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Chapter -2 Gene Transfer Methods in Plants

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 Chapter - 2
Gene Transfer Methods in Plants

Authors
Shivangi Rahangdale
Ph.D. Scholar
Department of Plant Breeding & Genetics
JNKVV, Jabalpur, Madhya Pradesh, India
Yogendra Singh
Assistant Professor (Senior Scale)-Biotechnology,
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur,
Madhya Pradesh, India
Deepak Katkani
Ph.D. Scholar
Department of Plant Breeding & Genetics
JNKVV, Jabalpur, Madhya Pradesh, India
Naveen Surjaye
M.Sc. Scholar
Department of Plant Breeding& Genetics
JNKVV, Jabalpur, Madhya Pradesh, India

Page | 19
Page | 20
 Chapter - 2
Gene Transfer Methods in Plants
Shivangi Rahangdale, Yogendra Singh, Deepak Katkani and Naveen Surjaye

Abstract
The continuous improvement in gene transfer approaches changed the
path of crop modification and resulted in significant advancements in crop
improvement, crop protection and agricultural production. Genetic
engineering that has revolutionized the crop improvement pathway involves
identifying and transferring novel genes to existing elite cultivars. Various
methods have been developed for transferring the gene into plant cells, and
continuous efforts have been made to increase its efficiency. Both direct and
indirect gene transfer approaches contain their own merits and demerits.
Continuous efforts were made to eliminate drawbacks and develop an easy,
elite and environmentally sound method for gene transfer. In recent year
studies, Agrobacterium-mediated gene transfer and gene gun transformation
method have proven to be doing well. The methodology concerned, the
merits and demerits of the various methods were discussed briefly as a
whole.
Keywords: agrobacterium, electroporation, gene gun, microinjection,
microprojectile, PEG, transformation
Introduction
Plant breeders have developed elegant schemes for crossing plants to
transmit and maintain the necessary and desirable traits in inbred lines.
Classical plant breeding method is, however, unpredictable and sluggish. A
mutual cross between two lines and then repeated back crossing between the
hybrid offspring and one of the parents is needed to transfer a specific gene
of interest through classical methods until a plant with the desirable
characteristic is produced. Plant breeding is a lengthy process, which takes
10 to 15 years to produce and release a new variety. Nevertheless, this
method is restricted to those plants that can hybridize sexually, and genes
will be passed in addition to the target allele.
Recombinant DNA technology by allowing plant genetics to recognise
and clone specific genes for desirable traits to overcome certain constraints.

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Plants have many distinct biological characteristics that can be examined
through recombinant DNA technology. Basic techniques used for genetic
manipulation of plants are discussed in this correspondence. Plant genetic
engineering not only improved the cycle of plant breeding but also helped to
introduce new genes, either by removing the obstacles of sexual
incompatibility by somatic hybridization or by introducing necessary genes
into plant cells utilizing different methods of transformation.
Genetic transformation is a powerful tool and a significant strategy for
studying plant functional genomics, i.e. gene exploration, new insights into
gene regulation, and the analysis of genetically regulated characteristics.
Furthermore, the work of isolated genes utilizing map-based cloning of
mutant alleles has been verified through functional complementation via
genetic transformation. In addition, genetic engineering allows the insertion
of alien genes into crop plants and the accelerated creation of new
genetically modified organisms. Gene transformation and genetic
engineering lead to the overall production of crops. (Sinclair et al., 2004).
Agrobacterium-mediated gene transfer is an indirect method and is highly
efficient compared with other methods that have been discovered
unexpectedly during the search for the causal organism for crown gall
disease (Que et al., 2019).
Importance of gene transfer technologies to plants
i. Provide resistance against viruses
ii. Acquire insecticidal resistance
iii. To strengthen the plant to grow against bacterial diseases
iv. Develop the plants to grow in draught
v. Engineering plants for nutritional quality
vi. Make the plants to grow in various seasons
vii. Herbicide resistant plant can be made
viii. Resistance against fungal pathogens
ix. Engineering of plants for abiotic stress tolerance
x. Delayed ripening can be done
Gene transfer technologies in plants
The process of transfer, integration and expression of transgene in the
host cells is known as genetic transformation. A foreign gene (transgene)
encoding the trait must be incorporated into plant cells, along with a
"cassette" of extra genetic material to add a desirable trait to a crop. The

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cassette includes a sequence of DNA called a "promoter", which determines
where and when the foreign gene is expressed in the host, and a "marker
gene" which allows breeders to determine by screening or selection which
plants contain the inserted gene. For example, marker genes may make
plants resistant to antibiotics not used routinely (e.g., agrimycin, kanamycin)
or tolerant of some herbicides.
Various genetic transfer techniques are grouped into two main
categories.
1) Vector mediated gene transfer (Indirect method)
2) Vectorless gene transfer (Direct method)
Table 1: Different gene transfer methods with properties

Transformation method Properties References

Ti-plasmid containing T-DNA
Agrobacterium-mediated
is used in the transfer of the Slater et al., 2009
gene transfer
DNA segment
Non-Agrobacterium Microbes containing plasmid of
Mullins, 2018
mediated gene transfer order Rhizobiales are used
Fiandaca and Federoff,
Viral method of gene Integration of gene into the
2014, Patel and Misra,
transfer viral genome
2011
Microcarrier coated with DNA
Gene gun is directly delivered into target Rajasekaran, 2013
under high pressure
Electrical charges are used to
Electroporation make pores in the membrane Young and Dean, 2015
that helps in the entry of DNA
Micropipette loaded with
Microinjection aqueous DNA deliver the target Saito, 2005
genes to nucleus or cytoplasm
Akowuah et al., 2005;
Ultrasound waves play a role in
Sonoporation Cochran and Wheatley,
gene delivery
2013
Hydro-dynamic gene Gene delivery is by the Jinturkar, Rathi and Misra,
transfer hydrostatic pressure 2011; Weiwei Wang, 2013
Polyethylene glycol Polyether compound PEG
Sahab, Hayden, Mason
(PEG)-induced DNA adhere to DNA and transport it
and Spangenberg, 2019
uptake into a target
Liposome-mediated gene Cationic liposome deliver DNA Kulkarni, Greiser, O'Brien
delivery into the host cell and Pandit, 2010
Fibre mediated gene Silicon carbide fiber facilitated Simon and Foroughi-
delivery the transfer Wehr, 2000

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Vector mediated gene transfer (indirect method)
Vector-mediated gene transfer is carried out either by Agrobacterium-
mediated transformation or by use of plant viruses as vectors. In this
approach the transgene is combined with a vector which takes it to the target
cells for integration. The term plant gene vector applies to potential vectors
both for transfer of genetic information between plants and the transfer of
genetic information from other organisms (bacteria fungi and animals) to
plants. The vector mediated transfer is strongly linked to regeneration
capabilities of the host plant.
The plant gene vectors being exploited for transfer of genes are plasmids
of Agrobacterium viruses and transposable elements.
Agrobacterium mediated transformation
The Agrobacterium system was historically the first successful plant
transformation system, marking the breakthrough in plant Genetic
engineering in 1983. The Agrobacterium is naturally occurring gram
negative soil bacterium with two common species A Tumifacience and a
rhizogenes there are known as natural gene engineers for their ability to
transform plants. A tumifacience induces tubers called crown galls, whereas
a rhizogenes causes hairy root diseases. Large plasmids in these bacteria are
called tumour inducing (Ti plasmid) and root inducing (Ri plasmid)
respectively. The major credit for the development of plant transformation
techniques goes to the natural unique capability of A. tumefaciens.
The Ti plasmid has two major segments of interest in transformation
that is T DNA and virus region. The T DNA region of the Ti plasmid is the
part which is transferred to plant cell and incorporated into nuclear genome
of cells. The transfer of T DNA is mediated by genes in the another region of
Ti plasmid called virs genes (virulence genes). Modified Ti plasmid are
constructed that lack of undesirable Ti genes but contain a foreign gene
(resistant to a disease) and a closely linked selectable marker gene (E.g.: - for
antibiotic resistance). Within the T DNA region any gene put in T DNA
region of plasmid cysts transferred to the plant genome. The T DNA is
generally integrated in low copy number per cell. Transfer of gene through
to wounded plant organs A. tumifacience has limited range of host. It can
infest about 60% gymnosperms and Angiosperm. Hence Agrobacterium
mediated transformation is the method of choice in dicotyledonous plant
species, where plant regeneration system are well established, However,
Monocotyledons could not be successfully utilized for Agrobacterium
mediated gene transfer.

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Plant transformation technique using agrobacterium
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.
acetosyringone) for activation of virulence genes
ii) Transformed cells/tissues should be capable to regenerate into
whole plants

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 In general, most of the Agrobacterium-mediated plant transformations
have the following basic protocol:
1. Development of Agrobacterium carrying the co-integrate or binary
vector with the desired gene
2. Identification of a suitable explant e.g. cells, protoplasts, tissues,
calluses, organs
3. Co-culture of explants with Agrobacterium
4. Killing of Agrobacterium with a suitable antibiotic without harming
the plant tissue
5. Selection of transformed plant cells
6. Regeneration of whole plants
Advantages
i. It is a natural means of gene transfer
ii. Agrobacterium is capable of infecting plant cells and tissue and
organs
iii. Agrobacterium is capable of transfer of large fragments of DNA
very efficiently
iv. Integration of T DNA is a relative precise process
v. The stability of gene transferred in excellent
vi. Transformed plants can be regenerated effectively
Limitations
i. Host specificity: There is a limitation of host plants for
Agrobacterium, since many crop plants (monocotyledons e.g.
cereals) are not infected by it. In recent years, virulent strains of
Agrobacterium that can infect a wide range of plants have been
developed
ii. Inability to transfer multiple genes: The cells that regenerate
more efficiently are often difficult to transform, e.g. embryonic
cells lie in deep layers which are not easy targets for Agrobacterium
iii. Somaclonal variation
iv. Slow regeneration
Vectorless gene transfer (direct method)
When the foreign DNA is directly inserted into the plant genome, the
word direct or vector less DNA transmission is used. Direct DNA transfer

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methods rely on naked DNA being delivered into the plant cells. This
contrasts with the transfer of agrobacterium or vector-mediated DNA that
can be considered indirect methods. The majority of the methods for direct
transfer of DNA are simple and effective. And in addition to this process has
been used to develop other transgenic plants. The introduction of DNA into
plant cells without biological agents such as Agrobacterium being involved
and leading to stable transformation is called direct gene transfer.
Types of direct DNA transfer
The direct DNA transfer can be broadly divided into three categories.
1. Physical gene transfer methods:
1.1 Electroporation
1.2 Particle bombardment
1.3 Micro injection
1.4 Macro injection
1.5 Liposome-Mediated Transformation
1.6 Silicon Carbide Fibre-Mediated Transformation
2. Chemical gene transfer methods:
2.1 Poly-ethylene glycol (PEG)-mediated
2.2 Diethyl amino ethyl (DEAE) dextran-mediated
2.3 Calcium phosphate precipitation
3. DNA imbibition by cells/tissues/organs
4. Pollen transformation
5. Delivery via growing pollen tubes
6. Laser induced transformation
7. Etc.
1. Physical gene transfer methods
1.1 Electroporation
Electroporation is the incorporation of DNA into the cell by exposing
them to high voltage electrical pulses for a very short period of time to cause
temporary pores in the plasma lemma. Plant cell electroporation generally
uses protoplast, while thick plant cell walls restrict the movement of
macromolecule (Bates, 1999).
The plant material is incubated in a buffer solution containing the
desired foreign/target DNA, and subjected to high voltage electrical

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impulses. The electric current leads to the formation of small temporary
holes in the membrane of the protoplasts through which the DNA can pass.
After entry into the cell, the Foreign DNA gets incorporated with the host
genome, resulting the genetic transformation the protoplasts are then
cultured to regenerate in to whole plants. This method can be used in those
crop species in which regeneration from protoplast is possible.
In the early years, only protoplasts were used for gene transfer by
electroporation. Now a day, 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.

Fig 2: Plant transformation process using particle bombardment includes the

following steps: (1) Isolate protoplasts from leaf tissues. (2) Inject DNA-coated
particles into the protoplasts using particle gun. (3) Regenerate into whole plants. (4)
Acclimate the transgenic plants in a greenhouse

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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 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
Particle bombardment/microprojectile/biolistic/gene gun/particle
acceleration
Particle bombardment is a technique used to introduce foreign DNA into
plant cells (Birch & Franks, 1991; Christou, 1992, 1995; Gan, 1989;
Takeuchi et al., 1992; Yao et al., 2006). 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. The process of transformation
employs foreign DNA coated with minute 0.2-0.7 µm gold (or) are tungsten
particles to deliver into target plant cells. The coated particles are loaded into
a particle gun and accelerated to high speed-
 By using pressurized helium gas
 By electro static energy released by a droplet of water exposed to a
high voltage
The target could be plant cell suspensions, callus cultures, or tissues.
The projectiles penetrate the plant cell walls and membranes. As the micro
projectiles enter the cells, transgenes are released from the particle surface
for subsequent incorporation into the plant’s chromosomal DNA.
Plant material used in bombardment: Two types of plant tissue are
commonly used for particle bombardment:
1. Primary explants which can be subjected to bombardment that are
subsequently induced to become embryo genic and regenerate
2. Proliferating embryonic tissues that can be bombarded in cultures
and then allowed to proliferate and regenerate

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 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
list of the transgenic plants (developed by biolistics) along with
Table 2: List of transgenic plants (along with cell sources) developed by
microprojectile bombardment

Plant Cell Source(s)

Rice Embryonic callus, Immature zygotic embryos
Wheat Immature zygotic embryos
Barley Cell suspension, Immature zygotic embryos
Corn Immature zygotic embryos, Embryonic cell suspension
Sorghum Immature zygotic embryos
Peas Zygotic embryos
Cotton Zygotic embryos
Tobacco Pollen
Sweet potato Callus cells
Groundnut Embryonic callus
Alfalfa Embryonic callus
Grape Embryonic callus
Banana Embryonic cell suspension
The sources of the plant materials used is given in Table-2.

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Factors affecting bombardment
i) Nature of micro particles: Inert metals such as tungsten, gold and
platinum are used as micro particles to carry DNA. These particles
with relatively higher mass will have a better chance to move fast
when bombarded and penetrate the tissues.
ii) Nature of tissues/cells: The target cells that are capable of
undergoing division are suitable for transformation.
iii) Amount of DNA: The transformation may be low when too little
DNA is used. On the other hand, too much DNA may result is high
copy number and rearrangement of transgenes. Therefore, the
quantity of DNA used should be balanced. 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.
iv) Environmental parameters: Many environmental variables are
known to influence particle bombardment. These factors
(temperature, humidity, photoperiod etc.) influence the physiology
of the plant material, and consequently the gene transfer. It is also
observed that some explants, after bombardment may require
special regimes of light, humidity, temperature etc.
The technology of particle bombardment has been improved in recent
years, particularly with regard to the use of equipment. A commercially
produced particle bombardment apparatus namely PDS-1000/HC is widely
used these days.
Advantages of particle bombardment
i) Gene transfer can be efficiently done in organized tissues
ii) Different species of plants can be used to develop transgenic plants
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

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1.2 Microinjection
Microinjection is a direct physical method involving the mechanical
insertion of the desirable DNA into a target cell. The target cell may be the
one identified from intact cells, protoplasts, callus, embryos, meristems etc.
Microinjection is used for the transfer of cellular organelles and for the
manipulation of chromosomes.
The DNA solution is injected directly inside the cell using capillary
glass micropipettes (0.5-10.0 pm tip) with the help of micromanipulators of a
microinjection assembly. It is easier to use protoplast than cells since cell
wall interferes with the process of microinjection. The protoplast are usually
immobilized in agarose (or) on a glass slides coated with polylysine or by
holding them under suction by a micropipette.
As the process of microinjection is complete, the transformed cell is
cultured and grown to develop into a transgenic plant. In fact, transgenic
tobacco and Brassica napus have been developed by this approach. The
major limitations of microinjection are that it is slow, expensive, and has to
be performed by trained and skilled personnel. The process of microinjection
is technically demanding and time consuming a maximum of 40-50
protoplasts can be microinjected in one hour.
1.3 Microinjection
The injection of plasmid DNA into the lumen of developing
inflorescence using hypodermic syringe is known as macro injection. An
aqueous solution of DNA was introduced into the developing floral tillers 14
days prior to meiosis. Transformed seeds were obtained from these injected
tillers after cross pollination with other and injected tillers. However, the
mechanism by which the DNA entered the zygotic tissue yet unknown.

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

Page | 33
Advantages of SCF-mediated transformation
i) Direct delivery of DNA into intact walled cells. This avoids the
protoplast isolation
ii) Procedure is simple, rapid and does not involve costly equipment
Disadvantages of SCF-mediated transformation
i) Silicon carbide fibres are carcinogenic and therefore have to be
carefully handled
ii) The embryonic plant cells are hard and compact and are resistant to
SCF penetration
In recent years, some improvements have been made in SCF-mediated
transformation. This has helped in the transformation of rice, wheat, maize
and barley by using this technique.
2. Chemical gene transfer methods
2.1 Polyethylene glycol (PEG)-mediated transfer
Polyethylene glycol (PEG), in the presence of divalent cations (using
Ca2+), destabilizes the plasma membrane of protoplasts and renders it
permeable to naked DNA. In this way, the DNA enters nucleus of the
protoplasts and gets integrated with the genome. The procedure involves the
isolation of protoplasts and their suspension, addition of plasmid DNA,
followed by a slow addition of 40% PEG-4000 (w/v) dissolved in mannitol
and calcium nitrate solution. As this mixture is incubated, protoplasts get
transformed.
Advantages of PEG-mediated transformation
i) A large number of protoplasts can be simultaneously transformed
ii) This technique can be successfully used for a wide range of plant
species
Limitations of PEG-mediated transformation
i) The DNA is susceptible for degradation and rearrangement
ii) Random integration of foreign DNA into genome may result in
undesirable traits
iii) Regeneration of plants from transformed protoplasts is a difficult
task
2.2 DEAE dextran-mediated transfer
The desirable DNA can be complexed with a high molecular weight
polymer diethyl amino ethyl (DEAE) dextran and transferred. The efficiency

Page | 34
increased to 80% when DMSO shock is given. The major limitation of this
approach is that it does not yield stable trans-formants.
2.3 Calcium phosphate co-precipitation-mediated transfer
The DNA is allowed to mix with calcium chloride solution and isotonic
phosphate buffer to form DNA-calcium phosphate precipitate. When the
actively dividing cells in culture are exposed to this precipitate for several
hours, the cells get transformed. The success of this method is dependent on
the high concentration of DNA and the protection of the complex precipitate.
Addition of dimethyl sulfoxide (DMSO) increases the efficiency of
transformation.
2.4 DNA imbibition by cells tissue, embryos and seeds
When dry isolated embryos of wheat barley, rye, pea and bean are
imbibed in a DNA solution, they take up the DNA and show the expression
of marker gene. Dry seeds, whose seed coats have been removed also take
up DNA when imbibed in a DNA solution. The imbibed seeds or embryos
are germinated on appropriate selective medium to isolate the transformed
embryos. It was thought that the DNA is taken up by the embryos through
the cells injured during their isolation. The DNA then moves through
plasmodesmata to other cells of embryos.
2.5 Pollen transformation
Involves the gene transfer by soaking the pollen grains in DNA solution
prior to their use for pollination. The method is highly attractive in view of
its simplicity and general applicability but so far there is no definite evidence
for a transgene being transferred by pollen soaked in DNA solution.
2.6 DNA delivery via growing pollen tubes
The stigmas were cut after pollination exposing the pollen tubes, the
DNA was introduced onto the cut surface that presumably diffused through
the germinating pollen tube into the ovule. This method is simple easy and
very promising provided consistent result and stable transformations are
achieved the mechanism of DNA transfer into zygote through this method is
not yet established.
2.7 Laser induced transformation
It is method of introducing DNA into plant cells with a laser micro
beam. Small pores in the membrane are created by laser micro beam. The
DNA from the surrounding solution may than enter into the cell cytoplasm
through the small pores. Laser-induced stress waves facilitate targeted gene

Page | 35
transfer. Pressure waves caused by nanosecond laser pulses can be used to
deliver macromolecules to cells and tissues. It is well established that a
strong pressure wave, known as a photomechanical or laser-induced stress
wave (LISW), accompanies laser-induced plasma.
Each and every approach has its own benefit and drawback in terms of
stability, specificity of the host and number of copies etc., as shown in Table
3 as suggested by Jinturkar et al., 2011. Recently two DNA delivery systems
viz. Agrobacterium mediated gene transfer and particle bombardment are
widely used for the development of transgenic embryos.
Table 2: Merits and demerits of different gene transformation methods

Transformation method Merits Demerits

Stable transformation and
Agrobacterium-mediated
high transformation Host-specific
gene transfer
frequency
Non-agrobacterium An alternate approach to Limited host range and low
mediated gene transfer Agrobacterium frequency of transformation
Viral method of gene Only transient
Wide host range
transfer transformation
Gene gun Easier and highly adaptable High copy number
Applicable to many species Standardization of dose is
Electroporation
and equipment is simple difficult
Microinjection Easy method Time-consuming
Eco-friendly approach Under research and need
Sonoporation
development more
Hydro-dynamic gene
Easy and reliable Need more development
transfer
Polyethylene glycol No need for specialized Preparation of transgenic
(PEG)-induced DNA equipment. Simple plant from protoplast is
uptake procedure difficult.
Liposome-mediated gene Easy to formulate, less Possible only in a limited
delivery toxicity number of species
Fibre mediated gene Less Transformation
Technically simple
delivery frequency

Application of gene transfer technology in crop improvement

Biotechnological strategies for crop improvement demand efficient
procedures for routine introduction of defined foreign genes into plant
genome. Successful genetic manipulation requires the ability to deliver
biologically active and functional DNA into plant cells followed by recovery
of transgenic plants expressing a foreign gene. Gene transfer technology is
playing significant role in improving plants and their yields.

Page | 36
Transformation of rice
Biolistic was successfully used for transformation of immature embryos
of rice. Reports were also made regarding the transformation of indica and
javanica rice in addition to other japonica rice. Fujimoto et al. (1993) were
the first to engineer japonica rice through electroporation with modified d-
endotoxin gene (cry) from Bacillus thuringiensis. It was found that the R2
generation of transgenic rice was more resistant to insects than wild type
plants. Later, Wu¨nn et al. (1996) obtained transgenic indica rice cultivar
IR58 expressing a synthetic cryIA(b) gene driven by 35S promoter through
particle bombardment. The rice Xa21 gene which confers resistance to blight
pathogen, Xanthomonas oryzae was cloned by Song et al. (1995).
Transgenic rice plants harbouring the cloned gene displayed high levels of
resistance. The gene has been found to be effective against several isolates.
Shimada et al. (1993) produced transgenic rice plants with antisense
construct of rice waxy gene coding for granule-bound starch synthase under
the control of 35S promoter. A significant reduction in amylose content of
grain starch was observed in the seeds of these plants. Most interestingly, to
confer the capability of producing precursor (β-carotene) of vitamin A in rice
endosperm, Burkhardt et al. (1997) engineered rice with the cDNA coding
for phytoene synthase from daffodil, the first of the four specific enzymes
involved in β-carotene (provitamin A) biosynthesis in plants. In the
endosperm of these transgenic plants, phytoene synthase accumulation was
observed indicating that engineering of provitamin A biosynthesis pathway
is possible in non-photosynthetic, carotenoid lacking tissue. Recently, the
same group reported Agrobacterium-mediated transformation of rice with all
the genes necessary for the accumulation of provitamin A in transgenic rice
seeds. Hayakawa et al. (1992) engineered the coat protein (Cp) gene of rice
stripe virus into two japonica rice varieties by electroporation of protoplasts
resulting in significant levels of resistance against the virus in the transgenic
plants.
Genetically engineered maize
A coat protein-mediated resistance to viruses, introduced in rice via
protoplast transformation, was transferred to maize and barley via particle
gun bombardment. The resistance to sulfonylurea (herbicide) conferred by
the ALS gene of Arabidopsis thaliana was also transferred to maize by
particle gun technology. Maize has been reported to get transformed by
Silicon carbide fiber-mediated DNA delivery system. Whiskers-mediated
maize transformation has also been reported.

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 Genetically engineered wheat
Wheat is a member of the Triticeae group of cereals. It is indisputably
one of the major food crops of the world and a foundation of human
nutrition. Genetic improvement of wheat has received considerable attention
worldwide over the years with the purpose of increasing the grain yield to
minimize crop loss due to unfavorable environmental conditions and
development of resistance against various pests and pathogens. The first
transgenic wheat plants were produced by Vasil et al. (1992), followed by
Vasil et al. (1993), Weeks et al. (1993), Nehra et al. (1994), and Altpeter et
al. (1996) employing microprojectile bombardment as a method of DNA
delivery. Subsequently, the development of methodology for the delivery of
genes into intact plant tissues by bombardment of DNA-coated gold or
tungsten particles has revolutionized the field of wheat transformation. In
recent years, sincere efforts are being made to transform wheat genetically
with different alien genes of agronomically importance. However, in
majority of reports, genetic transformation with a single target gene has been
used for the production of transgenic wheat expressing tolerance to
herbicide, resistance to fungal and viral diseases.
References
1. Akowuah EF, Gray C, Lawrie A, Sheridan PJ, Su CH, Bettinger T et al.
Ultrasound Mediated delivery of TIMP-3 plasmid DNA into saphenous
vein leads to increased lumen size in a porcine interposition graft model.
Gene Ther. 2005; 12(14):1154-1157. doi: 10.1038/ sj.gt.3302498.
2. Altpeter F, Vasil V, Srivastava Stroger E, Vasil IK. Accelerated
production of transgenic wheat (Triticum aestivum L.) plants. Plant Cell
Rep. 1996; 16:12-17.
3. Barro F, Rooke L, Bekes F, Gras P, Tatham AS, Fido R et al.
Transformation of wheat with high molecular weight subunit genes
results in improved functional properties. Nature Biotech. 1997;
15:1295-1299.
4. Bates GW. Plant transformation via protoplast electroporation. In Plant
Cell Culture Protocols, 1999, 359-366
5. Birch RG, Franks T. Development and optimization of microprojectile
systems for plant genetic transformation. Australian Journal of Plant
Physiology. 1991; 18:453-469. ISSN 0310-7841.
6. Bullock W, Dias D, Bagnal S, Cook K, Teronde S, Ritland J. A high
efficiency maize “whisker” transformation system. Plant and Animal
Genomes IX Conference, San Diego, CA, 2001, 148.

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