ASSIGNMENT

ON

Direct gene transfer methods in plants

Submitted to: Dr. Veena Jain Submitted By: Punesh

2009BS36D

Department of BiochemistryCCS 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.

References

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