ASSIGNMENT

ON

Direct gene transfer

methods in plants

Submitted to: Dr. Veena Jain

Submitted By: Punesh
2009BS36D

Department of Biochemistry
CCS Haryana Agriculture University, Hisar
Haryana (125001)
Introduction

Plant transformation, as we think of it today, started in the early 1980s with the first
conclusive demonstration that the causative agent of crown gall disease, Agrobacterium
tumefaciens, could be harnessed by researchers to introduce defined fragments of DNA into
plant cells. The last two decades have subsequently seen a burgeoning of publications on all
aspects of plant transformation so that an enormous body of literature has now accumulated.
Areas of this vast subject have been reviewed many times over the years and from diverse
viewpoints and at least two comprehensive books on the subject have recently been
published.
Plant transformation refers to the introduction and integration of “foreign” DNA in plant cells
and the consequent regeneration of transgenic plants. Transfer of DNA into plant cells can
lead to transient or stable expression of the introduced DNA. Transient expression, as its
name suggests, usually lasts for a few days only, but occupies a useful niche in such areas as
development of transformation methodology or metabolic studies, since it allows the effects
of experimental manipulations to be seen in a short time. Stable transformation, on the other
hand, is often a time-consuming process involving tissue culture techniques that facilitate the
growth of whole plants from treated cells or tissue explants. As a result of stable
transformation the introduced DNA is integrated into the host cell DNA and is thereby
eligible to be passed on to succeeding generations.

Transformation advancement

Currently, numerous transformation methods are available. They can be divided into two
main groups: indirect and direct ones. The indirect methods of plant transformation are based
on the introduction of a plasmid-carrying gene construct into the target cell by means of
bacteria Agrobacterium tumefaciens or Agrobacterium rhizogenes.
Direct methods do not use bacteria cells as mediators. Agrobacterium mediated
transformation is the main method used in the field of biotechnology, where the most often
applied direct methods are protoplast transformation or microprojectile bombardment. In the
case of Agrobacterium-mediated transformation, the efficiency for monocots is still
unsatisfactory. However, in recent years, it has become the method of choice for this group of
plants. The general disadvantages of direct protoplast transformation are problems with plant
regeneration (especially in monocotyledonous plants), and a low transient expression of
transgenes as compared with organized tissues. The electrical field and chemical substances
applied to disorganize cell walls strongly reduced the viability of protoplasts and their
capability of division. The most distinctive factor limiting the use of the gene gun is the
presence of multiple copies of introduced genes, which can lead to various unprofitable
effects like their suppressed or changed expression. The high expenses of gene gun
accessories should also be taken into consideration. Many methods of plant transformation
require the employment of in vitro culture, at least during some procedural steps.
During in vitro regeneration, some somaclonal changes may arise. This fact can make an
analysis of transformants difficult, and limits use in further study as well. All the above-
mentioned limitations inspired investigators to search for new alternative transformation
procedures. Up till now, several such methods have been developed. Among them, the most
often listed ones are 1) infiltration, 2) silicon carbide fibre mediated transformation, 3)
electroporation of cells and tissues, 4) electrophoresis of embryos, 5) microinjection, 6)
transformation via the pollen-tube pathway and 7) liposome-mediated transformation. The
majority of these were thought to be solutions for the effective transformation of recalcitrant
species, such as monocots or some legumes, as Agrobacterium-mediated transformation was
at that time not available for this group of plants.

Direct gene transfer methods

It has been sub divided into three categories:

1. Physical gene transfer method

2. Chemical gene transfer method

3. DNA imbibitions by cell, tissue and organs

1. Physical gene transfer methods

The species and genotype independent transformation methods wherein no natural vector is
involved but which are based on direct delivery of naked DNA to the plant cells have been
guided under this category. This is also referred to as DNA mediated gene transfer.

A). Electroporation:-

Electroporation is a technique that uses an electrical pulse to render cell walls or protoplast
membranes permeable, so that DNA can be taken up into the cells. A high-voltage electrical
pulse of short duration causes the formation of temporary pores, which allow cells to take up
plasmid DNA; this may lead to stable or transient DNA expression. The method was
originally applied to protoplasts, but has been found applicable to cells and even tissues.
Transfer of DNA by electroporation into tissues as opposed to protoplasts circumvents the
need for a protoplast culture system and may simplify the recovery of regenerated plants.
This method has been used successfully with immature zygotic embryos and embryogenic
callus to produce transgenic maize and with intact nodal meristems to produce transgenic
legumes.

It uses relatively high field strength (1-1.5 kV) with a low capacitance and therefore a short
decay time. In this procedure, a protoplast is pulsed with high/low voltage pulses in a
chamber of an electropoator. The chamber is cylindrical in form with a distance of 1cm
between parallel electrodes. The pulse is applied by discharge of capacitor across the cell. It
has been reported that using linear DNA rather than circular DNA, a field strength of
1.25kV/cm and employing polyethylene glycol (PEG) can increase protoplast transformation
efficiency. PEG is believed to assist the association of DNA with the membrane.

The range of tissues that can be transformed by electroporation seems to be narrower. For
tissues that are susceptible to DNA uptake by electroporation, this method is convenient,
simple, fast, low cell toxicity, and inexpensive to obtain transient and stable transformation in
different tissues. The disadvantage of the technique is the difficulty in regenerating plants
from protoplasts.

B). Particle Bombardment/microprojectile/biolistics:-

This technique has been shown to be most versatile and effective way for the creation of
many transgenic organisms, including microorganisms, mammalian cells and plant species.
As plant transformation gathered momentum, it became clear that an alternative method,
which avoided the host-range restrictions of Agrobacterium and the regeneration problems
encountered by protoplast systems, would be highly advantageous. To this end, a system
using high velocity microprojectiles for delivering nucleic acids into plant cells was
successfully demonstrated by Klein et al.(1987).

The basic system that has received attention has employs PDS 1000(gun powder driven
device) or PDS-1000/He (Helium driven particle gun). The DNA bearing tungsten or gold
particles (1-3µm) referred to as microprojectiles, is carried by a macroprojectile or
macrocarrier and is accelerated into living plant cells. The DNA bearing particles are placd
on the leading surface of the macrocarrier and release from the macrocarrier upon impact
with a stopping plate or screen. The stopping plate is designed to halt the motion of the
moving macroprojectile while permitting the passage of the microprojectiles. In this
procedure when Helium gas is released from the tank, a disc known as rupture disc blocks its
entry to the chamber. These discs are available with various strengths to resist the pressure of
the gas, which varies from 5000 to 700 psi when the disc rupture, compressed Helium gas is
suddenly released, which accelerates a thin plastic sheet carrying microprojectiles into a
metal screen, the macroprojectile movement is stopped, but this permits the passage of
microprojectiles through the mess screen. The microprojectile then travel through a partial
vacuum until they reach the target tissue. The partial vacuum is used to reduce the
aerodynamic drag upon the microprojectile and decrease the force of shock wave created
when the macrocarrier impact the stopping plate.

The use of particle bombardment requires a careful consideration of a number of parameters.

These can be classified under three categories:

Physical parameters: - Nature, chemical and physical properties of the metal particles
utilized to carry the foreign DNA: particles should be of high mass in order to possess
adequate momentum to penetrate into the appropriate tissue. Eg. Tungsten, gold, platinum,
palladium, rhodium, iridium, and possibly other 2nd and 3rd row transition metals can be used.
Size range is ca. 1µm.

Nature, preparation and binding of DNA on the particles: - the nature of DNA, i.e. as single
or double stranded, may be important under some conditions but this may not be a significant
variable in specific cases. In the process of coating metal particles with DNA, certain
additives such as spermidine or calcium chloride appear to be useful.

Target tissues: - it is apparent that different tissues have different requirements, thus
extensive study needs to be performed in order to ascertain the origin of regenerating tissue in
a particular transformation study.

Environmental parameters: - these include variables such as temperature, photoperiod and

humidity of donor plants, explants, and bombarded tissues. These parameters effect
physiology of tissues, influence receptiveness of the target tissue to foreign DNA delivery
and also affect its susceptibility for damage and injury that may adversely affect the outcome
the transformation process.

Biological parameters: - Choice and nature of explants, and pre- and post-bombardment
culture conditions determine whether experiments utilizing particle bombardments are
successful. The explants derived from a plant that are under stress or infected by bacteria or
fungi, over or under-watered will be inferior for bombardment. Osmotic pre- and post-
treatment of explants with mannitol has been shown to be important in transformation.
Experiments performed with synchronized culture cells indicate that the transformation
frequencies may be also influenced by cell cycle stage.

The DNA of choice is precipitated onto microscopic particles, which are then accelerated into
plant cells: as suspension cultures, tissues in culture, whole plant parts, etc. The main
advantage of this system is that it is species independent and avoids the complex interaction
between bacterium and plant tissue, with the result that the DNA to be introduced does not
need to contain the sequences necessary for TDNA replication and transfer. Particle
bombardment is thus a simple physical process compared with Agrobacterium-mediated
DNA transfer, where transformation occurs by way of a complex process that is still not
thoroughly understood. Refinements in particle bombardment technology have involved a
variety of alterations in the apparatus used to bombard selected tissue.

Transfer of DNA into plants by particle bombardment has become a major method of choice
alongside A. tumefaciens-mediated DNA delivery for the production of transgenic plants. It
has been used successfully in a wide range of species and has been instrumental in
transforming species that are not readily amenable to transformation with Agrobacterium or
that are recalcitrant to other direct transfer methods; it has also been instrumental in the
transformation of mitochondria and chloroplasts. The use of the particle gun is only limited
by the regeneration capacity of the tissue being bombarded and the efficiency of stable
integration of DNA. Plant apical meristems have been investigated as targets for particle
bombardment in a number of species, as they could potentially avoid the need for extensive
tissue culture manipulation. The use of meristems, however, generally leads to chimeric
plants with sectors of transgenic tissue that are not necessarily manifested in the germline,
and further tissue culture manipulations are indeed necessary to increase the chances of the
introduced transgene being inherited. Particle gun delivery of DNA has been shown to result
in integration of rearranged and/or truncated DNA sequences, as well as multiple copies or
concatamers (head-to-head or head-to-tail ligation of several transgenes) at sites of
integration. Depending upon the desired outcome, this may be a limitation, but, on the other
hand, the propensity for particle bombardment to integrate multiple copies may lend itself to
exploitation; in rice, for example, multiple genes have recently been co-bombarded and
integrated into one or a few loci, resulting in stable and predictable inheritance patterns.
Traditionally, DNA has been coated onto gold or tungsten particles for delivery, but
biological projectiles such as bacteria (Escherichia coli), yeast, and phage have been
complexed with tungsten and used as particles with some success as well as Agrobacterium.
Novel transformation technologies are being developed that combine elements of
Agrobacterium-mediated transformation with species independent direct gene transfer, by
using virD genes from Agrobacterium in concert with particle bombardment.

C). Microinjection:-

Transformation via microinjection is based on introducing DNA into the nucleus or

cytoplasm by means of a glass micro capillary-injection pipette. This operation requires a
micromanipulator. During the introduction of DNA into the nucleus, cells are immobilized
with a holding pipette and gentle suction. Both pipettes contain mineral oil, which works as a
cylinder. Microinjection is mainly used for the transformation of large animal cells. Its
importance for plant transformation is rather limited due to the characteristics of plant cell
walls, which contain a thick layer of lignins and cellulose. The plant cell wall is a barrier for
glass micro tools. The microinjection of protoplast could theoretically resolve this limitation,
but it carries with it the danger of releasing of hydrolases and other toxic compounds from the
vacuole to the cytoplasm, which can cause rapid death of the protoplast. Although it is
possible to remove vacuoles before microinjection without any consequences for protoplast
viability, their loss significantly decreases the capability for division and plant regeneration.
The microinjection of protoplasts requires different methods of immobilization—instead of
using a sucking capillary; protoplasts are attached to glass by coating them by poly-L-lisine
or agarose. None of these solutions has proved useful, as poly-L-lisine can be toxic for some
species and agarose (even a very thin layer) reduces visibility in the area of manipulation.
Currently, microinjection is widely used for the transformation of large animal cells e.g. frog
egg cells or the cells of mammalian embryos, whereas it has not been developed into a
routine transformation method for plants. The procedure is very slow and requires an
expensive micromanipulator. However, one of the unquestionable improvements of
microinjection was that it allowed the introduction not only of DNA plasmids but also of
whole chromosomes into plant cells.
Microinjection technique was used to study the cellular functions of plant cells and plastid
physiology, e.g. in tobacco and Vicia faba. Transgenic plants, however, were only recovered
in several studies involving such species as tobacco, petunia, rape, and barley, and usually at
very low frequency.

D). Liposome-mediated transformation:-

The idea of a method of direct plant transformation elaborated in the middle eighties was to
introduce DNA into the cell by means of liposomes. Liposomes are microscopic spherical
vesicles that form when phospholipids are hydrated. Liposomes are circular lipid molecules
with an aqueous interior that can carry nucleic acids. Liposomes encapsulate the DNA
fragments and then adhere to the cell membranes and fuse with them to transfer DNA
fragments. Thus, the DNA enters the cell and then to the nucleus. Lipofection is a very
efficient technique used to transfer genes in bacterial, animal and plant cells. They can be
loaded with a great variety of molecules, including DNA. In the case of protoplasts, the
transfection (lipofection) occurs through the membrane fusion and endocytosis. When pollen
grains are transformed, liposomes are delivered inside through pores. The efficiency of
bioactive-beads-mediated plant transformation was improved using DNA-lipofection
complex as the entrapped genetic material instead of naked DNA used in the conventional
method. Liposome-mediated transformation is far from routine, in spite of the low expense
and equipment requirement. A probable reason is its laboriousness and low efficiency. Only
several reports on the integration of genes introduced by means of liposomes followed by
transgenic plant regeneration for tobacco and wheat have been published thus far.

E). Silicon carbide fiber mediated transformation:-

SCMT is one of the least complicated methods of plant transformation. Silicon carbide fibers
are simply added to a suspension containing plant tissue (cell clusters, immature embryos,
callus) and plasmid DNA, and then mixed in a vortex, or in other laboratory apparatus such
as commercial shakers, blenders etc. DNA-coated fibers penetrate the cell wall in the
presence of small holes created in collisions between the plant cells and fibers. The most
often used fibers in this procedure are single crystals of silica organic minerals like silicon
carbide, which have an elongated shape, a length of 10–80 mm, and a diameter of 0.6 mm,
and which show a high resistance to expandability. Fiber size, the parameters of vortexing,
the shape of the vessels used, the plant material and the characteristics of the plant cells,
especially the thickness of the cell wall are the factors depending on the efficiency of SCMT.
The main advantages of this easy and quick procedure are the low expenses and usefulness
for various plant materials. The main disadvantages of this method is low transformation
efficiency, damage to cells negatively influencing their further regeneration capability, and
the necessity of obeying extraordinarily rigorous precaution protocols during lab work, as
breathing the fibers in, especially asbestos ones, can lead to serious sicknesses. There are
several known examples of deriving transgenic forms—cell colonies or plants—in maize,
rice, Wheat, tobacco, Lolium multiflorum, Lolium perenne, Festuca arundinacea, and
Agrostis stolonifera by SCMT. Kaeppler et al. (1992). transformed a cell suspension of the
Black Mexican Sweet (BMS) variety of maize with the plasmid-carrying genes bar and uidA.
The authors obtained approximately 3.4% transgenic cell lines expressing both transgenes
from a 300 ml of packed cell volume, which means that the integration of transgenes
occurred in one per one million cells. The efficiency was significantly lower than that
described earlier by the same team or other authors (among others Klein and co-workers
when micro bombardment was applied. One of the reasons for such a low efficiency could be
the notable reduction of cell viability, up to 29%, caused by damages during vortexing with
silicon carbide fibers. Frame et al. (1994) obtained first fertile transgenics for maize in 1994.
Three hundred and eleven transgenic plants were derived from 22 independent transgenic cell
lines, and eight of those turned out to be stable transformants. However, the efficiency was
significantly lower (5–10 fold) than that obtained earlier by gene gun-mediated
transformation in the same lab. A similar observation the efficiency being much lower in
comparison with micro bombardment was reported by Petolino et al. (2000). The authors also
considered the serious damage to transformed tissue by silicon carbide fibers to be the main
reason for the unsatisfactory results. In a study a silicon carbide whisker-mediated gene
transfer system with recovery of fertile and stable transformants in cotton (Gossypium
hirsutum L.) cv. Coker-312 resulted 94% transformation efficiency. The above-mentioned
reports clearly indicate a low efficiency of silicon carbide fiber-mediated transformation as
the main limitation for its practice. However, SCMT is an easy, fast and inexpensive
procedure. Therefore, it could be an attractive alternative method of plant transformation in
particular situations, e.g. when a gene gun is not available and Agrobacterium-mediated
transformation is difficult or not possible (as in the case of numerous monocots). Moreover,
the SCMT system of using commercial paint shakers, which has been reported for maize,
seemed to be very good for commercial large-scale transformation.

Silicon carbide whiskers have been used to introduce DNA into plant cells. Advantages of the
SCF-mediated method over other procedures includes the ability to transform walled cells
thus avoiding protoplast isolation, relative ease of the procedure and very low equipment
costs. This method is limited, however, to fine suspension cultures of cells that can be readily
penetrated by the whiskers, and thus has the associated problems of regeneration into whole
plants. Another disadvantage is that silicon fibers have similar properties to asbestos fibers
and care must be taken when working with them. While not widely used, the technique is a
useful alternative to particle bombardment if the latter is not appropriate for any reason.
Silicon carbide has some carcinogenic properties.

F).The pollen-tube pathway method:-

The transformation method via pollen-tube pathway has great function in agriculture
molecular breeding. Foreign DNA can be applied to cut styles shortly after pollination. The
DNA reaches the ovule by flowing down the pollen-tube. This procedure, the so-called
pollen-tube pathway (PTP), was applied first time for the transformation of rice. The authors
obtained transgenic plants at remarkably high frequency. Afterward PTP was used for other
species e.g. wheat, soybean, Petunia hybrida and watermelon. A bacterial inoculum or
plasmid DNA can also be injected into inflorescence with pollen mother cells in the pre-
meiotic stage without removing the stigma. In that case, it is expected that foreign DNA will
be integrated with the gamete genome. Such an approach has been employed for rye. Pollen
collected from inflorescences injected with a suspension of genetically engineered A.
tumefaciens strain was predestined for the pollination of the emasculated spikes of the
maternal plant. But the transformation efficiency was about 10-fold lower than that
approximately reached for this species via microprojectile bombardment. Shou et al. (2002)
also reported they were unable to reproduce the pollen-tube pathway transformation for
delivering plasmid DNA into soybean. They concluded that the pollen-tube pathway
transformation in cotton and soybean was not reproducible. This might have been because of
the manipulation of transformation, the growth stage of plants, the effects of environment and
weather.
G). Electroporation of intact plant cells and tissues:-

The electroporation of plant cells and tissues is very similar in its principles to the
electroporation of protoplasts. The main difference lies in the use of other plant material, such
as pollen, microspores, leaf fragments, embryos, callus, seeds or buds. For transformation,
both plasmid DNA and Agrobacterium inoculum can be applied. The first attempts to adopt
methods employed in protoplasts for organized plant tissues were reported in the early
nineties, and the main idea was to check the transient expression of transgenes under different
organo- or tissue-specific promoters. Efficient protocols for the electroporation of cell
suspensions have been worked out for many species, e.g. tobacco, rice, and wheat.
Experiments on obtaining transgenic plants also started in the early nineties. So far, the best
results have been obtained for maize. Deshayes et al. (1985) transformed immature embryos
and embryogenic callus type I, which were briefly digested in a solution of pectolytic
enzymes, followed by transfer into electroporating cuvettes. The electroporation efficiency
was relatively high: 90 transgenic plants were regenerated from 1440 embryos (6.25%) and
31 plants from 55 callus clusters (54.6%), which is fully comparable with the best results
obtained for this species after micro bombardment. Similar results for this species were
obtained by Laursen et al. (1994). Authors calculated that the integration of transgenes took
place approximately in one per 10,000 cells. A much lower efficiency ~3 transgenic plants
from 1080 immature embryos (0.28%), was stated in the case of wheat electroporation. The
post pulse addition of ascorbic acid or another ascorbate could significantly increase the
transformation efficiency without any negative influence on cell viability, as shown for a
maize BMS cell suspension. In a study the culture of electroporated tissues in liquid media
with 8mg/l benzyl adenine conducted to maximal regeneration through secondary somatic
embryogenesis. The secondary somatic embryos regenerated from electroporated torpedo
shape somatic embryos were positive for gus expression, and also in the PCR analysis for the
genes gus and bar. Although electroporation seems to be an extremely simple and effective
method, for at least some species, it has not yet been widely used for plant transformation.

H). Electrophoresis:-

At the end of the eighties, a method employing electrophoresis was developed for the
transformation of immature embryos, especially for the embryos of monocotyledonous
plants. It was proposed as an alternative method of transformation too expensive and not
always efficient microprojectile bombardment. Transfected embryos placed between the tips
of two pipettes connected to electrodes. The pipette connected to the anode is filled in its
narrow part with agar (or agarose) followed by an electrophoresis buffer containing EDTA.
The pipette connected to the negative electrode contains agar mixed with DNA and an
electrophoresis buffer. This pipette is in contact with the apical meristem of the embryo,
whereas the second one is located near its basal apical part. Flowing of DNA from cathode to
the anode through the embryo slows after switching on current (from the apical meristem to
its base part). The efficiency of electrophoresis-mediated transformation depends on
numerous factors, mainly on the parameters of the electrical field, the duration of
electrophoresis, the contents of the electrophoresis buffer, and the physicochemical properties
of the embryo tissue. A voltage of 25 mV and an amperage of 0.5 mA for 15 min are the most
often used parameters for electrophoresis. Electrophoresis has a rather inconsiderable
importance in plant transformation in spite of its simplicity and relatively low cost. The main
reason is the poor viability of the treated embryos. Although the first attempts of Ahokas
(1989) resulted in the derivation of plants from embryos of barley, none of them expressed
the uidA gene carried by the plasmid taken for transformation. Up till now, the only
successful transgenic plants were obtained for Calnthe orchid L.

2. Chemical gene transfer method

This involves plasma membrane destabilizing nad/or precipitating agents. Protoplasts are
mainly used which are incubated with DNA in buffers containing PEG, poly L-ornithine,
polyvinyl alcohol or divalent ions. The chemical transformation techniques work for a broad
spectrum of plants.

A). Polybrene–Spermidine Treatment:-

The combination polybrene–spermidine treatment greatly enhanced the uptake and

expression of DNA and hence the recovery of nonchimeric germline transgenic cotton plants.
The major advantages of using the polybrene–spermidine treatment for plant genetic
transformation are that polybrene is less toxic than the other polycations; spermidine protects
DNA from shearing because of its condensation effect; and because no carrier DNA is used,
and the integration of plasmid DNA into the host genome should enable direct analysis of the
sequences surrounding the site of integration. To deliver plasmid DNA into cotton suspension
culture obtained from cotyledon-induced callus, polybrene and/or spermidine treatments were
used. The transforming plasmid (pBI221.23) contained the selectable hpt gene for
hygromycin resistance and the screenable gus gene. Primary transformant cotton plants were
regenerated and analyzed by DNA hybridization and b-glucuronidase assay.

B). PEG mediated gene transfer:-

In this method protoplasts are isolated and a particular concentration of protoplast suspension
is taken in a tube followed by addition of plasmid DNA (donor or carrier). To this 40% PEG
4000(w/v) dissolved in mannitol and calcium nitrate solution is slowly added because of high
viscosity, and this mixture is incubated for few minutes (ca.5 min.). As per the requirements
of the experiments, transient or stable transformation studies are conducted.

Among the most important parameters that affect the efficiency of PEG-mediated gene
transfer are the concentration of calcium and magnesium ions in the incubation mixture, and
the presence of carrier DNA. The linearized dsDNA are more efficiently expressed and
integrated in the genome than the supercoiled forms.

The advantage of the method is that the form of DNA applied to the protoplast is controlled
entirely by the experimenter and not by intermediate biological vector. Main disadvantage is
that the system requires a protoplast.

C). Calcium-Phosphate co-precipitation:-

DNA when mixed with calcium chloride solution isotomic phosphate buffer DNA-CaPO4
precipitate. The precipitate is allowed to react with actively dividing cells for several hours,
washed and then incubated in the fresh medium. Giving them a physiological shock with
DMSO can increase the efficiency of transformation to a certain extent. Relative success
depends on high DNA concentration and its apparent protection in the precipitate.

D).DEAE dextran procedure:-

Transformation of cells with DNA complexed to the high molecular weight diethyl amino
ethyl (DEAE) dextran is used to obtain efficient transient expression. The efficiency increase
when 80% DMSO shock is given. But this technique does not produce stable transformants.

E).The polycation DMSO technique:-

It involves use of a polycation, polybrene, to increase the absorption the absorption of DNA
to the surface followed by a brief treatment by 25-30% DMSO to increase the membrane
permeability and enhance the uptake. The major advantage of polybrene is that it is less toxic
than other polycations and a high transformation efficiency requires very small quantities of
plasmid DNA to be used.

3. Direct gene transformation through imbibition

During imbibition the uptake of exogenous DNA of dehydrated plant tissues is a direct gene
transfer method which has been studied since the 1960s and for which the literature contains
a number of both claims and refutations. The physical and biochemical changes which are
already known occur in plant tissues during dehydration (e.g. a large water potential between
the dry tissue and external solution, rapid cell expansion, cell wall rupture, cell membrane
structural changes and leakiness; suggest that under these conditions DNA uptake might be
possible. DNA uptake and expression was observed under simple dehydration conditions, but
was stimulated by the presence of 20% DMSO, suggesting that membrane permeabity was an
important factor in the process. A number of lines of evidence supported the conclusion that
reporter gene expression was the result DNA uptake into cells and plants were recovered
from treated embryos, but no evidence of stable transformation was presented. Subsequent
research on the imbibition transformation has extended its application to dessicated somatic
embryos of alfalfa, which showed transient GUS expression at frequencies upto 70%. The
stable transformation of rice by embryo imbibition was also reported. The frequency of
transient expression of gusA and hpt genes using the CaMV35S promoter was about 30 to
50%. The main sites of gusA gene expression were meristems of roots and vascular bundles
of leaves. Also, DNA uptake, integration and expression of the hpt gene in selected rice were
investigated by various PCR methods and Southern blot analysis of genomic DNA. It was
shown that the hygromycin phosphotransferase (HPT) DNA was present in the rice genome
in an integrated form and not as a plasmid form. These methods are technically the most
simple of DGT methods, as they require no specialist equipment and the preparation of target
plant tissues generally simple. This simplicity constitutes the advantage of these techniques,
while their limitations are i) they cannot be applied only to very specific organs or tissues (i.e.
newly pollinated flowers or hydrating embryos) and ii) it is still not clear that they lead to
stable, and heritable transformation. While they add support to the observation that many
different plant cells may be amenable to DNA uptake and expression, at present these
techniques are subjects for further analysis and development rather than usable gene transfer
methods.

Conclusion

As mentioned above, none of the alternative transformation methods found a wide

employment in the laboratory. A low efficiency of transformation in almost all of these
procedures, a consequence of the decreased viability of cells, is one the most often listed
limitations of their application. However, it seems that at least some of the described
methods, SAAT, Shoot apex, Pollen-tube pathway, infiltration and silicon carbide fiber-
mediated transformation, can gain greater interest. All of them are characterized by
simplicity, reduced costs and a low equipment requirement. As concerned with application,
Agrobacterium-mediated transformation and biolistic method are most widely used methods.
The infiltration is observed to become the main transformation method for Arabidopsis,
whereas SCMT is for maize. With optimizing studies these experimental procedures might
soon be available for a broad spectrum of plant.

Future Direction

The technology for gene transfer has developed to the stage whereby almost any plant of
choice can be transformed, although there are still practical limitations in many species
because of low transformation efficiency and less than optimal transgene expression. While
there is still scope for improving gene transfer methodology in those species that remain
recalcitrant to some degree, the main focus of research in plant transformation is now on
issues concerned more with expression and stability of the transgenes once they are in the
host plant, than on the methodology of actually introducing foreign DNA. Removal of
extraneous DNA that is not necessary in the final product, such as the selectable marker
genes, understanding why and how transgenes are silenced through sense- and co-suppression
mechanisms and eliminating the position effects thought to be responsible for variable
expression levels of transgenes in different transgenic plants are all key areas for future
development. The potential advantages of plastid transformation, as a means of
circumventing some of the difficulties encountered with nuclear gene expression, have been
touched upon already and will no doubt continue to be examined as plastid transformation
becomes routine for species other than model systems. The first transgenic plants released for
commercial production expressed one or two genes of interest conferring a dominant
phenotype alongside the selectable marker gene. Many traits of commercial significance,
however, require expression of several transgenes, a situation that current transformation
technology cannot adequately address. The first step toward routine multigene transformation
has been taken recently with the successful demonstration that up to 13 different transgenes
could be introduced via particle bombardment into rice. Fertile plants containing up to 12
transgenes were recovered and protein expression was shown for the four selectable and
scorable marker genes that were tested. It was estimated that segments of DNA up to 300 kb
in length had been integrated, double the previous estimate of 150 kb introduced into plant
genomic DNA by BAC vectors. Chloroplast transformation has also been proposed as a
possible mechanism for obtaining polycistronic expression and warrants further investigation.
The ability to integrate many genes and ensure their stable inheritance and expression is
essential for the successful manipulation of complex biosynthetic pathways and polygenic
agronomic traits in the future.

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