Methods of Gene Transfer

Lesson Prepared Under MHRD project “ National Mission on

Education Through ICT”

Discipline: Botany
Paper: Plant Biotechnology

National Coordinator: Prof. S.C. Bhatla

Lesson:Methods of Gene Transfer

Lesson Developer: Dr Neetu Chaudhary1, Dr. Parul Agarwal2

College: 1Acharya Narender Dev College,2Department of Genetics,
University of Delhi, South Campus

Lesson Reviewer: Prof. Suresh Chand, Professor and Head, School of

Life Sciences, Devi Ahilya Vishwavidyalaya, Indore

Language Editor: Namrata Dhaka

Department/College: Department of Genetics, University of Delhi,
South Campus

Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL

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 Methods of Gene Transfer

Chapter: Methods of Gene Transfer

Table of Contents

• Introduction
• Indirect Methods
• Agrobacterium-mediated genetic transformation
• Structure of Ti Plasmid
• Use of Ti plasmid in genetic transformation

• Steps involved in Agrobacterium-mediated genetic

transformation of plants by ‘Wounded explant’
method

• Direct Method
• Microprojectile/particle Bombardment (biolistics)

• Electroporation

• Microinjection

• Chemical mediated gene transfer

• Liposome mediated gene transfer

• Silicon carbide method

• Selection of transformants
• Selectable marker
• Screening marker

• Summary

• Exercise/practice

• Glossary

• References/Bibliography/Further Reading

• Web links

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 Methods of Gene Transfer

Introduction

Foreign genes are introducedartificially into crops by overcoming the fertility [Link]
process, also known asgenetic transformation,is a very important step in genetic engineering.

Horizontal vs vertical gene transfer

The natural transfer of genetic material from one organism to another is referred to as
horizontal gene transfer or the lateral gene transfer. The foreign DNA is either randomly
inserted into the host genome or recombines if there is sequence homology between the two
genomes. This is different from the vertical gene transfer where the genetic material is
transferred from the parents to the offsprings, through sexual reproduction.

Horizontal gene transfer is facilitated by various mechanisms. In prokaryotes mainly

transformation (intake of genetic material from surrounding), conjugation (exchange of
genetic material with the physical union of two cells) and transduction (transmission of DNA
through bacteriophages from one cell to another) are responsible for the transfer of the gene
within organisms. In eukaryotes, the presence of the outer cell membrane and the nuclear
membrane makes transfer of DNA difficult between organisms. Horizontal gene transfer plays
important role in evolution of both prokaryotes and eukaryotes.

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Video: Horizontal vs Vertical gene transfer [Link]

Plant transformation systems generally include following steps,

• Introduction of a DNA segment into totipotent cells.

• Its integration into host cells genome.
• Subsequent regeneration from transformed cellto produce whole plant.

Plant transformation methods therefore require an efficient wayto introduce DNA into cell and
the regeneration of the transformed cells or tissues into whole plants.

TheDNA segment which is introduced in this process contains the gene of interestanda
cassette containing additional genetic material. Additional genetic material includes

• A promoter which determines the site and timing of expression of the introduced gene
• A terminator to identify the end of transcription and
• Amarker gene which allows selection of plants having the introduced gene.

Various desirable traits have been efficiently introduced and stably expressed in almost 150
plant species.

Different methods are available to achieve genetic transformation of plants i.e. the delivery of
the foreign DNA into the host plant. These are divided into two main groups

• Indirect methods: In this case vector is needed for insertion of the foreign DNA into the
host genome.
• Direct methods: This method is vector independent. The DNA is directly inserted into the
host genome.

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Figure: Types of methods for gene transfer in plants

Source: Namrata Dhaka, Research Scholar, Department of Genetics, University of Delhi,
South Campus.

In-direct Methods

Agrobacterium-mediated genetic transformation

The method of genetictransformation, which employs bacteria as a vector to introduce the

gene construct into the target cell,isknown as indirect method. Thismethod uses
Agrobacterium (a gram-negative soil bacteria which causes crown gall disease in many
plants) for the plant transformation experiments.

The most commonly studied species of this genus is Agrobacterium tumefaciens, which forms
an efficient delivery system for genetic transformationin [Link] bacteria harbor a large
plasmid called Ti plasmid (tumour inducing) having tumor-inducing genes (T-DNA) and other
genes involved in integration of T-DNA into host [Link] plants secrete a sap with
high content of phenolic compounds which serve as chemical attractants for Agrobacteria and
stimulateexpression of virgenes. It results ininfection of plantby Agrobacterium, insertion of
T-DNA region at a random site inhost genome and proliferation of plant cells to form crown
gall growth.
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Another commonly used species is Agrobacterium rhizogens, which induces hairy roots in
[Link] contains Ri plasmid (root inducing plasmid). The genus Agrobacterium has a wide
host range and can infect a number of dicots and some monocots.

Figure: Crown gall disease caused by Agrobacterium on rose stem.

Source:[Link] (CC)

Figure:Ti plasmid with T-DNA region.

Source: [Link] (CC)

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 Methods of Gene Transfer
Structure of Ti Plasmid
In transformation experiments, vector is the genetic vehicle needed to transport the gene of
interest, promoter, terminator and selectable marker genes to DNA of host plant. Virulence of
Agrobacterium is conferred by Ti plasmid having genes for tumor-induction, T-DNA
integration and synthesis of plant hormones and opines.

Figure: An illustration showing the important regions of a Ti plasmid.

Source: Namrata Dhaka, Department of Genetics, University of Delhi, South Campus.

Link for animation-

[Link]
0072437316/120078/[Link]::The+Ti+Plasmid

• Origin or replication
This region is responsible for the replication of Ti plasmid independent of the bacterium
cell.

• Virulence region
This region contains genes called vir genes whose products enable processing and
transfer of the T-DNA from bacterium to plant cells. Their expression is triggered by
certain phenolic compounds like acetosyringone, which are secreted by plants in
response to wounding.

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Figure:Figure showing a model of how different vir proteins interact with T-DNA to mediate
its transfer from the bacterium to the host cell.
Source: Gelvin, Stanton B. "Traversing the cell: Agrobacterium T-DNA’s journey to the host
genome." Frontiers in plant science 3 (2012).
[Link]
(cc)

• T-DNA region
It is a region of Ti plamid which contains genes from induction of tumour. It is flanked
by 25 bp direct repeat sequences on both sides. These repeats are called as Left border
(LB) and Right border (RB). The different genes present in this region are –
iaaM and iaaH genes - responsible for synthesis of indole acetic acid (an auxin),
ipt gene - responsible for synthesis of an enzyme isopentenyl adenine (a cytokinin)
tml gene – another gene involved in formation of tumours.
Opine biosynthesis genes–lead to synthesis of opines.
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All these genes result in overgrowth of tissue in the plant cells leading to tumour
formation.

Figure: Arrangements of different genes in T-DNA region. Arrows represent the

direction of transcription.
Source: [Link]
(cc)

• Region of opine catabolism

It contains several other genes involved in metabolism of opines. This region of the
plasmid is not transferred to the plant cells during infection.

For further information, read -

[Link]

Use of Ti plasmid in genetic transformation

For its use in genetic transformation as a vector, most of the T-DNA region of bacterial
plasmid is replaced with the gene of interest while leaving the left and right border
sequences. The T-DNA region is defined not by its sequence but by its borderswhich enables
itsinsertion into host plant genome.

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Figure: An illustration showing that the genes of Ti plasmid can be replaced with any gene of
interest and selectable marker gene for selection of the transformed cells.
Source: Namrata Dhaka, Department of Genetics, University of Delhi, South Campus.

Figure: An illustration showing arrangement of different motifs in a cassette carrying gene of

interest, used for [Link]: Left Border; RB: Right Border
Source: Namrata Dhaka, Department of Genetics, University of Delhi, South Campus.

Wild type Ti plasmid from Agrobacterium cannot be directly used as vector system due to
their large size, presence of tumor causing genes and absence of marker genes and unique
restriction sites within T-DNA region. Therefore they are genetically engineered to incorporate
these [Link] are two types of genetically engineered Ti plasmid based vectors.

1. Binary vectors

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In this system, there is a pair of plasmids, which are independent of each other and present
in the same Agrobacterium cell. This vector system is based on the fact that vir genes
need not be present in the same plasmid with T-DNA for its transfer.

• Helper Ti plasmid

The vir regionrequired for T-DNA integrationis present in this plasmid.

• A disarmed Ti plasmid (Mini Ti plasmid)

T-DNA region having the gene of interest and plant selectable marker gene with
promoter and terminator sequences + ori for both Agrobacterium and E. coli are
present in a separate disarmed Ti plasmid.

A B

Figure:A binary vector system (A) A helper plasmid containing vir region but no T-
DNA, (b) Another plasmid containinggene of interest within the border sequences of T-
DNA and selectable marker gene but no virregion.
Source: Namrata Dhaka, Department of Genetics, University of Delhi, South Campus.

2. Co-integrate vectors

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These vectors are also known as hybrid Ti plasmids. In these vectors both T-DNA
region (having gene of interest) and vir genes are present in the same plasmid used
for transformation.

Figure: Co-integrate vectors contain vir genes as well as T-DNA region.

Source: Namrata Dhaka, Department of Genetics, University of Delhi, South Campus.

Figure: Vector map of co-integrated plasmid

Source: [Link] (CC)

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Steps involved inAgrobacterium-mediated genetic transformation of plants by
‘Wounded explant’ method

• Gene of interest isisolated/amplified from a source organism.

• An expression cassetteis developed including gene of interest flanked by promoter and

terminator sequences to drive expression and marker genes to facilitate selection of
transformed plants by tracking introduced genes in the host plant.

• Insertion of cassette into T-DNA region of the binary vector/co-integrate vector is carried
out.

• Transformation of the above expression vector into Agrobacteriumis done.

• Explants are obtained from plant that is to be transformed. They are wounded and then
co-cultured for a brief period for Agrobacterium infection. This is done using tissue
culture methods.

• The transformed explants are then grown in presence of a bacteriostatic agent to prevent
the growth of Agrobacterium and in presence of a selective antibiotic which prevents
survival of any untransformed explants.

• Positive transformants are tested by PCR analysis to check for the presence of desired
gene.

• The transformed explants are maintained in vitro until they are ready for transplantation.
After the transformed plants are obtained, DNA is isolated and Southern hybridization is
carried out to confirm the site of integration and the copy number of the desired gene.

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Figure: Figure showing how a desired gene is inserted into Ti plasmid, which is then used to
transfer it to a plant.

Source:Narusaka, Yoshihiro, et al. "Methods to Transfer Foreign Genes to Plants." IN:

Transgenic Plants–Advances and Limitations, Yelda Ozden Çiftçi (Ed.), ISBN (2012): 978-953.

[Link]
transfer-foreign-genes-to-plants (cc)

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Figure: Steps in screening of tranformants by PCR analysis

Source:Narusaka, Yoshihiro, et al. "Methods to Transfer Foreign Genes to Plants." IN:

Transgenic Plants–Advances and Limitations, Yelda Ozden Çiftçi (Ed.), ISBN (2012): 978-953.

[Link]
transfer-foreign-genes-to-plants (cc)

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Figure:Diagram illustrating Agrobacterium-mediated transformation of sugarcane.

[Link]
[Link]?aid=18824 (CC)

For further details visit: [Link]

Direct Methods

Direct methods are those methods which do not use bacteria as mediators for integration of
DNA into host genome. These methods include microprojectile bombardment,electroporation
and microinjection.

Microprojectile/particle Bombardment (biolistics)

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 Methods of Gene Transfer
Biolistics is a method where cells are physically impregnated with nucleic acids or other
biological [Link] particle delivery system is a device for plant transformation
where cells are bombarded with heavy metal particles coated with DNA/RNA. This technique
was invented by John Stanford in 1984 for introduction of DNA into cells by physical means to
avoid the host-range restrictions of [Link]-mediated genetic
transformation system works well for dicotyledonous plants but has
low efficiency for [Link] particle delivery system provides an effective and
versatile way to transform almost all type of cells. It has been proven to be a successful
alternative for creating transgenic organisms inprokaryotes, mammalian and plant species.

Figure: A biolistic microprojectile gun.

Source: [Link] (cc)

In this process, construct having gene of interest is coated on the surface oftiny particles of
gold or tungsten (0.6 – 1 mm in size). Prior to coating, DNA is precipitated with calcium
chloride, spermidine and polyethylene [Link] coated microparticles are loaded on to the
macrocarrier and accelerated to high speed by using pressurized helium [Link] cell
suspensions, callus cultures, or tissues could be used as the target of thesemicroprojectiles.
As the microprojectiles penetrate the plant cell walls and membranes to enter the cells,
coated DNAis released from its surface and incorporated into the plant’s [Link] biolistics,
use of binary vectors with T-DNA border sequences is not required.

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This method is especially importantformonocots, for which efficiency of othertransformation
methods is not satisfactory. A wide range of tissues such as apical and floral meristems,
embryos, seedlings, leaves, cultured cells and floral tissues could be used as target in this
method.

Figure:Particle bombardment method of Plant transformation (1) Isolation of protoplasts.

(2)Injection of DNA-coated particles using particle gun. (3) Regeneration of transformed
protoplasts into plantlets. (4) Acclimatizationof regenerated plantlets in a greenhouse.

Source: Narusaka, Yoshihiro, et al. "Methods to Transfer Foreign Genes to Plants." IN:
Transgenic Plants–Advances and Limitations, Yelda Ozden Çiftçi (Ed.), ISBN (2012): 978-953.

[Link]
transfer-foreign-genes-to-plants (cc)

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A number of parameters should be carefully considered before using particle bombardment.
These can be classified under three categories:

• Physical parameters
Nature, chemical and physical properties of the metal particles utilized to carry the
foreign DNA. The nature and preparation of DNA,binding of DNA on the particles and
target tissues.

• Environmental parameters
Variables such as temperature, photoperiod and humidity of donor plants, explants, and
bombarded tissues affect physiology of tissues and influence receptiveness of the target
tissue.

• Biological parameters
Choice and nature of explants, pre- and post bombardment culture conditions, osmotic
pre- and post-treatment of explants.

Advantages of particle bombardment over Agrobacterium-mediated DNA transfer:

• This system is species independent and can been used successfully for a wide range of
organisms.
• Many species which are recalcitrant to other direct transfer methods or are not readily
amenable to Agrobacterium-mediated transformation have been transformed by this
technique.
• Introduced DNA does not need sequences necessary for T-DNA replication and transfer as
complex interaction between bacterium and plant tissue does not take place.
• Transformation of organelle DNA (mitochondria and chloroplasts) has also been achieved
by this method.
• Multiple genes can be introduced in a single plant.
• Particles can be coated with DNA/RNA/siRNA/large fragments of nucleic acids.

Limitations of particle bombardment method :

• Limited regeneration capacity of tissue being bombarded
• Efficiency of stable integration of DNA.
• Insertion of multiple copies of the gene
• Integration of rearranged and/or truncated DNA sequences

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• Damage to the cellular tissue.
• Specialized and expensive equipments are required

Electroporation

Electoporation 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, thorough which DNA seems to enter inside the cell and then
nucleus.

Figure:Electroporation (A) Diagram showing formation of transient pores in cell

membrane on applying electrical pulse, entry of DNA inside thecell and sealing of
pores afterwards.

Source: [Link]
Santra, Tuhin Subhra, and Fang Gang Tseng. "Recent trends on micro/nanofluidic single cell
electroporation." Micromachines 4.3 (2013): 333-356. (cc)

A suspension of cells with plasmid DNAis taken in an electroporation cuvette placed between
electrodesand electrical pulses are [Link] micropores are formed in cell
membranes whichallow cells to take up plasmid DNA leading to stable or transient DNA
expression.

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A B

Figure:(A) Main components of an electroporator. (B) Cuvettes used for electroporation.

These are plastic cuvettes with lid and aluminium electrodes, having a maximum capacity of
400 μl.
Source:
[Link] (cc)

[Link] (cc)

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 frequencyespecially
in monocotyledonous plants. The electrical field and
chemicalsubstancesapplied to disorganize cell walls strongly reducethe viability and capability
of divisionof protoplasts.

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Electoporation 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 [Link] 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 intothe nucleus or cytoplasm

using a glass microcapillary injectionpipette. The protoplasts are immobilized in low melting
agar, while working under a microscope, using a holding pipette and suction force. DNA is
then directly injected into the cytoplasm or the nucleus. Theinjected cells are then cultured
invitro and regenerated into plants. Successful examples of this process has been shown in
rapeseed, tobacco and various other plants.

Stable transformants can be achieved through this method but it requires technical expertise
and is atime consuming process. Also,microinjection has achieved only limited success in
plant transformationdue to the thick cell walls of plants and a lack of availability of a single-
cell-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
40-50x)is [Link] the microscope target cell is positioned and cell membrane and nuclear
envelope are penetrated with the help of two [Link] micromanipulator holds
the pipette and another holds the microcapillary needle.

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Figure: Illustration of microprojectile method

Source: [Link] (cc)

There are two types of microinjection systems; constant flow system and pulsed flow system.

• Inthe constant flow system the amount of sample injected is determined by the
duration for which needle remains in the [Link] constant flow system is relatively
simple and inexpensive but outdated.
• The pulsed flow systemhas greater control over thevolume of substance delivered,
needle placement and movement and has better precision. This technique results in
less damage to the receiving cell, however, the components of this system are quite
expensive.

Chemical mediated gene transfer

Cells or protoplasts can be stimulated to take up foreign DNA using some chemicals.
Polyethylene glycol (PEG) is the most commonly used chemical for this purpose. It helps in
precipitation of DNA, which can then be taken up by the calls through the process of
endocytosis.

Liposome mediated gene transfer

Plasmid containing foreign desired gene can be enclosed in small lipid bags called lipososmes,
which can then be fused with protoplasts using chemicals like PEG.

Silicon carbide method

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In this method, fibres of organic material like silicon carbide are used for gene transfer. These
fibres, when mixed with plasmid DNA and plant tissue or cells, help in penetration of the
foreign DNA into the plant tissue.

Figure: An illustration showing different techniques used for transformation of tree species.

Source: Castellanos-Hernández, Osvaldo A., et al. "Genetic Transformation of Forest Trees."

[Link]
trees

(cc)

Selection of transformants
In a genetic transformation experiment, only one in a several million to billion cells may take
up the transgene depending upon the efficiency of transformation. Rather than checking
every single cell/organism, a selective agent that kills or gives a different phenotype to all the
cells not carrying foreign DNA can be employed. These selective genes are called as marker
genes. These genes also help in assessing the success rate of a genetic transformation study.

Marker gene is a gene introduced into cell along with the transgene. It is used to determine if
the transgene has been successfully inserted into host organism's genome as marker gene’s
presence can be seen or [Link] are two types of marker genes:

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• Selectable marker
• Screening marker.

Selectable marker
A selectable marker is a gene that confers a trait suitablefor artificial selection as it protects
the organism from a selective agent that would normally kill it or prevent its [Link] most
of the genetic transformation experiments only one in a several million or billion cells will take
up the transgene. In order to find out transformed cell/organism a selective agent is utilized
which kills all the cells withouttransgene, leaving only the transformed ones.

Selectable marker genes can be divided into several categories depending on whether they
confer positive or negative selection and whether selection is conditional or non-conditional on
the presence of external substrates.

Negative selectable marker genes result in death of the untransformed tissue whereas
transformed cells are able to survive .e.g., antibiotics, herbicides,

Antibiotics or herbicides are the most common selective [Link] grown on medium
containing antibiotic or herbicide the non-recombinants die due to lack of resistance. In
bacteria, antibiotics are used almost exclusively. In plant systemnptII, hpt II, and bar
contribute to production of over 95% transgenic plants. In mammals, resistance to antibiotics
that would kill the mitochondria is used as a selectable marker. All the regenerants obtained
after a plant transformation experiment are grown on a medium containing an antibiotic, and
those plants that can grow have successfully taken up and expressed the introduced gene.
Efficiency, biosafety, scientific applications and commercialization issues for almost fifty
marker genes used for transgenic and transplastomic plant research have been assessed.

Examples of selectable markers

• hptII gene codes for hygromycin phosphotransferase (HPT) enzyme, which detoxifies the
aminocyclitol antibiotic hygromycin B. Hygromycin Bcauses mistranslocation of Mrna
thereby inhibiting protein synthesis. hptII gene has been used to transform a large
numberof plants and including monocots. It has been found to be more effective than
kanamycin in cereals. hptII gene is also used for selecting transformed mammalian cells.

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• NptII (neomycin phosphotransferase)gene from Tn5 confers resistance to kanamycin in
bacteria and geneticin in eukaryotes.
• bar gene confers resistance to phosphinothricinin plants.
• Beta-lactamase gene confers resistance to ampicillin in bacterial system.
• Mutant FabI gene (mFabI) from E. coli which confers resistance to triclosan.

Figure:(A) Herbicide resistant transgenic coffee plant and (B) non-transformed coffee
plant one week after spraying with herbicide (Ammonium glufosinate)

Source:[Link]
04202006000100007&script=sci_arttext (cc)

A B C

Figure: Petriplates showing result of a transformation experiment in Brassica juncea. A

shows a plate used as negative control. In this no transformation has been carried out but
the explants are grown in presence of selective antibiotic, therefore, none of them
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survives. B shows a plate in which transformed explants are grown. Only the positive
transformants are resistant to antibiotic and can grow, the untransformed explants die. C
is a positive control used to ascertain that the explants are healthy. These are
untransformed explants and the plate does not contain selective antibiotic, therefore all
the explants show growth.

Source: Manisha Sharma, Department of Genetics, University of Delhi, South Campus.

Positive selectable marker genes are defined as those that promote the growth of
transformed tissue however, untransformed tissue is not killed in the process.

Initially positive selectable marker genes conditional on the use of toxic agents such as
antibiotics, herbicides or drugs were developed. Recently positive selectable marker genes
which are conditional on non-toxic agents (compounds which induce growth and
differentiationof transformed tissuesas theyare used as substrates)have been developed. The
new generation of positive selectable marker genes is not conditional on external substrates
but they modify the normal physiological processes involved in plant [Link]
gene(derived from E. coli) coding for phosphomannose isomerase enzyme is used as a
positive selection marker. Transformants carrying transgene and pmi gene are able to use
mannose as a carbon source and grow in its presence. However, the untransformed tissue is
unable to metabolize mannose and its growth is arrested.

Figure: Mannose-based positive selection system in plants

Source: [Link] (cc)

Although a large number of selectable marker genes for plant system are known, only a few
are used for crop development and plant research due to practical issues. Many of the marker

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genes have not been thoroughly tested or displayspecific limitations. Biosafety issues are also
considered while choosing markers for future crop developmentand widespread use.

Screening marker
A marker for screening is one which will make cells containing the gene look different and
allows the researcher to distinguish between wanted and unwanted cells/organisms is known
as screening marker. These markers do not provide a cell with a selective advantage, but are
used to identify transgenic events by manually separating transgenic and non-transformed
[Link] systems have been used to determinethe intracellular localization of a gene
product, efficiency of gene delivery systems, detection of protein-protein or protein-DNA
interactions and activity of promoter

A common example of reporter system is blue and white selection in bacteria. This assay is
based upon insertional inactivation of Lac-z-gene which produces beta-galactosidase enzyme.
This enzyme converts a colorless substrate into blue colored product which is responsible for
blue color of colonies. Non recombinant colonies have intact lac-zgene thus they are blue
colored, whereas recombinants have insertionally inactivated lac-zgeneand colorless colonies.

There are three more types of screening markers commonly used:

GUS Assay

GUS assay is a simple method for detection oftransformed cell without needing any
complicated equipment. β-glucuronidase, an enzyme from the bacterium Escherichia coli is
utilized in this technique. This enzyme can transform colorless or non-fluorescent substrates
into coloured or fluorescent products thereby giving the transformed cell a different
phenotype.

Histochemical gusstaining is done using Xgluc i.e. 5-bromo-4-chloro-3-indolyl glucuronide,

which produces blue color. Tissueexpressing the gus gene when incubated with Xgluc
produces blue color. For fluorimetric estimation 4-methylumbelliferyl-beta-D-glucuronide
(MUG) is used. In the reactionMUG acts as a substrate, which upon hydrolysis, produces
glucuronic acid and the fluorescent 4-methylumbelliferone. For 4-MU the excitation is at 365
nm and emission at 455nm.

A B C
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 Methods of Gene Transfer

Figure: GUS histochemical assay in rice, (A) embryo, (B) anthers and style and (C) seed
aleurone layer showing GUS expression.

Source: [Link]

[Link]

[Link] (cc)

This technique is used to analyze the activity of regulatory elements by estimating expression
of GUS gene either quantitativelyor bystudying activity in different tissues. It is very
commonly used but the biggest drawback is that the cells are killed in the process.

GFP

The 2008 Nobel Prize in Chemistry was awarded to Martin Chalfie, Osamu Shimomura and
Roger Y. Tsein for discovery and development of the green fluorescent protein (GFP). GFPis a
protein which exhibits bright green fluorescence after exposure to light in the blue
to ultraviolet range. The cells containing GFP glow green under UV light and can be seen with
the help of a specialized microscope. GFP was first isolated from the jellyfish Aequorea
victoria where it is expressed in small granules around the rim of the jellyfish [Link] made
up of 238 amino acid residues and displays a major excitation peak at 395 nm and a minor
one at 475 nm. It gives a green fluorescence as its emission peak (509 nm) falls in the lower
green portion invisible region of [Link] versions of this protein giving yellow and red

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 Methods of Gene Transfer
fluorescence are also available, so that multiple genes in one organismcan be followed at the
same time.

A B

Figure:GFP expression in (A) mouse embryo (B) Enhanced fluorescence of super folder GFP
mutant (C) Variants of GFP that emit different colors of light.

Source:

[Link]
[Link]

[Link]

[Link] (cc)

GFP gene is frequently used as a reporter which confirms expression of atransgene

throughout the organism. It can be introduced into organisms through breeding, cell
transformationor injection with a viral vector. This gene has been introduced and expressed in
many bacteria, fungi, plant, fish, fly and mammalian cells.

Many different mutants of GFP have been engineered.

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 Methods of Gene Transfer

Examples of GFP

1. A single point mutation (S65T) in GFP dramatically improved the spectral characteristics
of GFP, and resulted in increased fluorescence as well asphoto stability. The improved
version is named as EGFP and is credited with use of GFPs in mammalian cells.

2. Super folder GFP, having a series of mutations that allow its rapid folding even when it is
fused to poorly folding peptides, was reported in 2006.

3. Many other mutations resulting in color mutants have also been made e.g., derivatives of
yellow fluorescent protein (YFP, Citrine, Venus, YPet), blue fluorescent protein (EBFP,
EBFP2, Azurite, mKalama1) and cyan fluorescent protein (ECFP, Cerulean, CyPet).

Source: [Link] (cc)

A B

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Figure: (A) GloFish, the first pet sold with artificially expressing fluorescent proteins, are
available in bright red, green, orange-yellow, blue, and purple fluorescent colors. (B)
Transgenic mice glowing green under UV light (left & right), compared to a normal mouse
(center).
Source:[Link]
[Link] (cc)

Luciferase

Production and emission of light by a living organism is known as [Link] is a type

of chemiluminescence where a chemical reaction results in release of light energy.
Bioluminescence in nature is exhibited by both simple unicellular organisms (e.g., bacteria,
dinoflagellates) and higher-order organisms (e.g., fish, insects). This phenomenon is more
commonly seen in marine animals living in depths of the ocean, as compared to terrestrial
organisms. It serves many natural purposes for these organisms. e.g., defense, camouflage,
feeding, mating etc.

Luciferases are enzymes which emit light. Luciferase-mediated bioluminescence is used by

many organisms to attract prey or mates or for protection from predators. Firefly luciferase
from the firefly Photinus pyralisreleases green light during the oxidation of its chemical
substrate, luciferin. Other organisms which express the luciferase (LUC) gene will also glow
faintly green when supplied with luciferin.

A B

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 Methods of Gene Transfer

Figure:Luciferase-mediated bioluminescence. (A) marine dinoflagellate protist N. scintillans

(known as sea sparkle) causing phosphorescent tide (B) glowing tobacco transformed with
firefly luciferase gene.

Source: [Link] (cc)

[Link] (CC)

Bioluminescence reaction takes place in the presence of two agents:the enzyme (firefly
luciferase) which catalyzes the reaction, and thesubstrate(luciferase). Thisreaction takes place
in two steps:

Luciferin + ATP → Luciferyl Adenylate + O2 → Oxyluciferin + light

Oxyluciferin,formed in the end of this reaction in an electronically excited state and releases a
photon of light while returning to the ground [Link] sensitive apparatus such as
aluminometer or modified optical microscopes are used to detectthis photon
[Link] bioluminescence does not require light excitation resulting in almost
negligible autofluorescence which gives background-free [Link] glow is utilized as an assay
for LUC(Luciferase gene) expression, which acts as a "reporter" to assessthe activity of
regulatory [Link] is inherent variability in light emission form Luciferase enzymes
isolated from different sources, therefore two or more luciferase enzymes can be used in
combination to perform analysis of multiple genes.
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 Methods of Gene Transfer

Summary

1. The artificial or natural introduction of foreign genetic material into a cell is known as
gene transfer.
2. Foreign genes can be introduced artificially into crops by a process known as genetic
transformation.
3. Plant transformation systems generally include introduction of a DNA segment into plant
cells or explants, its integration into host cells genome and subsequent regeneration
from transformed cell to produce whole plant.
4. The DNA segment which is introduced in this process contains the gene of interest with
flanking regions containing promoter, terminator and marker gene.
5. Methods available to achieve genetic transformation of plants can be divided into two
main types; direct and indirect.
6. Methods of genetic transformation which employ bacteria as a vector to introduce the
gene construct into the target cell are known as indirect methods. These methods use
Agrobacterium, whichharbor Ti plasmid and causes crown gall disease in a number of
plants. The genes to be integrated are present in T-DNA (transfer DNA), a specific
region of the Ti plasmid which is transferred to the plant genome. This transfer is
controlled by vir (virulence) genes present in some other region of Ti plasmid.
7. There are two types of genetically engineered Ti plasmid based vectors: Binary and co-
integrate.
8. Direct methods are those methods which do not use bacteria as mediators for
integration of DNA into host genome. These methods include microprojectile
bombardment,electroporation and microinjection.
9. Biolistics is a method for plant transformation where cells are bombarded with heavy
metal particles coated with DNA/RNA. As the microprojectiles penetrate the plant cell
walls and membranes to enter the cells, coated DNA is released from its surface and
incorporated into the plant’s genome. It provides an effective and versatile way to
transform almost all type of cells, including genetic material of the nucleus as well as
organelles.
10. Electroporation is a method of transformation where transfection 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

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 Methods of Gene Transfer
the plasma membrane, thorough which DNA seems to enter inside the cell and then
nucleus.

11. Microinjection is the process of using a fine glass micropipette to manually inject
transgene at microscopic or borderline macroscopic level. Selection of transgenics is
needed in a transformation experiment as only one in many may take up the transgene
depending upon the efficiency of transformation. Rather than checking every single
cell/organism a marker gene is introduced into cell along with the transgene to
determine if the transgene has been successfully inserted into host organism's genome,
as marker gene’s presence can be seen or detected. There are two types of marker
genes: selectable and screening marker.

12. A selectable marker is a gene that protects the organism from a selective agent that
would normally kill it or prevent its growth. In order to find out transformed
cell/organism a selective agent is utilized which kills all the cells withouttransgene,
leaving only the transformed ones. These genes can be divided into several categories
depending on whether they confer positive or negative selection and whether selection is
conditional or non-conditional on the presence of external substrates e.g., herbicides,
antibiotics etc.
13. A marker for screening which will make cells containing the gene look different and
allows the researcher to distinguish between wanted and unwanted cells/organisms is
known as screening marker. These markers do not provide a cell with a selective
advantage, but are used to identify transgenic events by manually separating transgenic
and non-transformed material e.g., Gus, GFP, Luciferase.

Exercise
• How is Agrobacterium-mediatedtransformation is different from other genetic
transformation systems?

• What is the role of Ti plasmid in manifestation of crown gall disease?

• How is Ti plasmid is modified to function as a vector for genetic transformation?

• Discuss the merits and demerits of particle bombardment methodof genetic

transformation.

• What is Electroporation? How it is different from microinjection?

• Why do we need a marker gene in plant transformation experiment?

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 Methods of Gene Transfer
• Differentiate between selectable and screening marker? Give examples.

• What is the difference between positive and negative selection system?

Glossary
Crown gall disease:Adisease that affects many species of plants and is caused by bacteria
(Agrobacteriumspecies) which forms tumorous enlargements (several inches or more in
diameter) usually at the junction of stem and root.

Fluorescence:Fluorescence is the emission of light by a substance that has absorbed light or

other electromagnetic radiation.

Homologous recombination:Homologous recombination is a type of

genetic recombination in which nucleotide sequences are exchanged between homologous
chromosomes.

Horizontal gene transfer:The acquisition by an organism of genetic information by transfer,

for example via the agency of a virus, from an organism that is not its parent and is typically
a member of a different species.

Provirus: A form of virus that is integrated into the genetic material of a host cell and by
replicating with it can be transmitted from one cell generation to the next without causing
lysis.

Reporter genes:A gene which is used to `tag' another gene or DNA sequence of interest,
such as a promoter. Expression of the reporter is easily monitored, and permits the function
or whereabouts of the `target' sequence to be tracked.

Retrotransposons:These aretransposable elements (transposons) that involve a retrovirus-

like process of reverse transcription. The DNA element is transcribed into RNA, reverse-
transcribed into DNA, and then inserted at a new site in the genome.

RNAi:A post-transcriptional genetic mechanism that suppresses gene expression and in which
double-stranded RNA cleaved into small fragments initiates the degradation of a
complementary messenger RNA.

Totipotent:Totipotency is the ability of a single cell to divide and produce all of the
differentiated cells in an organism.

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 Methods of Gene Transfer
Transplastomic plant:A transplastomic plant is a genetically modified plant in which the
new genes have not been inserted in the nuclear DNA but in the DNA of the chloroplasts.

References/Bibliography/Further Reading

• Chawla, H. S. Introduction to plant biotechnology. Science Publishers, Inc., 2000.

• Gelvin, Stanton B. "Agrobacterium-mediated plant transformation: the biology behind the
“gene-jockeying” tool." Microbiology and molecular biology reviews 67.1 (2003): 16-37.
• Mantell, Sinclair H., John A. Matthews, and R. A. McKee. Principles of plant biotechnology:
an introduction to genetic engineering in plants. Blackwell Scientific Publications, 1985.
• Primrose, Sandy B., and Richard Twyman. Principles of gene manipulation and genomics.
John Wiley & Sons, 2009.
• Slater, Adrian, Nigel W. Scott, and Mark R. Fowler. "Genetic manipulation of plants." Plant
Biotechnology Oxford, England: Oxford University Press (2003).

Web links

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ng_Crops

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