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Gene Transfer Technologies in plants: Roles in improving Crops

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MOLECULAR BIOLOGY & BIOTECHNOLOGY

GENE TRANSFER TECHNOLOGIES IN PLANTS: ROLES IN IMPROVING CROPS

Abstract
Gene is a segment of nucleic acid that encodes a functional protein or RNA and is the unit of inheritance. The principle objective of
plant biotechnology is to create new varieties of cultivated plants by manipulating DNA molecule. Plant transformation technology
has become a versatile platform for cultivar improvement as well as for analysis of gene function in plants. This article discuses
and summarizes important work in the literature regarding the gene transfer technologies in plants. The main techniques focused
in this article are gene transfer by Agrobacterium tumefaciens, microprojectile bombardment, electroporation of protoplast,
polyethylene glycol method, microinjection, silicon carbide mediated transformation, liposome mediated gene transfer and
sonication assisted Agrobacterium-mediated transformation. Moreover the application of gene transfer technologies related to the
improvement of crops was also focused. This article will help the reader to have an idea on gene transfer technologies and also to
the researcher working on plant genetic engineering.

Key Words: Gene; Gene transfer; Transformation technology; Crops improvement.

1. Introduction
Genetic manipulation of plants has been done by breeding, but also helps the introduction of new genes,
plant breeders for years with great success. Elegant either by overcoming the barriers of sexual incompatibility
schemes have been developed by plant breeders for through somatic hybridisation or by the insertion of
crossing plants in order to transmit and maintain the required genes into plant cells using various
required and desirable traits in inbred lines. However, the transformation methodologies. This article will be helpful
process of classical plant breeding are uncertain and for those researchers who are working in the field of plant
slow. To transmit a required gene of interest by classical genetic engineering.
methods requires a sexual cross between two lines and
then repeated back crossing between the hybrid offspring 2. Gene transfer technologies in plants
and one of the parents until a plant with the desired Presently, a number of methods exist for the genetic
characteristic is obtained. Plant breeding is a lengthy manipulation of plant cells. These procedures range from
process, taking ten to fifteen years to produce and to exploitation of the natural gene transfer system
release a new variety. This process, however, is limited of Agrobacterium to the chemical treatment of isolated
to those plants which can sexually hybridize, and genes protoplasts by polyethylene glycol. It also includes
in addition to the desired gene will be transferred. physical procedures of DNA introduction, including
Recombinant DNA technologies circumvent these electroporation of protoplasts and tissues, microinjection
limitations by enabling plant geneticts to identify and and silicon carbide fibre-mediated transformation.
clone specific genes for desirable traits. Plants have a Moreover microprojectile bombardment has also received
number of unique biological features that can be explored much attention as a physical method of DNA transfer and
with recombinant DNA technologies. In this in many laboratories, is now a routine and reliable
communication basic techniques used to manipulate technique for the production of transgenic plants. The
plants genetically are discussed. The progresses in importance of gene transfer technologies to plants are
developing agriculturally important plants by recombinant listed in Table 1. A number of gene transfer technologies
DNA technologies are reviewed. Plant genetic are discussed below.
engineering not only elevated the process of plant


Corresponding Author, Email: hamkishwar191@yahoo.co.in
 K. H. Khan/Rec Res Sci Tech 1 (2009) 116-123

Table 1: Benefits of gene transfer technologies. three years later [10]. In the same year, microprojectile-
S.No Importance of gene transfer technologies to plants mediated delivery of plasmid DNA resulted in
1. Provide resistance against viruses. the introduction of a foreign gene, also in onion cells [11].
2. Acquire insecticidal resistance. Microprojectile bombardment has got much attention
3. To strengthen the plant to grow against bacterial and attraction as a physical procedure of DNA transfer
diseases. in many research laboratories during the past years. This
4. Develop the plants to grow in draught. method is routine and reliable way of producing
5. Engineering plants for nutritional quality. transgenic plants. The method relies on a device which
6. Make the plants to grow in various seasons.
utilizes a propelling force, such as compressed gas or
7. Herbicide resistant plant can be made.
8. Resistance against fungal pathogens.
gunpowder, to accelerate inert (usually metal) particles
9. Engineering of plants for abiotic stress tolerance. (the microprojectiles), coated with DNA, into target cells.
10. Delayed ripening can be done. This technique is also referred to as particle
bombardment, particle gun method, particle acceleration
2.1. Gene transfer by Agrobacterium tumefaciens and Biolistics (Biological ballistics). A number of
Agrobacterium tumefaciens has been extensively applications of this method in plant science have been
used to introduce gene into plant cells. This bacterium is listed in Table 2.
responsible for crawn gall disease in a variety of
Table 2: List of applications of microprojectiles.
dicotyledonous plants. A plsmid carried within this S.No Applications of microprojectile bombardment
bacterium cause crown gall disease [1][2]. This plasmid 1. Highly versatile and adaptable technique which
is called tumor inducing plasmid (Ti). Ti plasmid is upto can be applied to a wide range of cells and
200 bp large and carries genes that are required for tissues.
infection. This plasmid has T-DNA that becomes 2. Method is simple and efficient.
integrated into plant genome at an apparently random 3. The process of microprojectile bombardment has
position through non homologous recombination. The also led to an increased understanding of the
size of T-DNA is approximately 23 kbp and is responsible mechanisms of gene expression and regulation.
for the cancerous properties of the transformed cells. It 4. Microprojectile bombardment can even be used to
also synthesizes opines. In the T plasmid, T-DNA is wound plant tissues, allowing more efficient
flanked by two 25 bp imperfect direct repeats. These transformation via Agrobacterium.
sequences play roles in the integration of T-DNA into the 5. This method permits the transformation of cells
plant genome [3]. Agrobacterium has proved to be an from a wide range of sources including cell
incredible useful tool for the integration of genes into suspensions, callus, meristematic tissues,
plants [4]. immature embryos, protocorms, coleoptiles and
The most widely used technique for plant pollen.
transformation is based on Agrobacterium, in which novel 6. Microprojectile technique significantly reduces the
genes, linked to the Ti or Ri plasmid T-DNAs, are inserted time required for the production of genetically
into the host plant cells during T DNA transfer modified plants.
7. This method help in the transformation of several
[5]. This approach has been used to transform numerous
major cereals, including barley, maize, wheat, rice,
plants, but is almost exclusively restricted to dicotyledons
pearl millet, together with other monocotyledons
[6]. Despite recent reports using a strain of A.
such as tulip and orchids.
tumefaciens carrying a vector with the vir B and vir G
genes from the supervirulent Ti plasmid pTiBo542 to
2.3. Electroporation of protoplast
transform rice [7], attempts to infect monocotyledonous
High concentration of plasmid DNA containing the
plants, which constitute some of the world's most
gene of interest is added to a suspension of protoplast
important food crops, with Agrobacteria have been rarely
and the mixture is given a shock with an electric field of
successful [8].
200-600 V/cm. The protoplasts are then grown in tissue
culture for a period of one or two weeks. The selection
2.2. Gene transfer by microprojectile bombardment
pressure is then applied to select the transformed one.
The cocept of transfering DNA-coated particles
Both maize and rice protoplast have been successfully
directly into cells was first conceived by Sanford and co-
transformed with efficiencies of between 0.1 and 1%.
workers in 1984 [9]. The first results using a gunpowder-
Introduction and expression of transgenes in plant
driven device to deliver tungsten microprojectiles coated
protoplasts were also reported [12]. Moreover transient
with viral RNA into onion epidermal cells were published

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expression of fluorescent fusion proteins in protoplasts of expandability. In this method silicon carbide fibers’ are
suspension cultured cells were also explained [13]. added to a suspension containing plasmid DNA and plant
tissue (immature embryos, callus, cell cluster). It is then
2.4. Gene transfer by polyethylene glycol mixed in commercial shakers or in vortex. Fibres coated
This technology is applicable for protoplast only. The with DNA penetrate the plant cell wall in the presence of
chemical used is polyethylene glycol. It stimulates small holes produced at the time of collision between
endocytosis and thereby causing the uptake of DNA. In fibres and plant cells [21]-[23].
this method protoplast are kept in polyethylene glycol The factors on which the efficiency of transformation
(PEG) solution. The concentration of PEG used is 15% depends are the plant material, fiber size, parameters of
having 8000 dalton molecular weight. After exposure of vortexing, shape of the vessels used, and the
protoplasts to exogenous DNA in presence of PEG and characteristics of the plant cells, especially the thickness
other chemicals, PEG is removed and intact protoplast of the cell wall. This process is easy and quick. It is not
are then cultured to form cells with walls and colonies in so expensive and useful for various plant materials. The
turn [14]. Selection pressure is then applied to get the main drawback of this technique is low transformation
transformants. The transfer of gene across the protoplast efficiency, damage to cells negatively influencing their
membrane can be initiated by a number of chemicals of further regeneration capability, and the need of following
which polyethylene glycol is the most important. It has extraordinarily rigorous precaution protocols during
become the most widely used due to the availability of laboratory work, as breathing the fibers in, especially
simple transformation protocol. Method was developed asbestos ones, can lead to serious sicknesses [24].
using calcium alginate micro beads to immobilize DNA Silicon carbide whisker-mediated embryogenic callus
molecules in combination with polyethylene glycol transformation of cotton (Gossypium hirsutum L.) and
treatment also [15]. regeneration of salt tolerant plants were also reported
[25].
2.5. Gene transfer through microinjection
Transformation through microinjection is based on 2.7. Liposome mediated gene transfer
introducing DNA into the cytoplasm or nucleus by using a Liposomes are circular lipid molecules with an
glass micro capillary-injection pipette [16]-[17]. This aqueous interior that can carry nucleic acids. It
operation requires a micromanipulator. During the encapsulates the DNA fragments and then adheres to
introduction of DNA into the nucleus, cells are the cell membranes and fuse with them to transfer DNA
immobilized with a holding pipette and gentle suction. fragments. Thus, the DNA enters the cell and then to the
Microinjection is mainly used for the transformation of nucleus. It is a very efficient technique used to transfer
large animal cells. Its importance for plant transformation genes in bacterial, animal and plant cells. Various reports
is rather limited due to the characteristics of plant cell on the integration of genes introduced by means of
walls, which contain a thick layer of lignins and cellulose. liposomes followed by transgenic plant regeneration for
The plant cell wall is a barrier for glass micro tools. The tobacco [26] and wheat [27] have been published so far.
method allowed the incorporation not only of DNA
plasmids but also of whole chromosomes into plant cells 2.8. Pollen tube pathway method
[18]-[19]. The transformation method via pollen-tube pathway
Although it has a fairly high transformation frequency has great significance in agriculture molecular breeding
(20–50%), microinjection is a time consuming process [28]. After pollination the styles were cut. The DNA was
that requires specific equipment and considerable then applied. The DNA reaches the ovule by flowing
training. This technique was used to study the cellular down the pollen-tube. This procedure, the so-called
functions of plant cells and plastid physiology, e.g. in pollen-tube pathway (PTP), was applied first time for the
tobacco and Vicia faba [20]. transformation of rice [29]. Here the transgenic plants
were obtained at remarkably high frequency. Afterward
2.6. Silicon carbide mediated transformation PTP was used for other species e.g. wheat [30], soybean
Silicon carbide mediated method is also one of the [31], Petunia hybrida [32] and watermelon [33].
transformation method used to transform plants. This
method is least complicated. In this technique fibres are 2.9 Sonication assisted Agrobacterium mediated
used which are single crystals of silica organic minerals transformation
like silicon carbide which possess an elongated shape, Sonication-assisted Agrobacterium-mediated transformation
having a diameter of 0.6 mm and a length of 10–80 mm. (SAAT) is an efficient transformation technology, reported
Moreover it also exhibits a high resistance to by Trick and Finer [34]. It is Agrobacterium based

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technology. This method consists of subjecting the target cloned by Song et al. [51]. Transgenic rice plants
plant tissue to brief periods of ultrasound while immersed harbouring the cloned gene displayed high levels of
in an Agrobacterium suspension. SAAT overcomes resistance. The gene has been found to be effective
certain barriers such as the host specificity and the against several isolates [52].
inability of Agrobacterium to reach proper cells in the Shimada et al. [53] produced transgenic rice plants
target tissues. This method also enhances DNA with antisense construct of rice waxy gene coding for
integration in many plant groups including dicots, granule-bound starch synthase under the control of 35S
monocots, and gymnosperms. It is likely that the promoter. A significant reduction in amylose content of
enhanced transformation rates using SAAT result from grain starch was observed in the seeds of these plants.
micro-wounding both on the surface and deep within the Most interestingly, to confer the capability of producing
target tissue. Therefore, unlike other transformation precursor (β-carotene) of vitamin A in rice endosperm,
methods, this system also has the potential to transform Burkhardt et al. [40] engineered rice with the cDNA
meristematic tissue buried under several cell layers [30]. coding for phytoene synthase from daffodil, the first of the
Cotton transformation based on cavitations caused by four specific enzymes involved in β-carotene (provitamin
sonication which results in thousands of micro wounds on A) biosynthesis in plants. In the endosperm of these
and below the surface of plant tissue and allows transgenic plants, phytoene synthase accumulation was
Agrobacterium to travel deeper and completely observed indicating that engineering of provitamin A
throughout the tissue. This wounding fashion biosynthesis pathway is possible in non-photosynthetic,
increases the probability of infecting plant cells lying carotenoid lacking tissue. Recently, the same group
deeper in tissue. reported Agrobacterium-mediated transformation of rice
with all the genes necessary for the accumulation of
provitamin A in transgenic rice seeds [54]. Hayakawa et
3. Roles in improving crops al. [55] engineered the coat protein (Cp) gene of rice
stripe virus into two japonica rice varieties by
Biotechnological strategies for crop improvement electroporation of protoplasts resulting in significant
demand efficient procedures for routine introduction of levels of resistance against the virus in the transgenic
defined foreign genes into plant genome. Successful plants.
genetic manipulation requires the ability to deliver
biologically active and functional DNA into plant cells 3.2. Genetically engineered maize
followed by recovery of transgenic plants expressing a A coat protein-mediated resistance to viruses,
foreign gene. Gene transfer technology is playing introduced in rice via protoplast transformation [55], was
significant role in improving plants and their yields. transferred to maize and barley via particle gun
bombardment [56]-[57]. The resistance to sulfonylurea
3.1. Transformation of rice (herbicide) conferred by the als gene of Arabidopsis
Rice is the staple food for more than one third of thaliana was also transferred to maize [58] by particle
world’s population. To feed the growing world population gun technology. Maize has been reported to get
it is the requirement to increase the total food transformed by Silicon carbide fiber-mediated DNA
production. Although the world food supply has more delivery system [59]-[62]. Whiskers-mediated maize
than doubled since the onset of the green revolution but transformation has also been reported [63].
still there is a need to improve the quantity as well as
quality. Biolistic was successfully used for transformation 3.3. Genetically engineered wheat
of immature embryos of rice [35]. Reports were also Wheat is a member of the Triticeae group of cereals.
maderegarding the transformation of indica and javanica It is indisputably one of the major food crops of the world
rice in addition to other japonica rice [36]-[48]. and a foundation of human nutrition. Genetic
Fujimoto et al.[49] were the first to engineer japonica improvement of wheat has received considerable
rice through electroporation with modified d-endotoxin attention worldwide over the years with the purpose of
gene (cry) from Bacillus thuringiensis. It was found that increasing the grain yield to minimize crop loss due to
the R2 generation of transgenic rice was more resistant unfavorable environmental conditions and development
to insects than wild type plants. Later, Wu¨nn et al. [50] of resistance against various pests and pathogens.
obtained transgenic indica rice cultivar IR58 expressing a The first transgenic wheat plants were produced by
synthetic cryIA(b) gene driven by 35S promoter through Vasil et al. [64], followed by Vasil et al. [65], Weeks et al.
particle bombardment. The rice Xa21 gene which confers [66], Nehra et al. [67], and Altpeter et al. [68] employing
resistance to blight pathogen, Xanthomonas oryzae was microprojectile bombardment as a method of DNA

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delivery. Subsequently, the development of methodology still there with regards to the transformation technology it
for the delivery of genes into intact plant tissues by self. In future, a combination of improved transgene
bombardment of DNA-coated gold or tungsten particles engineering, a reliable genetic recombination systems
has revolutionized the field of wheat transformation. In and efficient DNA delivery procedures should give rise to
recent years, sincere efforts are being made to transform a new generation of transformation technologies.
wheat genetically with different alien genes of
agronomically importance [69]-[75]. However, in majority Acknowledgements
of reports, genetic transformation with a single target The first and corresponding author Dr. Kishwar
gene has been used for the production of transgenic Hayat Khan working as an Assistant Professor at Medical
wheat expressing tolerance to herbicide, resistance to Biotechnology Division, School of Biosciences and
fungal and viral diseases [76]. Technology, VIT University, Vellore, Tamil Nadu, India
wishes to thank this University for providing facilities and
3.4. Tobacco plant support. The name of S.K. Jain is highly acknowledged.
Tobacco has been found to be the key plant model
for the development of transformation technology. This
probably reflects the fact that tobacco was the first plant
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