.Under the microscope target cell is positioned and cell membrane and
nuclear Envelope are penetrated with the help of two micromanipulators.One
micromanipulator holds the pipette and another holds the micro capillary needle.

PARTICLE BOMBARDMENT (BIOLISTIC/GENE GUN):

Particle bombardment employs high-velocity micro projectiles to deliver
substances into cells and tissues. For genetic transformation, foreign DNA is coated
onto the surface of micron-sized tungsten or gold particles by precipitation with
calcium chloride and spermidine. Once inside the cells, the DNA elutes off the
particles. If the foreign DNA reaches the nucleus, then transient expression will likely
result and the transgene may be stably incorporated into host chromosomes.
Regeneration system must be applied to have genetic modified plantlets.
In this technique, foreign DNA consists of a coding region of a certain gene Particle
bombardment employs high-velocity micro projectiles to deliver
Substances into cells and tissues. For genetic transformation, foreign DNA is coated
Onto the surface of micron-sized tungsten or gold particles by precipitation with
Calcium chloride and spermidine. Once inside the cells, the DNA elutes off the
Particles. If the foreign DNA reaches the nucleus, then transient expression will likely
Result and the transgene may be stably incorporated into host chromosomes.

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Regeneration system must be applied to have genetic modified plantlets.
In this technique, foreign DNA consists of a coding region of a certain gene between
promoter and terminator few terminal DNA bases.

SILICON CARBIDE FIBREMEDIATED TRANSFORMATION:

The silicon carbide fibers (SCF) are about 0.30.6 pm in diameter and 10100 pm in
Length. These fibers are capable of Penetrating the cell wall and plasma Membrane, and
thus can deliver DNA into The cells. The DNA coated silicon carbide Fibers are vortexes
with ‘plant material suspension culture, calluses). During the Mixing, DNA adhering to the
fibers enters the cells and gets stably integrated with the genome. The silicon carbide
fibers with The trade name Whiskers are available in the market.
Advantages of SCFmediated Transformation:
i. Direct delivery of DNA into intact walled Cells. This avoids the protoplast isolation
Procedure is simple and does not involve Costly equipment.
Disadvantages of SCFmediated Transformation:
i. Silicon carbide fibers are carcinogenic and Therefore have to be carefully handled.

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 ii. The embryonic plant cells are hard and Compact and are resistant to SCF Penetration.
In recent years, some improvements have Been made in SCFmediated transformation.
This has helped in the transformation of Rice, wheat, maize and barley by using this
technique.

VACUUM INFILTRATION:
Vacuum infiltration is a physical method for plant transformation that uses a vacuum to drive
bacteria into the air spaces between plant cells. The process is often used in combination with
Agrobacterium, a bacterium that is prominent in plant biotechnology. Here are some steps for
vacuum infiltration: Immerse flower buds in an Agrobacterium suspension
Apply a vacuum to force the bacteria into the plant’s air spaces
Gently release the vacuum to avoid damaging the explants
Remove excess Agrobacterium with sterilized filter paper
Culture the explants in a cultivation medium in the dark for two days
Rinse the explants with sterile water and transfer them to a selection medium
Culture the explants in the selection medium for 3–4 weeks to assess the transformation effect
Vacuum infiltration is often used for small-sized plants, but there are protocols that can be used
for larger plants. For example, one protocol for cacao plants involves applying a vacuum to the
aerial parts of the leaves.
Another method for plant transformation is floral dip, which is a simpler, less labor-intensive
alternative to vacuum infiltration. In floral dip, developing floral tissues are dipped into a
solution containing Agrobacterium.

CHEMICAL METHODS OF GENE TRANSFER:

There are several chemical methods of gene transferring in plants including ,PEG,DEAD
Dextron, Calcium Phosphate, Artificial Lipid, Proteins, and Dendrimers.

1. POLYETHYLENE GLYCOL (PEG)-MEDIATED TRANSFER:

DNA molecules can be forced to enter the host genome only in those cells which do
not possess cell walls. The naked plant protoplasts are mixed with molecules of linearized
plasmid DNA containing the foreign gene. The two are mixed in a transformation medium
rich in Mg2+ ions in place of Ca2+ ions, following which 20% polyethylene glycol (PEG)
solution is added. After the treatment, the PEG concentration is reduced and Ca2+
concentration is enhanced. It promotes the frequency of transformation.

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The chemical method involving transformation in presence of polyethylene glycol is
convenient and simple but there are, however, some disadvantages:
1) Many cells are so sensitive that the chemical method cannot be applied whereas
some cells die during the treatment.
2) This method is not perfect because many treated cells do not contain any transfer DNA.

3) Sometimes the foreign DNA is degraded in the cytoplasm before reaching the nucleus.

2: DEAE-DEXTRAN (DIETHYLAMINOETHYL DEXTRAN) MEDIATED

DNA TRANSFER:
Diethylaminomethyl dextran (DEAE-dextran) is a soluble polycationic carbohydrate that
promotes interactions between DNA and endocytic machinery of the cell.
In this method, the negatively charged DNA and positively charged DEAE – dextran form
aggregates through electrostatic interaction and form a polyplex. A slight excess of DEAE –
dextran in mixture results in net positive charge in the DEAE – dextran/ DNA complex
formed. These complexes, when added to the cells, bind to the negatively charged plasma
membrane and get internalized through endocytosis. Complexed DNA delivery with DEAE
-dextran can be improved by osmotic shock using DMSO (Dimethyl sulfoxide) or glycerol.
Several parameters such as number of cells, polymer concentration, transfected DNA
concentration and duration of transfection should be optimized for a given cell line.

Advantages:

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• Simple and inexpensive
• More sensitive
• Can be applied to a wide range of cell types
• Can be used for transient transfection.

Disadvantages:
• Toxic to cells at high concentrations
• Transfection efficiency varies with cell type
• Can only be used for transient transfection but not for stable transfection
• Typically produces less than 10% delivery in prim

3: CALCIUM PHOSPHATE TRANSFECTION:

This method is based on the precipitation of plasmid DNA and calcium ions by their
interaction. In this method, the precipitates of calcium phosphate and DNA being small and
insoluble can be easily adsorbed on the surface of cell. This precipitate is engulfed by cells
through endocytosis and the DNA gets integrated into the cell genome resulting in stable or

permanent transfection
Advantages:
• Simple and inexpensive
• Applicability to generate stably transfected cell lines

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• Highly efficient (cell type dependent) and can be applied to a wide range of cell types.
Disadvantages:
• Toxic especially to primary cells
• Slight change in pH, buffer salt concentration and temperature can compromise the efficacy
• Relatively poor transfection efficiency compared to other chemical transfection methods like
lipofection.

4: LIPOSOME MEDIATED GENE TRANSFER (LIPOFECTION):

Lipofection, also known as “lipid transfection” or “liposome-based transfection,” uses a lipid
complex to deliver DNA to cells.
Liposomes are artificial phospholipid vesicles used for the delivery of a variety of
molecules into the cells. They may be multi-lamellar or unilamellar vesicles with a size
range of 0.1 to 10 micrometer or 20-25 nanometers respectively.
They can be preloaded with DNA by two common methods- membrane-membrane fusion and
endocytosis thus forming DNA- liposome complex. This complex fuses with the protoplasts to
release the contents into the cell. Animal cells, plant cells, bacteria, yeast protoplasts are
susceptible to lipofection method.
Advantages:
• Economic
• Efficient delivery of nucleic acids to cells in a culture dish.
• Delivery of the nucleic acids with minimal toxicity.
• Protection of nucleic acids from degradation.
Disadvantages:
• It is not applicable to all cell types

5.DENDRIMERS MEDIATED GENE TRANSFER:

. Dendrimers are highly branched, tree-like molecules that can be used in gene transfer
methods in plants. In gene transfer, dendrimers can act as carriers to deliver genetic
material (such as DNA or RNA) into plant cells.
The process typically involves the following steps:
1. Complex Formation: Dendrimers can form complexes with the genetic material to

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 protect it and facilitate its delivery into the plant cells. The genetic material binds to the
dendrimer through electrostatic interactions or other bonding mechanisms
2, Cellular Uptake: Once the dendrimer-genetic material complex is formed, it can be taken up
by the plant cells. Dendrimers can help in crossing the cell membrane barriers and reaching the
interior of the cell.
3.Release of Genetic Material: Inside the cell, the dendrimer releases the genetic material,
allowing it to function and carry out its intended role. This can involve the genetic material being
released into the cell's nucleus where it can interact with the plant's own genetic material.
4. Gene Expression: The transferred genetic material can then be expressed by the plant cell,
leading to the production of specific proteins or traits encoded by the transferred gene.

6. PROTEIN MEDIATED GENE TRANSFER:

Protein-mediated gene transfer in plants involves using proteins to deliver genetic material into
plant cells. One common method is using proteins called transcription factors that can bind to
specific DNA sequences and regulate gene expression. Transcription factors can be engineered to
carry the desired genetic material and deliver it to the plant cells.
The process typically includes the following steps:
1. Protein-DNA Interaction: The engineered transcription factor binds to the specific DNA
sequences in the plant genome, carrying the genetic material that needs to be transferred.
2. Cellular Uptake: The protein-DNA complex is taken up by the plant cells, often facilitated by
cell-penetrating peptides or other mechanisms that help the complex cross the cell membrane.
3. Release of Genetic Material: Once inside the cell, the protein releases the genetic material,
allowing it to interact with the plant's cellular machinery.
4. Gene Expression: The transferred genetic material is then expressed by the plant cell, leading
to the production of specific proteins encoded by the transferred gene.
This method of gene transfer using proteins provides a targeted and controlled way to introduce
new genetic material into plants, enabling researchers to modify plant traits for various purposes
such as improving crop characteristics or enhancing plant resistance to environmental stresses.

INDIRECT PLANT TRANSFORMATION METHODS:

BIOLOGICAL METHODS:
INTRODUCTION:
Biological methods of gene transfer refer to the processes by which foreign genetic material is
introduced into an organism's cells using biological agents or naturally occurring mechanisms.
These methods are widely used in genetic engineering, biotechnology, and research. They utilize
the natural ability of certain organisms or molecular tools to insert genes into host genomes,

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either for functional studies, therapeutic purposes, or to produce genetically modified organisms
(GMOs). There are various biological approaches to gene transfer, each with its own
mechanisms and applications. Below are some of the most common biological methods of gene
transfer:

Agrobacterium Mediated Gene Transfer Methods:

Plant transformation based on Agrobacterium-mediated transformation is a technique that
mimics the natural agrobacterium system for gene(s) introduction into crops. Through this
technique, various crop species have been improved/modified for different trait/s, showing a
successful genetic transformation so far. This technique has many advantages over other
transformation methods such as stable integration of transgene, cost effective. However, there are
many limitations of this technology such as mostly the crops are recalcitrant to agrobacterium,
low transformation efficiency, transgene integration as well as off targets. So, it’s very important
to explore the major limitations and possible solutions for Agrobacterium-mediated
transformation in order to increase its genetic transformation efficiency.

THROUGH AGAROBACTERIUM RHIZOGENES:

Agrobacterium rhizogenes is a soil bacterium that is closely related to Agrobacterium
tumefaciens, both of which belong to the family Rhizobiaceae. This bacterium is known for its
ability to induce root overgrowth in plants, a process that leads to the formation of "hairy roots."
Agrobacterium rhizogenes contains a root-inducing plasmid (Ri plasmid), which carries the T-DNA
(transfer DNA) that is responsible for the transformation. The bacterium infects plant tissue, particularly
at wound sites, and transfers T-DNA into the plant's genome. This results in the uncontrolled growth of
roots, which is beneficial in various plant biotechnology applications, such as plant transformation and
the production of secondary metabolites.

MECHANISM:
Plant Signal Recognition: Acetosyringone is a phenolic compound that is naturally produced by
plants, especially in response to wounding or stress. This compound is secreted by plants at the
site of injury, such as when a plant is damaged by herbivores, pathogens, or mechanical
wounding.
Recognition by A. rhizogenes: When A. rhizogenes comes into contact with plant tissue (e.g., at
a wound site), it recognizes acetosyringone as a key signal. The bacterium has specific receptors
that detect acetosyringone in the plant's extracellular environment. This recognition is critical for
the activation of the Vir genes (virulence genes) located on the Ri plasmid of A. rhizogenes. The
binding of acetosyringone to these receptors triggers a signal transduction pathway within the
bacterium.
Activation of Virulence Genes: The binding of acetosyringone activates the Vir genes in A.
rhizogenes, particularly the VirA and VirG proteins.
 VirA is a sensor kinase that detects acetosyringone and undergoes a conformational
change, becoming autophosphorylated in the presence of acetosyringone.

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  The phosphorylation of VirA then activates VirG, a response regulator, which in turn
activates the expression of other Vir genes (such as VirD, VirE, and others). These
proteins are essential for processing the T-DNA from the Ri plasmid and its transfer into
the plant cell.
T-DNA Transfer and Plant Transformation: The activated Vir proteins (such as VirD and VirE)
process and mobilize the T-DNA, which is then transferred into the plant cell via a type IV
secretion system. The T-DNA integrates into the plant genome, and the transferred genes,
including those responsible for root overgrowth, begin to express themselves, leading to the
formation of hairy roots.
Induced Root Formation: The genes transferred into the plant, particularly the rol genes (root-
inducing genes), stimulate the abnormal formation of roots. These roots, referred to as "hairy
roots," exhibit uncontrolled, rapid growth and often produce high levels of auxins, leading to the
characteristic appearance of dense, fibrous root systems.

THROUGH AGAROBACTERIUM TUMEFACIENS:

Plant genetic transformation heavily relies on the bacterial pathogen Agrobacterium tumefaciens
as a powerful tool to deliver genes of interest into a host plant. Inside the plant nucleus, the
transferred DNA can integrate into the plant genome for inheritance to the next generation (i.e.
stable transformation). Alternatively, the foreign DNA can transiently remain in the nucleus
without integrating into the genome but still be transcribed to produce desirable gene products
(i.e. transient transformation). From the discovery of A. tumefaciens to its wide application in
plant biotechnology, numerous aspects of the interaction between A. tumefaciens and plants have
been elucidated. This article aims to provide a comprehensive review of the biology and the
applications of Agrobacterium-mediated plant transformation, which may be useful for both
microbiologists and plant biologists who desire a better understanding of plant transformation,
protein expression in plants, and plant-microbe interaction.

MECHANISM:
Recognition and Attachment to Plant Cells: Agrobacterium tumefaciens typically infects
plants through wound sites, where the plant's defenses are weaker. The bacterium recognizes
specific plant signals, particularly phenolic compounds (such as acetosyringone) released by the
plant in response to wounding. These signals bind to receptors on the bacterial surface, which
trigger the activation of the Vir genes located on the Ti plasmid (tumor-inducing plasmid) of A.
tumefaciens.
2. Activation of Vir Genes: Upon detecting plant signals like acetosyringone, the bacterium
activates its Vir genes (VirA and VirG), which are essential for the T-DNA transfer process.
o VirA is a sensor kinase that detects plant signals and activates VirG, a
transcriptional regulator.
o VirG then activates the other Vir genes involved in processing the T-DNA and its
transfer to the plant cell.

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3. Processing of T-DNA: The T-DNA is a specific region of the Ti plasmid that carries the genes
responsible for the disease (tumor formation) and is eventually transferred to the plant genome.
VirD proteins cleave the T-DNA from the Ti plasmid, producing a single-stranded copy of the T-
DNA. The T-DNA is then bound by VirE proteins, which protect it during transfer and facilitate
its passage into the plant cell.
4. Transfer of T-DNA into Plant Cells: The T-DNA is transferred into the plant cell through a
specialized structure called the type IV secretion system, which forms a pilus-like structure that
facilitates the passage of T-DNA. The T-DNA is transferred into the plant cell's cytoplasm, where
it is delivered into the plant’s nucleus.
5. Integration of T-DNA into Plant Genome inside the plant cell, the T-DNA is integrated into the
plant’s genome at random locations. This integration is mediated by plant enzymes, and the T-
DNA is now a permanent part of the plant’s DNA. The T-DNA typically contains genes that code
for the production of plant hormones, such as auxins and cytokinins, leading to uncontrolled cell
division and the formation of crown galls (tumors) at the wound site.
6. Expression of T-DNA Genes: The genes within the T-DNA are expressed in the plant cells,
leading to the abnormal growth of plant tissues. This results in the formation of galls, which are
often seen as swollen masses of cells at the infection site. Additionally, some T-DNA regions
carry genes that provide the plant with resistance to antibiotics such as kanamycin, which are
often used as selectable markers in genetic experiments.
7. Tumor Formation and Symptom Development: The T-DNA includes genes like oncogenes
(rol genes) that induce the production of excessive growth regulators (auxins and cytokinins),
promoting the formation of tumors (crown galls). These galls are usually formed at the infection
site, but their uncontrolled growth is the result of the expression of foreign genetic material in the
plant genome.

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VIRUS-MEDIATED TRANSFORMATION:
The gene can be packed into a virus and allow it to infect the host cell without harming it in any
way. This method can be used both for the transformation of prokaryotic host cell as well as
transfection of eukaryotic host cells. In the case of bacterial host cells the recombinant DNA can
be packed into the empty head of a specially designed bacteriophage (e.g., lambda phage) and
allow the virion to infect the host cell.
Similarly, while transfecting the plant host cells we can follow the similar strategy by using plant
viruses like Caulimo virus and Gemini virus. In the case of animals, retrovirus infection of
embryos has been used to produce transgenic mice.
This virus has been found to be an efficient vector system for animals. The virus carrying the
gene of interest transfers it into the genome of embryonic cells leading to its integration and
production of transgenic animals.

PLANT VIRUSES AS VECTORS:

Plant viruses are considered as efficient gene transfer agents as they can infect the intact plants
and amplify the transferred genes through viral genome replication. Viruses are natural vectors
for genetic engineering. They can introduce the desirable gene(s) into almost all the plant cells
since the viral infections are mostly systemic.

CRITERIA FOR A PLANT VIRUS VECTOR:

An ideal plant virus for its effective use in gene transfer is expected to posses the following
characteristics:

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i. The virus must be capable of spreading from cell to cell through plasmodesmata.
ii. The viral genome should be able to replicate in the absence of viral coat protein and spread
from cell to cell. This is desirable since the insertion of foreign DNA will make the viral genome
too big to be packed.
iii. The recombinant viral genome must elicit little or no disease symptoms in the infected plants.
iv. The virus should have a broad host range.
v. The virus with DNA genome is preferred since the genetic manipulations involve plant DNA.

CAULIMOVIRUSES AS VECTORS:
The caulimoviruses contain circular double- stranded DNA, and are spherical in shape.
Caulimoviruses are widely distributed and are responsible for a number of economically
important diseases in various crops. The caulimovirus group has around 15 viruses and among
these cauliflower mosaic viruses (CaMV) is the most important for gene transfer. The other
caulimoviruses include carnation etched virus, dahlia mosaic virus, mirabilis mosaic virus and
strawberry vein banding virus.

CAULIFLOWER MOSAIC VIRUS (CAMV):

CaMV infects many plants (e.g. members of Cruciferae, Datura) and can be easily transmitted,
even mechanically. Another attractive feature of CaMV is that the infection is systemic, and large
quantities of viruses are found in infected cell.
Geminiviruses: It belongs to the genus Geminivirus within the family Geminiviridae, having
circular single-stranded DNA with the size of 3.2-5.2kb and can be engineered to carry
heterologous coding sequences. The replication of geminiviruses relies primarily on the
replication-associated protein (Rep), which is essential for initiating rolling-circle replication.
This process allows for high viral genome copy numbers within infected cells. Geminivirus
vectors can be engineered using either an entire virus strategy, where most viral features are
retained, or a deconstructed approach that removes non-essential components, allowing for the
insertion of foreign DNA sequences. In the host cell nucleus, the single stranded DNA (ssDNA)
genome of geminiviruses undergoes replication through a rolling-circle mechanism, utilizing
double-stranded DNA (dsDNA) intermediates, similar to the replication process of ssDNA
bacteriophages. The International Committee on Virus Taxonomy (ICTV) classifies the
Geminiviridae family into fourteen genera: Becurtovirus, Begomovirus, Capulavirus, Curtovirus,
Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus, Turncurtovirus, Citlodavirus, Maldovirus,
Mulcrilevirus, Opunvirus and Topilevirus. This classification is based on factors such as vector
(insect pest), host range, genome organization and sequence identity. They are highly suitable for
creating viral vectors ingenetic engineering due to their ease of manipulation, ability to replicate
without integrating into the plant genome and capacity to deliver DNA repair templates.
However, their use is constrained by limited carrying capacity and a restricted host range (23,24).
Example: Wheat Dwarf India Virus (WDIV), The detection of WDIV in naturally infected wheat,
barley and sugarcane in the field-along with its demonstrated ability to systemically infect wheat,

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oat, barley, corn, soybean and tobacco under laboratory conditions-provides strong evidence that
WDIV is the first Gemini virus capable of infecting both monocot and dicot plant species by
using PDS as a reporter gene which results in 80-90% of plants shows symptoms.

IN PLANTA:
MERISTEM:
1. Target Tissue: Actively dividing meristematic tissues (e.g., shoot apical meristems) are
targeted for transformation.

2. GENE DELIVERY METHODS:

o Agrobacterium-mediated transformation: Agrobacterium tumefaciens carrying
the gene of interest is applied to the meristem (e.g., injection, soaking).
o Particle bombardment: DNA-coated microprojectiles are delivered directly to the
meristem.
o Electroporation or direct DNA application: DNA is introduced directly into
meristem cells.

3. INTEGRATION AND REGENERATION:

o The foreign gene integrates into the plant genome during cell division in the
meristem.
o The transformed cells give rise to shoots, leading to a fully developed transgenic
plant.

4. ADVANTAGES:

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 o Bypasses tissue culture.
o Saves time and reduces somaclonal variation.

Floral dip transfer method:

The floral dip method is a popular technique used for gene transfer in plants, especially in
Arabidopsis thaliana, a model plant species commonly used in research. Here's how the floral dip
method works:
1. Preparation of Agrobacterium tumefaciens: Agrobacterium tumefaciens is a bacterium
commonly used in genetic engineering to transfer DNA into plants. Scientists engineer this
bacterium to carry the desired gene(s) to be transferred.
2. Selection of Plants: The plants intended for gene transfer are usually in the flowering stage.
The flowers should be at a specific developmental stage to ensure successful transformation.
3. Dipping Process: The plants are inverted and immersed upside down into a solution
containing the Agrobacterium tumefaciens carrying the desired gene(s). The flowers are dipped
gently to ensure that the bacterial solution reaches the reproductive structures of the plant.
4. Recovery and Growth: After the dipping process, the plants are allowed to recover and grow
under controlled conditions. The transformed plants are then selected using appropriate markers
or selection agents.
This method is efficient for introducing genes into plants without the need for tissue culture. It
allows for the transformation of a large number of plants quickly

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Pollen transformation method:
The pollen transformation method is a technique used to introduce foreign genes in to plants by
targeting the pollen grains. This method is particularly useful for plants that are
difficult to transform using other techniques or for species that do not produce seeds easily.
Here's how the pollen transformation method generally works:
1. Isolation of Pollen: Pollen grains are collected from the flowers of the plant species being
studied. The pollen is usually isolated and purified to ensure that only the desired pollen grains
are used for transformation.
2. Gene Transfer: The foreign gene(s) of interest are introduced into the isolated pollen grains.
This can be achieved through various methods such as ballistics (gene gun), electroporation, or
using Agrobacterium tumefaciens.
3. Pollen Germination: The transformed pollen grains are then allowed to germinate at a suitable
growth medium. The pollen tubes grow and penetrate the stigma of the flower.
4. Fertilization: During the process of fertilization, the transformed pollen grains deliver the
foreign gene(s) to the ovule, which eventually develops into seeds containing the new genetic
material.
5. Seed Production: The seeds produced from this method carry the introduced gene(s) and can
be grown into plants for further study and analysis.

THE END

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