Dr.

Arundhati Banerjee (Notes)

Genetic engineering is the manipulation of DNA sequences in organisms using biotechnology.

This process involves knocking out DNA sequences from organisms, as well as changing and adding new
segments of DNA. Genetic engineering is widely used in biology research, food production, and medical
therapies to alter phenotypes and functions of target organisms for desired traits. Agrobacterium
tumefaciens is a rod-shaped and gram-negative soil bacterium. This species of bacteria infects plants by
inducing crown gall disease. A.tumefaciens could infect the healthy plants with a small segment of DNA
from the plasmids of bacteria called T-DNA. The infected DNA segment is tumor-inducing. It triggers
hormone release by the plant's cells and stimulates tumorous growth. Tumor cells in plants then release
“opines”, which can be used by A.tumefaciens as a source of carbons and nitrogen for metabolism.
The ability of A.tumefaciens to integrate DNA segments into plant cells is widely used by scientists in the
field of genetic engineering. In the engineered A.tumefaciens, the oncogene in the plasmids is knocked out
and replaced by the gene of interest. Without destroying the ability for A.tumefaciens to integrate its DNA
into a plant cell, the engineered plasmids successfully induce the gene of interest such as traits that
promote crops growth and make the fruits taste better.

Agrobacterium-mediated transformation

This illustration depicts the specific steps of Agrobacterium-mediated transformation. After target gene is
inserted into T-DNA, it integrated into chromosome of cultured plant cells, which is then transferred into
the target plants Agrobacterium-mediated transformation.

Agrobacterium genus is well-known for its ability to transfer genetic material, specifically DNA, into plant

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Dr. Arundhati Banerjee (Notes)

cells, which has made it an important tool for the production of genetically modified (GM) crops.
Agrobacterium tumefaciens is the most commonly studied species for genetic engineering.
Researchers have harnessed the ability of Agrobacterium. to introduce target DNA segments into plant
cells for genetic engineering purposes. The process of utilizing T-DNA of Agrobacterium genus is called
Agrobacterium-mediated transformation (AMT). This process requires researchers to expose the
engineered Agrobacterium cells to plant tissues. After Agrobacterium successfully transfers T-DNA to the
plants, the target DNA segments will be integrated into plant cells, and the target genes can be further
replicated by plant cells. A.tumefaciens is most frequently used species to conduct AMT because of its
relatively simple genetic makeup and well-characterized mechanisms for T-DNA transfer of DNA into
plants.
In addition, the research team led by Michielse have found that the efficiency of AMT increases when
several genes of Wild Type A.tumefaciens are mutated. They first discovered that mutations
in A.tumefaciens genes chvA, chvB, or exoC don’t affect the efficiency of AMT. These genes are
responsible for the production of synthesis and export of β-1,2-glucan, as well as the attachment and
subsequent transformation in the plant infection process. However, the mutated versions of chvA, chvB, or
exoC don’t affect AMT. The roles of these genes in AMT are rejected by Michielse and his research team.
Besides, they found that the inactivation of gene Cel1, which is responsible for the cellulose production,
has led to a seven-fold increase in AMT efficiency. Their result showed that the cellulose fibers have a
negative influence on the attachment and infection of A.tumefaciens to their hosts. The discovery led by
Michielse and his research team has helped the scientists to design specific mutated A.tumefaciens strain to
conduct AMT in a much more efficient way.
Genome Structure

The unique action mode of A.tumefaciens benefits from its genomic structure. A.tumefaciens contains a
mix of linear and circular chromosomes. Its circular plasmids promote the invasion of genetic material to
its host. Circular plasmids are smaller in size compared to the chromosome of bacteria. While genes in
chromosomes have instructions for basic functions of bacterial cells, such as cell division metabolis, genes
in plasmids usually code for secondary functions, such as antibiotics resistance and cell to cell
communication. Therefore, A.tumefaciens utilized the characteristics that plasmids are small, and the
transmission of plasmids genes are not lethal to achieve infections. T-DNA is the segment of DNA that is
excised from the Ti-plasmid of A.tumefaciens that is transferred into the target plants. T-DNA in plasmids
is transferred and incorporated into the host plants’ genome when the infection process is completed.
Two component regulators

Moreover, the infection process requires two component regulators of virA and virG in the vir operon
by A.tumefaciens cells. The wounded plant tissue is the environmental signal to trigger A.tumefaciens cells
to bind. A.tumefaciens cells could make complex biofilms at the colonization site on wounded tissues. The
monosaccharides of sugars released by plants are detected by virA transmembrane receptor kinase. The
receptor kinase then triggers autophosphorylation. Phosphate is transferred to virG, which is the activator
that initiates the transcription of vir operon. The proteins generated by vir operon facilitate the
transportation of T-DNA into the plant cells. T-DNA is then integrated into the plant genomes by non-
homologous recombination.
In addition, virA and virG two component systems are significant mediators of signal transduction that
enables A.tumefaciens to respond quickly to the environmental change. Recent evidence has shown that
virA and virG two component systems can adapt to function in tobacco protoplasts. The vir operon can be
efficiently transcribed with eukaryotic transcriptional apparatus in tobacco protoplasts. The system allows
the plants to react quickly to environmental changes such as pH.

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Dr. Arundhati Banerjee (Notes)

Genes in T-DNA

This picture depicts the Ti-plasmid of A.tumefaciens. The T-DNA segment contains right and left border
sequences along with plant hormone coding genes. Ti-Plasmid.

Genes in T-DNA of A.tumefaciens are mainly involved in transferring genetic materials into plants,
extracting nutrients from the plants and resulting in formation of tumors in the plants. Three components of
T-DNA helping A.tumefaciens to achieve these functions are border sequences, transfer genes and
oncogenes. Border sequences are the recognition sites for the excision of T-DNA from the Ti-plasmid.
The border sequence is significant in helping A.tumefaciens to excise all sequences of T-DNA, while not
cutting other parts of the plasmids. Transfer genes are mainly responsible for the transfer process of T-
DNA into the plant cells. Transfer genes include virA, virD1, virD2, virD3, VirF and virG. After Vir A
detects the sugar released by plants, Vir G is auto phosphorylated, which triggers increased Vir operon
expression. As a result, virD1, virD2, virD3, VirF are produced. Vir D1 and Vir D2 are involved in the
protection of T-DNA and integration of T-DNA into plants' genomes. VirD3 and Vir F play an important
role in the transfer process of T-DNA.. Oncogenes of T-DNA directly result in the uncontrolled cell
growth and tumor formation in the plants. The oncogenes could trigger the synthesis of many growth
hormones by the infected plants, such as the release of auxins and cytokinin. The normal developmental
cycle of plants is disrupted, and tumors can form as a result of uncontrolled development.
Researchers have identified some oncogenes. Two A.tumefaciens T-DNA auxin biosynthesis genes
products called iaaM and iaaH are identified to trigger the auxin overproduction and uncontrolled cell
growth in plants. These two genes work together to encode enzymes that produce indoleacetic acid (IAA),
which is a common plant hormone of auxin class. In particular, iaaM encodes tryptophan monooxygenase
to convert tryptophan to indoleacetamide (IAM). iaaH then encodes indoleacetamide hydrolase to convert
IAM to IAA. The release of IAA promotes into plant tissues causes cell elongation and cell growth in the
plants. Auxin is essential for the regulation of plant growth and development. Most plants are very
sensitive to the change of Auxin. Changes in micromolar amounts of auxins can cause dramatic changes in
plant phenotypes and development, while changes in millimolar amounts of auxins can be lethal to plants.
This also explains why infection by A.tumefaciens induces tumor formation in plants. Severe infections
by A.tumefaciens to susceptible plant species can even lead to plant death.

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Dr. Arundhati Banerjee (Notes)

Besides transferring genetic materials to the plant cells, A.tumefaciens can also transfer its genetic
materials to other bacteria by conjugation. Conjugation is a type of horizontal gene transfer, which is the
transfer of genetic material between bacteria that are not parent and offspring. During
conjugation, A.tumefacienss can transfer the Ti plasmid to other bacteria in close proximity through a
specialized structure called a conjugative pilus, which forms a physical bridge between the donor
bacterium and the recipient bacterium. The Ti plasmid is then transferred from the donor to the recipient,
and the recipient bacterium can acquire the ability to cause crown gall disease in plants if it receives the
complete Ti plasmid, including the T-DNA region. The mechanism of conjugation allows A.tumefaciens to
spread its virulence genes to other A.tumefaciens cells. The recipient cells can then gain the ability to infect
crown gall disease to different plant tissues.
Genetic engineering in crop improvement

In addition to medical use, A.tumefaciens genetic engineering has also been utilized as a powerful tool for
crop improvement. By exploiting its natural ability to transfer DNA into the genomes of
plants, A.tumefaciens has been employed to introduce desirable traits into crops, leading to improved
agricultural production. For instance, genes that confer resistance to pests, diseases, or environmental
stress have been successfully inserted into crops, providing increased yield and reduced crop losses.
Additionally, A.tumefaciens has been used to develop herbicide-tolerant crops, enabling more efficient
weed control. These advancements in crop improvement using A.tumefaciens have the potential to
contribute to sustainable agriculture by enhancing crop productivity and reducing the reliance on chemical
pesticides.
Usually, selectable marker genes are integrated into the T-DNA of A.tumefaciens. Different crop species
need different marker genes to increase its viability and production in the environment. Researchers
carefully select the corresponding marker genes based on the crop species. For example, the most
commonly used markers in cereals to confer resistance to antibiotics and herbicides hygromycin
phosphotransferase (hpt) and phosphinothricin acetyltransferase (pat). Gene hpt is the best selectable
marker for rice and barley, while gene pat is most selectable for maize.
Furthermore, most marker genes are tissue-specific, and T-DNA of A.tumefaciens must be transferred to
the specific tissue type to induce the gene expression. For example, rice globulin (Glb) promoter and wheat
glutenin gene promoter (Glu) are used for endosperm-specific gene expression. Chlorophyll a/b binding
(cab) gene is leaf-specific for rice. The tissue-specific induction of gene expression is also seen in other
techniques of genetic engineering, such as promoter engineering.
When transferring T-DNA of A.tumefaciens into crops, several key factors should be considered, which
include type of stage of the plants, the strain of Agrobacterium, type of vectors, acetosyringone and co-
cultivation temperature. According to the previous experiments, plant tissues in active dividing stages are
most suitable for the transformation since there will be higher frequencies of expression for the
incorporated genes. For Agrobacterium strains, it has been shown that the super-virulent strain is most
suitable for the plant transformation.
Conclusion

In conclusion, A.tumefaciens has revolutionized genetic engineering and biotechnology by serving as a

powerful tool for introducing foreign genes into plants. Its natural ability to transfer DNA to plant cells has
been harnessed to create genetically modified organisms (GMOs) with desired traits, such as resistance to
pests, and tolerance to environmental conditions. This has led to significant advancements in agriculture,
including increased crop yields, and improved food quality. Genetic engineering of A.tumefaciens is also
utilized to design medicines with higher tolerance to temperature variation and increase the cost-
performance ratio for pharmaceutical industries.
However, the use of A.tumefaciens in genetic engineering also raises ethical, social, and environmental
concerns. Critics argue that GMOs may have unknown long-term effects on ecosystems and human health
and may contribute to the loss of biodiversity and the dependence on large seed corporations. Therefore, it
is crucial to conduct thorough risk assessments and adopt responsible practices when
using A.tumefaciens and other genetic engineering techniques.

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Dr. Arundhati Banerjee (Notes)

Despite the controversies surrounding GMOs, A.tumefaciens remains a valuable tool in plant
biotechnology, with immense potential for addressing global challenges such as food security, health
issues, and sustainable agriculture. Continued research and regulatory oversight are essential to ensure the
safe and responsible use of A.tumefaciens and other genetic engineering technologies, while balancing the
benefits and risks associated with GMOs. Overall, A.tumefaciens has paved the way for genetic
engineering to become a powerful tool in modern agriculture, offering a promising future for crop
improvement and medical treatments.

Agrobacterium rhizogenes is the etiological agent for hairy-root disease (also known as root-mat disease).
This bacterium induces the neoplastic growth of plant cells that differentiate to form “hairy roots.”
Morphologically, A. rhizogenes-induced hairy roots are very similar in structure to wild-type roots with a
few notable exceptions: Root hairs are longer, more numerous, and root systems are more branched and
exhibit an agravitropic phenotype. Hairy roots are induced by the incorporation of a bacterial-derived
segment of DNA transferred (T-DNA) into the chromosome of the plant cell. The expression of genes
encoded within the T-DNA promotes the development and production of roots at the site of infection on
most dicotyledonous plants. A key characteristic of hairy roots is their ability to grow quickly in the
absence of exogenous plant growth regulators. As a result, hairy roots are widely used as a trans-genic tool
for the production of metabolites and for the study of gene function in plants. Researchers have utilized
this tool to study root development and root–biotic interactions, to overexpress proteins and secondary
metabolites, to detoxify environmental pollutants, and to increase drought tolerance. In this review, we
provide an up-to-date overview of the current knowledge of how A. rhizogenes induces root formation, on
the new uses for A. rhizogenes in tissue culture and composite plant production (wild-type shoots with
transgenic roots), and the recent development of a disarmed version of A. rhizogenes for stable transgenic
plant production.

GENE TRANSFER IN PLANTS:

Gene Transfer Methods:

The gene transfer techniques in plant genetic transformation are broadly grouped into two
categories:
I. Vector-mediated gene transfer

II. Direct or vector less DNA transfer

The salient features of the commonly used gene (DNA) transfer methods are given in Table 49.1.

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Dr. Arundhati Banerjee (Notes)

Method # I. Vector-Mediated Gene Transfer:

Vector-mediated gene transfer is carried out either by Agrobacterium-mediated transformation or by use of
plant viruses as vectors.

Agrobacterium-Mediated Gene Transfer:

Agrobacterium tumefaciens is a soil-borne, Gram-negative bacterium. It is rod shaped and motile, and
belongs to the bacterial family of Rhizobiaceae. A. tumefaciens is a phytopathogen, and is treated as the
nature’s most effective plant genetic engineer.

Some workers consider this bacterium as the natural expert of inter-kingdom gene transfer. In fact, the
major credit for the development of plant transformation techniques goes to the natural unique capability
of A. tumefaciens. Thus, this bacterium is the most beloved by plant biotechnologists.

There are mainly two species of Agrobacterium:

i. A. tumefaciens that induces crown gall disease.

ii. A. rhizogenes that induces hairy root disease.

Crown Gall Disease and Ti Plasmid:

Almost 100 years ago (1907), Smith and Townsend postulated that a bacterium was the causative agent of
crown gall tumors, although its importance was recognized much later. As A. tumefaciens infects wounded
or damaged plant tissues, in induces the formation of a plant tumor called crown gall (Fig. 49.1). The entry

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Dr. Arundhati Banerjee (Notes)

of the bacterium into the plant tissues is facilitated by the release of certain phenolic compounds
(acetosyringone, hydroxyacetosyringone) by the wounded sites.

Crown gall formation occurs when the bacterium releases its Ti plasmid (tumor- inducing plasmid) into the
plant cell cytoplasm. A fragment (segment) of Ti plasmid, referred to as T-DNA, is actually transferred
from the bacterium into the host where it gets integrated into the plant cell chromosome (i.e. host genome).
Thus, crown gall disease is a naturally evolved genetic engineering process.

The T-DNA carries genes that code for proteins involved in the biosynthesis of growth hormones (auxin
and cytokinin) and novel plant metabolites namely opines — amino acid derivatives and agropines —
sugar derivatives.

The growth hormones cause plant cells to proliferate and form the gall while opines and agropines are
utilized by A. tumefaciens as sources of carbon and energy. As such, opines and agropines are not
normally part of the plant metabolism (neither produced nor metabolised). Thus, A. tumefaciens
genetically transforms plant cells and creates a biosynthetic machinery to produce nutrients for its own use.

As the bacteria multiply and continue infection, grown gall develops which is a visible mass of the
accumulated bacteria and plant material. Crown gall formation is the consequence of the transfer,
integration and expression of genes of T-DNA (or Ti plasmid) of A. tumefaciens in the infected plant.

The genetic transformation leads to the formation of crown gall tumors, which interfere with the normal
growth of the plant. Several dicotyledonous plants (dicots) are affected by crown gall disease e.g. grapes,
roses, stone-fruit trees.

Organization of Ti plasmid:
The Ti plasmids (approximate size 200 kb each) exist as independent replicating circular DNA molecules
within the Agrobacterium cells. The T-DNA (transferred DNA) is variable in length in the range of 12 to
24 kb, which depends on the bacterial strain from which Ti plasmids come. Nopaline strains of Ti plasmid

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Dr. Arundhati Banerjee (Notes)

have one T-DNA with length of 20 kb while octopine strains have two T-DNA regions referred to as
TL and TR that are respectively 14 kb and 7 kb in length.
A diagrammatic representation of a Ti plasmid is depicted in Fig. 49.3. The Ti plasmid has three important
regions.

1. T-DNA region:
This region has the genes for the biosynthesis of auxin (aux), cytokinin (cyt) and opine (ocs), and is
flanked by left and right borders. These three genes-aux, cyto and ocs are referred to as oncogenes, as they
are the determinants of the tumor phenotype.

T-DNA borders — A set of 24 kb sequences present on either side (right and left) of T-DNA are also
transferred to the plant cells. It is now clearly established that the right border is more critical for T-DNA
transfer and tumori-genesis.

2. Virulence region:
The genes responsible for the transfer of T-DNA into the host plant are located outside T-DNA and the
region is referred to as vir or virulence region. Vir region codes for proteins involved in T-DNA transfer.
At least nine vir-gene operons have been identified. These include vir A, vir G, vir B1, vir C1, vir D1,
D2 and D4, and vir E1, and E2.
3. Opine catabolism region:
This region codes for proteins involved in the uptake and metabolisms of opines. Besides the above three,
there is ori region that is responsible for the origin of DNA replication which permits the Ti plasmid to be
stably maintained in A. tumefaciens.

T-DNA transfer and integration:

The process of T-DNA transfer and it integration into the host plant genome is depicted in Fig. 49.4, and is
briefly described.

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Dr. Arundhati Banerjee (Notes)

1. Signal induction to Agrobacterium:

The wounded plant cells release certain chemicals- phenolic compounds and sugars which are recognized
as signals by Agrobacterium. The signals induced result in a sequence of biochemical events in
Agrobacterium that ultimately helps in the transfer of T-DNA of T-plasmid.

2. Attachment of Agrobacterium to plant cells:

The Agrobacterium attaches to plant cells through polysaccharides, particularly cellulose fibres produced
by the bacterium. Several chromosomal virulence (chv) genes responsible for the attachment of bacterial
cells to plant cells have been identified.

3. Production of virulence proteins:

As the signal induction occurs in the Agrobacterium cells attached to plant cells, a series of events take
place that result in the production of virulence proteins. To start with, signal induction by phenolics
stimulates vir A which in turn activates (by phosphorylation) vir C. This induces expression of virulence
genes of Ti plasmid to produce the corresponding virulence proteins (D1, D2, E2, B etc.). Certain sugars
(e.g. glucose, galactose, xylose) that induce virulence genes have been identified.
4. Production of T-DNA strand:
The right and left borders of T-DNA are recognized by vir D1/vir D2 proteins. These proteins are involved
in the production single-stranded T-DNA (ss DNA), its protection and export to plant cells. The ss T-DNA
gets attached to vir D2.
5. Transfer of T-DNA out of Agrobacterium:
The ss T-DNA — vir D2 complex in association with vir G is exported from the bacterial cell. Vir B
products form the transport apparatus.
6. Transfer of T-DNA into plant cells and integration:
The T-DNA-vir D2 complex crosses the plant plasma membrane. In the plant cells, T-DNA gets covered
with vir E2. This covering protects the T-DNA from degradation by nucleases; vir D2 and vir E2 interact
with a variety of plant proteins which influences T-DNA transport and integration.

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Dr. Arundhati Banerjee (Notes)

The T-DNA-vir D2-vir E2 — plant protein complex enters the nucleus through nuclear pore complex.
Within the nucleus, the T-DNA gets integrated into the plant chromosome through a process referred to
illegitimate recombination. This is different from the homologous recombination, as it does not depend on
the sequence similarity.

Hairy Root Disease of A. Rhizogenes — R1 Plasmids:

Agrobacterium rhizogenes can also infect plants. But this results in hairy roots and not crown galls as is the
case with A. tumefaciens. The plasmids, of A. rhizogenes have been isolated and characterized. These
plasmids, referred to as Ri plasmids, (Ri stands for Root inducing) are of different types. Some of the Ri
plasmid strains possess genes that are homologous to Ti plasmid e.g. auxin biosynthetic genes.

Instead of virulence genes, Ri plasmids contain a series of open reading frames on the T-DNA. The
products of these genes are involved in the metabolism of plant growth regulators which gets sensitized to
auxin and leads to root formation.

Vectors of A. rhizogenes:
As it is done with A tumefaciens, vectors can be constructed by using A. rhizogenes. These vectors are
alternate strategies for gene transfer. However, employment of A. rhizogene-based vectors for plant
transformation is not common since more efficient systems of A. tumefaciens have been developed.

Importance of hairy roots:

Hairy roots can be cultured in vitro, and thus are important in plant biotechnology. Hairy root systems are
useful for the production of secondary metabolites, particularly pharmaceutical proteins.

Ti Plasmid-Derived Vector Systems:

The ability of Ti plasmid of Agrobacterium to genetically transform plants has been described. It is
possible to insert a desired DNA sequence (gene) into the T-DNA region (of Ti plasmid), and then use A.
tumefaciens to deliver this gene(s) into the genome of plant cell.

In this process, Ti plasmids serve as natural vectors. However, there are several limitations to use Ti
plasmids directly as cloning vectors:
i. Ti plasmids are large in size (200-800 kb). Smaller vectors are preferred for recombinant experiments.
For this reason, large segments of DNA of Ti plasmid, not essential for cloning, must be removed.

ii. Absence of unique restriction enzyme sites on Ti plasmids.

iii. The phytohormones (auxin, cytokinin) produced prevent the plant cells being regenerated into plants.
Therefore auxin and cytokinin genes must be removed.

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Dr. Arundhati Banerjee (Notes)

iv. Opine production in transformed plant cells lowers the plant yield. Therefore opine synthesizing genes
which are of no use to plants should be removed.

v. Ti plasmids cannot replicate in E. coli. This limits their utility as E. coli is widely used in recombinant
experiments. An alternate arrangement is to add an origin of replication to Ti plasmid that allows the
plasmid to replicate in E. coli.

Considering the above limitations, Ti plasmid- based vectors with suitable modifications have been
constructed.

These vectors are mainly composed of the following components:

1. The right border sequence of T-DNA which is absolutely required for T-DNA integration into plant cell
DNA.

2. A multiple cloning site (poly-linker DNA) that promotes the insertion of cloned gene into the region
between T-DNA borders.

3. An origin of DNA replication that allows the plasmids to multiply in E. coli.

4. A selectable marker gene (e.g. neomycin phosphotransferase) for appropriate selection of the
transformed cells.

Two types of Ti plasmid-derived vectors are used for genetic transformation of plants— co-integrate
vectors and binary vectors.

Co-integrate vector:
In the co-integrate vector system, the disarmed and modified Ti plasmid combines with an intermediate
cloning vector to produce a recombinant Ti plasmid (Fig. 49.5).

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Dr. Arundhati Banerjee (Notes)

Production of disarmed Ti plasmid:

The T-DNA genes for hormone biosynthesis are removed (disarmed). In place of the deleted DNA, a
bacterial plasmid (pBR322) DNA sequence is incorporated. This disarmed plasmid, also referred to as
receptor plasmid, has the basic structure of T-DNA (right and left borders, virulence genes etc.) necessary
to transfer the plant cells.

Construction of intermediate vector:

The intermediate vector is constructed with the following components:
i. A pBR322 sequence DNA homologous to that found in the receptor Ti plasmid.

ii. A plant transformation marker (PTM) e.g. a gene coding for neomycin phosphotransferase II (npt II).
This gene confers resistance to kanamycin in the plant cells and thus permits their isolation.

iii. A bacterial resistance marker e.g. a gene coding for spectinomycin resistance. This gene confers
spectinomycin resistance to recipient bacterial cells and thus permits their selective isolation.
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Dr. Arundhati Banerjee (Notes)

iv. A multiple cloning site (MCS) where foreign genes can be inserted.

v. A Co/E1 origin of replication which allows the replication of plasmid in E. coli but not in
Agrobacterium.
vi. An oriT sequence with basis of mobilization (bom) site for the transfer of intermediate vector from E.
coli to Agrobacterium.

Production and use of co-integrate vectors:

The desired foreign gene (target-gene) is first cloned in the multiple cloning site of the intermediate vector.
The cloning process is carried out in E. coli, the bacterium where the cloning is most efficient. The
intermediate vector is mated with Agrobacterium so that the foreign gene is mobilised into the latter.

The transformed Agrobacterium cells with receptor Ti plasmid and intermediate vector are selectively
isolated when grown on a minimal medium containing spectinomycin. The selection process becomes easy
since E. coli does not grow on a minimal medium in which Agrobacterium grows.

Within the Agrobacterium cells, intermediate plasmid gets integrated into the receptor Ti plasmid to
produce co-integrate plasmid. This plasmid containing plant transformation marker (e.g. npt II) gene and
cloned target gene between T-DNA borders is transferred to plant cells. The transformed plant cells can be
selected on a medium containing kanamycin when the plant and Agrobacterium cells are incubated
together.

Advantages of co-integrate vector:

i. Target genes can be easily cloned

ii. The plasmid is relatively small with a number of restriction sites.

iii. Intermediate plasmid is conveniently cloned in E. coli and transferred to Agrobacterium.

Binary vector:
The binary vector system consists of an Agrobacterium strain along with a disarmed Ti plasmid called vir
helper plasmid (the entire T-DNA region including borders deleted while vir gene is retained). It may be
noted that both of them are not physically linked (or integrated). A binary vector with T-DNA can replicate
in E. coli and Agrobacterium.

A diagrammatic representation of a typical binary vector system is depicted in Fig. 49.6. The binary vector
has the following components.

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Dr. Arundhati Banerjee (Notes)

1. Left and right borders that delimit the T-DNA region.

2. A plant transformation marker (PTM) e.g. npt II that confers kanamycin resistance in plant transformed
cells.

3. A multiple cloning site (MCS) for introducing target/foreign genes.

4. A bacterial resistance marker e.g. tetracycline resistance gene for selecting binary vector colonies in E.
coli and Agrobacterium.

5. oriT sequence for conjugal mobilization of the binary vector from E. coli to Agrobacterium.

6. A broad host-range origin of replication such as RK2 that allows the replication of binary vector in
Agrobacterium.
Production and use of binary vector:
The target (foreign) gene of interest is inserted into the multiple cloning site of the binary vector. In this
way, the- target gene is placed between the right and left border repeats and cloned in E. coli. By a mating
process, the binary vector is mobilised from E. coli to Agrobacterium. Now, the virulence gene proteins of
T-DNA facilitate the transfer of T-DNA of the vector into plant cells.

Advantages of binary vectors:

i. The binary vector system involves only the transfer of a binary plasmid to Agrobacterium without any
integration. This is in contrast to co-integrate vector system wherein the intermediate vector is transferred
and integrated with disarmed Ti plasmid.

ii. Due to convenience, binary vectors are more frequently used than co-integrate vectors.

Plant Transformation Technique Using Agrobacterium:

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Dr. Arundhati Banerjee (Notes)

Agrobacterium-mediated technique is the most widely used for the transformation of plants and generation
of transgenic plants. The important requirements for gene transfer in higher plants through Agrobacterium
mediation are listed.

i. The explants of the plant must produce phenolic compounds (e.g. autosyringone) for activation of
virulence genes.

ii. Transformed cells/tissues should be capable to regenerate into whole plants.

In general, most of the Agrobacterium-mediated plant transformations have the following basic protocol
(Fig. 49.7)

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Dr. Arundhati Banerjee (Notes)

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.

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Dr. Arundhati Banerjee (Notes)

4. Killing of Agrobacterium with a suitable antibiotic without harming the plant tissue.

5. Selection of transformed plant cells.

6. Regeneration of whole plants.

Advantages of Agrobacterium- mediated transformation:

i. This is a natural method of gene transfer.

ii. Agrobacterium can conveniently infect any explant (cells/tissues/organs).

iii. Even large fragments of DNA can be efficiently transferred.

iv. Stability of transferred DNA is reasonably good.

v. Transformed plants can be regenerated effectively.

Limitations of Agrobacterium- mediated transformation:

i. 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.

ii. 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.

Virus-Mediated Gene Transfer (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.

Plant viruses are non-integrative vectors:

The plant viruses do not integrate into the host genome in contrast to the vectors based on T-DNA of A.
tumefaciens which are integrative. The viral genomes are suitably modified by introducing desired foreign
genes. These recombinant viruses are transferred, multiplied and expressed in plant cells. They spread
systemically within the host plant where the new genetic material is expressed.

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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Dr. Arundhati Banerjee (Notes)

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.

The three groups of viruses — caulimoviruses, Gemini viruses and RNA viruses that are used as vectors
for gene transfer in plants are briefly described.

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 virus (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 cells.

A diagrammatic view of the CaMV genetic map is depicted in Fig. 49.8. The genome of CaMV consists of
a 8 kb (8024 bp) relaxed but tightly packed circular DNA with six major and two minor coding regions.
The genes II and VII are not essential for viral infection.

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Dr. Arundhati Banerjee (Notes)

Use of CaMV in gene transfer:

For appropriate transmission of CaMV, the foreign DNA must be encapsulated in viral protein. Further,
the newly inserted foreign DNA must not interfere with the native assembly of the virus. CaMV genome
does not contain any non-coding regions wherein foreign DNA can be inserted. It is fortunate that two
genes namely gene II and gene VII have no essential functions for the virus. It is therefore possible to
replace one of them and insert the desired foreign gene.

Gene II of CaMV has been successfully replaced with a bacterial gene encoding dihydrofolate reductase
that provides resistance to methotrexate. When the chimeric CaMV was transmitted to turnip plants, they
were systemically infected and the plants developed resistance to methotrexate.

Limitations of CaMV as a vector:

i. CaMV vector has a limited capacity for insertion of foreign genes.

ii. Infective capacity of CaMV is lost if more than a few hundred nucleotides are introduced.

iii. Helper viruses cannot be used since the foreign DNA gets expelled and wild-type viruses are produced.

Gemini Viruses as Vectors:

The Gemini viruses are so named because they have geminate (Gemini literally means heavenly twins)
morphological particles i.e. twin and paired capsid structures. These viruses are characterized by
possessing one or two single-stranded circular DNAs (ss DNA). On replications, ss DNA forms an
intermediate double-stranded DNA.

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Dr. Arundhati Banerjee (Notes)

The Gemini viruses can infect a wide range of crop plants (monocotyledons and dicotyledons) which
attract plant biotechnologists to employ these viruses for gene transfer. Curly top virus (CTV) and maize
streak virus (MSV) and bean golden mosaic virus (BGMV) are among the important Gemini viruses.

It has been observed that a large number of replicative forms of a Gemini virus genome accumulate inside
the nuclei of infected cells. The single-stranded genomic DNA replicates in the nucleus to form a double-
stranded intermediate.

Gemini virus vectors can be used to deliver, amplify and express foreign genes in several plants/ explants
(protoplasts, cultured cells). However, the serious drawback in employing Gemini viruses as vectors is that
it is very difficult to introduce purified viral DNA into the plants. An alternate arrangement is to take the
help of Agrobacterium and carry out gene transfer.

RNA Plant Viruses as Vectors:

There are mainly two type’s single-stranded RNA viruses:
1. Mono-partite viruses:
These viruses are usually large and contain undivided genomes for all the genetic information e.g. tobacco
mosaic virus (TMV).

2. Multipartite viruses:
The genome in these viruses is divided into small RNAs which may be in the same particle or different
particles, e.g. brome mosaic virus (BMV). HMV contains four RNAs divided between three particles.
Plant RNA viruses, in general, are characterized by high level of gene expression, good efficiency to infect
cells and spread to different tissues. But the major limitation to use them as vectors is the difficulty of
joining RNA molecules in vitro.

Use of cDNA for gene transfer:

Complementary DNA (cDNA) copies of RNA viruses are prepared in vitro. The cDNA so generated can
be used as a vector for gene transfer in plants. This approach is tedious and cumbersome. However, some
success has been reported. A gene sequence encoding chloramphenicol resistance (enzyme-
chloramphenicol acetyltransferase) has been inserted into brome mosaic virus genome. This gene
expression, however, has been confined to protoplasts.

Limitations of Viral Vectors in Gene Transfer:

The ultimate objective of gene transfer is to transmit the desired genes to subsequent generations. With
virus vectors, this is not possible unless the virus is seed-transmitted. However, in case of vegetatively
propagated plants, transmission of desired traits can be done e.g. potatoes. Even in these plants, there is
always a risk for the transferred gene to be lost anytime. For the reasons referred above, plant
biotechnologists prefer to insert the desired genes of interest into a plant chromosome.

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Dr. Arundhati Banerjee (Notes)

Method # II. Direct or Vector-less DNA Transfer:

The term direct or vector less transfer of DNA is used when the foreign DNA is directly introduced into
the plant genome. Direct DNA transfer methods rely on the delivery of naked DNA into the plant cells.
This is in contrast to the Agrobacterium or vector-mediated DNA transfer which may be regarded as
indirect methods. Majority of the direct DNA transfer methods are simple and effective. And in fact,
several transgenic plants have been developed by this approach.

Limitations of direct DNA transfer:

The major disadvantage of direct gene transfer is that the frequency of transgene rearrangements is high.
This results in higher transgene copy number, and high frequencies of gene silencing.

Types of direct DNA transfer:

The direct DNA transfer can be broadly divided into three categories.

1. Physical gene transfer methods—electro- portion, particle bombardment, microinjection, liposome

fusion, silicon carbide fibres.

2. Chemical gene transfer methods—Polyethylene glycol (PEG)-mediated, diethyl amino ethyl (DEAE)
dextran-mediated, calcium phosphate precipitation.

3. DNA imbibition by cells/tissues/organs.

The salient features of the different methods for direct DNA transfer are given in Table 49.1 .

(A) Physical Gene Transfer Methods:

An overview of the general scheme for the production of transgenic plants by employing physical transfer
methods is depicted in Fig. 49.9. Some details of the different techniques are described.

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Dr. Arundhati Banerjee (Notes)

1. Electroporation:
Electroporation basically involves the use of high field strength electrical impulses to reversibly
permeabilize the cell membranes for the uptake of DNA. This technique can be used for the delivery of
DNA into intact plant cells and protoplasts.

The plant material is incubated in a buffer solution containing the desired foreign/target DNA, and
subjected to high voltage electrical impulses. This results in the formation of pores in the plasma
membrane through which DNA enters and gets integrated into the host cell genome.

In the early years, only protoplasts were used for gene transfer by electroporation. Now a days, intact cells,
callus cultures and immature embryos can be used with suitable pre- and post-electroporation treatments.
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 cost-effective.

ii. The transformed cells are at the same physiological state after electroporation.

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Dr. Arundhati Banerjee (Notes)

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.

2. Particle Bombardment (Biolistics):

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 (1988).
Biolistics is a combination of biological and ballistics. There are other names for this technique- particle
gun, gene gun, bio blaster. A diagrammatic representation of micro projectile bombardment system for the
transfer of genes in plants is depicted in Fig. 49.10, and briefly described below.

Micro carriers (micro projectiles), the tungsten or gold particles coated with DNA, are carried by macro
carriers (macro projectiles). These macro-carriers are inserted into the apparatus and pushed downward by
rupturing the disc.

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Dr. Arundhati Banerjee (Notes)

The stopping plate does not permit the movement of macro carrier while the micro carriers (with DNA) are
propelled at a high speed into the plant material. Here the DNA segments are released which enter the
plant cells and integrate with the genome.

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.

A selected list of the transgenic plants (developed by biolistics) along with the sources of the plant
materials used is given in Table 49.2.

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Dr. Arundhati Banerjee (Notes)

Factors affecting bombardment:

Several attempts are made to study the various factors, and optimize the system of particle bombardment
for its most efficient use. Some of the important parameters are described.

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.

Nature of tissues/cells:
The target cells that are capable of undergoing division are suitable for transformation. Some more details
on the choice of plant material used in bombardment are already given.

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. Recently, some workers have started using the chemical aminosiloxane to coat the micro
particles with low quantities of DNA adequate enough to achieve high efficiency of transformation.

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

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Dr. Arundhati Banerjee (Notes)

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.

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.

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 technique of microinjection involves the transfer of the gene through a micropipette (0.5-10.0 pm tip)
into the cytoplasm/nucleus of a plant cell or protoplast. While the gene transfer is done, the recipient cells
are kept immobilized in agarose embedding, and held by a suction holding pipette (Fig. 49.11).

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.

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Dr. Arundhati Banerjee (Notes)

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 (Fig. 49.12).

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.

5. Silicon Carbide Fibre-Mediated Transformation:

The silicon carbide fibres (SCF) are about 0.3-0.6 pm in diameter and 10-100 pm in length. 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.

Advantages of SCF-mediated transformation:

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Dr. Arundhati Banerjee (Notes)

i. Direct delivery of DNA into intact walled cells. This avoids the protoplast isolation.

ii. Procedure is simple and does not involve costly equipment.

Disadvantages of SCF-mediated transformation:

i. Silicon carbide fibres are carcinogenic and therefore have to be carefully handled.

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.

(B) Chemical Gene Transfer Methods:

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.
ADVERTISEMENTS:

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. Deae Dextran-Mediated transfer:

The desirable DNA can be complexed with a high molecular weight polymer diethyl amino ethyl (DEAE)
dextran and transferred. The major limitation of this approach is that it does not yield stable trans-
formants.

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Dr. Arundhati Banerjee (Notes)

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.

Dna Imbibition By Cells/Tissues:

Some workers have seriously tried to transform cells by incubating cell suspensions, tissues, embryos and
even seeds with DNA. The belief is that the DNA gets imbibed, and the cells get transformed. DNA
imbibition approach has met with little or no success.

Plant Viruses as Vectors | Genetics

Exploitation of plant viruses as transformation vectors by massive infection may be harmful and even
deleterious to the target plants. It is still however able to express and produce foreign proteins.

Plant viruses must exhibit some of the exemplary features before they are considered as vectors. They
should extend their broader host-range, spread of seed transmission and carry additional copies of gene of
interest.

Several viral vectors require suitable modification in order to accommodate extra nucleic acid and also
aggressive in infection process. Although several groups of viruses have been identified, some moderate
progresses have been made only in two groups. These two groups are Caulimo virus and Gemini virus,
which have DNA genome as genetic material.

Cauliflower Mosaic Virus (Caulimovirus):

Cauliflower mosaic virus (CamV) belongs to the group caulimovirus, can be used as potential candidate to
deliver foreign gene into the plant. It is perhaps the best studied viruses among plant virus, which infects
several members belonging to Cruciferae family. Cauliflower mosaic virus contains circular double helical
DNA as genetic material.

As an infective agent, can cause disease in wide range of commercially important cultivated crops.
Cauliflower mosaic DNA has been subjected to a wide range of manipulation. This was the only and first
virus to be manipulated and used as a favourable choice for genetic engineering work. Elucidation of 8 kb
CamV reveals that, it contains six major and two minor reading frames (Fig. 14.12).

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Dr. Arundhati Banerjee (Notes)

Presence of ribonucleotide in DNA leads to the conclusion that CamV replication involves the synthesis of
negative DNA strand by employing reverse transcriptase and followed by synthesis of positive DNA
strand. Once the synthesis of double strand DNA completes, it is then packed into viral particles and
continue the cycles of transcription and translation.

CamV Vector:
Cauliflower mosaic virus can be used as a potential vector due to the infective nature of its genetic
material. This could be proved by applying viruses on the leaf rubbed with abrasive material. The CamV
cannot accommodate foreign DNA, if the size exceeds its normal size. The inserted DNA may destabilize
infectious nature of the virus.

Other constraints are the packaging of genome and limitation of the insertion of foreign DNA. Despite the
marginal constraints, CamV genome can be packed in nucleosome and is able to undergo transcription by
plant RNA polymerase II. The genome of CamV consists of six major and two minor open reading frames
(ORF), in tightly packed arrangements.

The two ORF regions, one (ORFII) codes for insect transmission factor and other (PRF VII) with unknown
functions can be replaced with gene of interest.

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Dr. Arundhati Banerjee (Notes)

Gemini Virus:
It is a DNA virus, known to infect wide range of economically important and agriculture crops of
monocotyledonous and dicotyledonous plants. Several diseases such as maize streak virus and curly top
virus are caused by Gemini virus. The genome is single Stranded Circular DNA and its replication takes
place by DNA immediately.

Tobacco Mosaic Virus Based Expression Vector:

Tobacco mosaic virus (TMV) is a RNA virus and shows several advantages by designing expression
vector. TMV was the first virus to be purified and sequenced. As far as biohazard is considered TMV
could be used as a comparatively safe recombinant virus in the field.

The coat protein of the TMV is one of the most accumulated proteins in plants reachable upto 10% of the
dry weight of infected plant. Approximately, under ideal condition, 2000 kg tobacco protein can be
produced per acre per year. Moreover, TMV can be purified in crystalline form in substantial quantity by
simple methodology.

The single stranded RNA genetic material of TMV encloses 6300 nucleotides with four open reading
frames (Fig. 14.13). The filamentous nature of the RNA virus is determined by the length of the viral
nucleic acid. Both 183 kD read through protein and 126 kD coat proteins are translated from the 5′ end of
the genomic RNA.

These two proteins form replicase complex. In addition to these two proteins, the 30-kD movement protein
and 17.5-kD capsid proteins are translated at 3′ region of sub-genomic mRNA during replication.

TMV can be subjected to a wide range of manipulation by replacing the viral coat protein with a foreign
protein, for example, replacement of coat protein with reporter gene chloramphenical acetyl transferase
(CAT) resulted in a free-RNA virus that generated high CAT activity. Improvisation of TMV vector was
achieved by placing CAT gene under the control of a coat protein sub-genomic promoter of TMV into the
entire TMV genome.

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Dr. Arundhati Banerjee (Notes)

This recombinant virus produced third sub-genomic mRNA and this was translated into CAT enzyme with
high activity. Similarly, another TMV hybrid expression vector TB2 was designed in which coat protein
gene and neomycin phosphotransferase marker gene was placed under the control of sub-genomic
promoters of TMV coat protein and ORSV, respectively.
This was referred as extra gene vector. TB2 effectively produced the foreign protein without any major
constraints. Another extra gene in TMV based vector, 4GD-PL, was developed from tomato green mosaic
virus. The 4GD-PL vector was able to express foreign proteins systematically throughout plants.
All these studies demonstrated that proximity of genes to the 3′-untranslated region of the genome
increases efficiency of their translation. Another improved TMV vector, 30B, was designed in which the
start codon (AUG) of the capsid protein was mutated to AGA, and restriction cloning sites were
engineered (40 nucleotide) to provide a full-size sub-genomic RNA promoter.

Possibility of satellite RNA to be used as vector has been considered. They vary in their size between 0.27
and 1.6 kb. They are not indispensable for virus replication. However, their functions can alter
pathogenecity of virus.

Cow Pea Mosaic Virus Expression Vector:

Cow pea mosaic virus (CpmV) is also a RNA virus and infects species of legumes. There are two separate
positive strand-RNA molecules present in the genetic material of CpmV. The number of nucleotides
present in the RNA I and RNA II strand is 5889 and 3480, respectively. Although RNA I alone can
replicate on its own but both RNAs are indispensable for infectivity.

The proteins involved in the replication of the virus are encoded by RNA I whereas movement proteins are
encoded by RNA II. CpmV capside of both large (L) and small (S) coat protein of 30 copies each are in
isohedral symmetry. The two capsid proteins are folded into three antiparallel β-barrel structures.

In the construction of CpmV expression vector, preference was given to the replacement of stable chimeras
by insertion of foreign sequences rather than replacement for native residues.

Therefore, in the construction of viable and well refined CpmV vector, precise site of insertion of foreign
sequence was given a prime choice by introducing foreign DNA sequence into βB-βC loop of the S protein
for most chimeras foreign sequences inserts immediately upstream of proline 23 of the S protein. In view
of propagating the chimeras, engineered pCP2 and pCP1 are linearised and inoculated on cow pea plants.
(Fig. 14.14)

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Dr. Arundhati Banerjee (Notes)

Gene targeting is a biotechnological tool used to change the DNA sequence of an organism (hence it
is a form of Genome Editing). It is based on the natural DNA-repair mechanism of Homology Directed
Repair (HDR), including Homologous Recombination. Gene targeting can be used to make a range of sizes
of DNA edits, from larger DNA edits such as inserting entire new genes into an organism, through to much
smaller changes to the existing DNA such as a single base-pair change. Gene targeting relies on the
presence of a repair template to introduce the user-defined edits to the DNA. The user (usually a scientist)
will design the repair template to contain the desired edit, flanked by DNA sequence corresponding
(homologous) to the region of DNA that the user wants to edit; hence the edit is targeted to a particular
genomic region. In this way Gene Targeting is distinct from natural homology-directed repair, during
which the ‘natural’ DNA repair template of the sister chromatid is used to repair broken DNA (the sister
chromatid is the second copy of the gene). The alteration of DNA sequence in an organism can be useful in
both a research context – for example to understand the biological role of a gene – and in biotechnology,
for example to alter the traits of an organism (e.g. to improve crop plants).

Methods
Wild-type Physcomitrella and knockout-mosses: Deviating phenotypes induced in gene-disruption library
transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to
induce differentiation and development of gametophores. For each plant, an overview (upper row, scale
bar corresponds to 1 mm) and a close-up (bottom row, scale bar equals 0.5 mm) is shown. A, Haploid
wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B-D,
Different Mutants.
To create a gene-targeted organism, DNA must be introduced into its cells. This DNA must contain all of
the parts necessary to complete the gene targeting. At a minimum this is the homology repair template,
containing the desired edit flanked by regions of DNA homologous (identical in sequence to) the targeted
region (these homologous regions are called “homology arms” ). Often a reporter gene and/or a selectable
marker is also required, to help identify and select for cells (or “events”) where GT has actually occurred.
It is also common practice to increase GT rates by causing a double-strand-break (DSB) in the targeted
DNA region. Hence the genes encoding for the site-specific-nuclease of interest may also be transformed

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Dr. Arundhati Banerjee (Notes)

along with the repair template. These genetic elements required for GT may be assembled through
conventional molecular cloning in bacteria.
Gene targeting methods are established for several model organisms and may vary depending on
the species used. To target genes in mice, the DNA is inserted into mouse embryonic stem cells in culture.
Cells with the insertion can contribute to a mouse's tissue via embryo injection. Finally, chimeric mice
where the modified cells make up the reproductive organs are bred. After this step the entire body of the
mouse is based on the selected embryonic stem cell.
To target genes in moss, the DNA is incubated together with freshly isolated protoplasts and
with polyethylene glycol. As mosses are haploid organisms, moss filaments (protonema) can be directly
screened for the target, either by treatment with antibiotics or with PCR. Unique among plants, this
procedure for reverse genetics is as efficient as in yeast. Gene targeting has been successfully applied to
cattle, sheep, swine and many fungi.
The frequency of gene targeting can be significantly enhanced through the use of site-
specific endonucleases such as zinc finger nucleases, engineered homing endonucleases, TALENS, or
most commonly the CRISPR-Cas system. This method has been applied to species including Drosophila
melanogaster, tobacco, corn, human cells, mice and rats.

Applications
Applications in mammalian systems
Gene targeting was developed in mammalian cells in the 1980s, with diverse applications possible as a
result of being able to make specific sequence changes at a target genomic site, such as the study of gene
function or human disease, particularly in mice models. Indeed gene targeting has been widely used to
study human genetic diseases by removing ("knocking out"), or adding ("knocking in"), specific mutations
of interest. Previously used to engineer rat cell models,[30][31] advances in gene targeting technologies
enable a new wave of isogenic human disease models. These models are the most accurate in vitro models
available to researchers and facilitate the development of personalized drugs and diagnostics, particularly
in oncology. Gene targeting has also been investigated for gene therapy to correct disease-causing
mutations. However the low efficiency of delivery of the gene-targeting machinery into cells has hindered
this, with research conducted into viral vectors for gene targeting to try and address these challenges.
Applications in yeast and moss
Gene targeting is relatively high efficiency in yeast, bacterial and moss (but is rare in higher eukaryotes).
Hence gene targeting has been used in reverse genetics approaches to study gene function in these systems.
Applications in plant genome engineering
Gene targeting (GT), or homology-directed repair (HDR), is used routinely in plant genome engineering to
insert specific sequences, with the first published example of GT in plants in the 1980s. However, gene
targeting is particularly challenging in higher plants due to the low rates of Homologous Recombination,
or Homology Directed Repair, in higher plants and the low rate of transformation (DNA uptake) by many
plant species. However, there has been much effort to increase the frequencies of gene targeting in plants
in the past decades, as it is very useful to be able to introduce specific sequences in the plant genome for
plant genome engineering. The most significant improvement to gene targeting frequencies in plants was
the induction of double-strand-breaks through site specific nucleases such as CRISPR, as described above.
Other strategies include in planta gene targeting, whereby the homology repair template is embedded
within the plant genome and then liberated using CRISPR cutting; upregulation of genes involved in the
homologous recombination pathway; downregulation of the competing Non-Homologous-End-Joining
pathway; increasing copy numbers of the homologous repair template; and engineering Cas variants to be
optimised for plant tissue culture. Some of these approaches have also been used to improve gene targeting
efficiencies in mammalian cells.
Plants that have been gene-targeted include Arabidopsis thaliana (the most commonly used model plant),
rice, tomato, maize, tobacco and wheat.

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Dr. Arundhati Banerjee (Notes)

Technical challenges
Gene targeting holds enormous promise to make targeted, user-defined sequence changes or sequence
insertions in the genome. However its primary applications - human disease modelling and plant genome
engineering - are hindered by the low efficiency of homologous recombination in comparison to the
competing non-homologous end joining in mammalian and higher plant cells.[50] As described above, there
are strategies that can be employed to increase the frequencies of gene targeting in plants and mammalian
cells. In addition, robust selection methods that allow the selection or specific enrichment of cells where
gene targeting has occurred can increase the rates of recovery of gene-targeted cells.

Protein engineering is the process of developing useful or valuable proteins through the design and
production of unnatural polypeptides, often by altering amino acid sequences found in nature. It is a young
discipline, with much research taking place into the understanding of protein folding and recognition
for protein design principles. It has been used to improve the function of many enzymes for industrial
catalysis.
There are two general strategies for protein engineering: rational protein design and directed evolution.
These methods are not mutually exclusive; researchers will often apply both. In the future, more detailed
knowledge of protein structure and function, and advances in high-throughput screening, may greatly
expand the abilities of protein engineering. Eventually, even unnatural amino acids may be included, via
newer methods, such as expanded genetic code, that allow encoding novel amino acids in genetic code.

Examples of engineered proteins

Computing methods have been used to design a protein with a novel fold, named Top7, and sensors for
unnatural molecules. The engineering of fusion proteins has yielded rilonacept, a pharmaceutical that has
secured Food and Drug Administration (FDA) approval for treating cryopyrin-associated periodic
syndrome.
Another computing method, IPRO, successfully engineered the switching of cofactor specificity
of Candida boidinii xylose reductase. Iterative Protein Redesign and Optimization (IPRO) redesigns
proteins to increase or give specificity to native or novel substrates and cofactors. This is done by
repeatedly randomly perturbing the structure of the proteins around specified design positions, identifying
the lowest energy combination of rotamers, and determining whether the new design has a lower binding
energy than prior ones.
Computation-aided design has also been used to engineer complex properties of a highly ordered nano-
protein assembly. A protein cage, E. coli bacterioferritin (EcBfr), which naturally shows structural
instability and an incomplete self-assembly behavior by populating two oligomerization states, is the
model protein in this study. Through computational analysis and comparison to its homologs, it has been
found that this protein has a smaller-than-average dimeric interface on its two-fold symmetry axis due
mainly to the existence of an interfacial water pocket centered on two water-bridged asparagine residues.
To investigate the possibility of engineering EcBfr for modified structural stability, a semi-empirical
computational method is used to virtually explore the energy differences of the 480 possible mutants at the
dimeric interface relative to the wild type EcBfr. This computational study also converges on the water-
bridged asparagines. Replacing these two asparagines with hydrophobic amino acids results in proteins
that fold into alpha-helical monomers and assemble into cages as evidenced by circular dichroism and
transmission electron microscopy. Both thermal and chemical denaturation confirm that, all redesigned
proteins, in agreement with the calculations, possess increased stability. One of the three mutations shifts
the population in favor of the higher order oligomerization state in solution as shown by both size
exclusion chromatography and native gel electrophoresis.

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