Gene delivery to a potential transgenic plant
Various methods of gene delivery to a potential transgenic plant.
The
uptake of foreign DNA or transgenes by plant cell is called transformation. A
variety of techniques have been used to introduce transgene into plant cell.
This can be grouped into following two major categories:-
a) Agrobacterium mediated gene
transfer.
b) Direct gene transfer.
a)Agrobacterium mediated gene
transfer:- Agrobacterium tumefaciens is a remarkable species of soil-dwelling bacteria that has the ability
to infect plant cells with a piece of its DNA. When the bacterial DNA is
integrated into a plant chromosome, it effectively hijacks the plant’s cellular
machinery and uses it to ensure the proliferation of the bacterial population.The
gene transfer through Agrobacterium is achieved in the two
ways –
(i) Co culture with tissue explants
(ii) In plant a transformation
In the 1st method Agrobacterium
is co cellular with tissue explants (generally leaf discs).The transgene is
delivered by this bacterium into the explants cell with the help of
acetosyringone. The transformed cells are screened by Kanamycin and the
Agrobacterium cells are killed by carbenicillim. The transformed cells are
transferred to the regeneration media where it produces whole plants.
In the 2nd
method the seed or flower of plants are imbibed into Agrobacterium culture. The
bacterium then infects the zygote or pollens which in appropriate media produce
the plant.
b)
Direct gene transfer:-Induction of DNA into plant cell
without the involvement of biological agent and leading to stable
transformation is known as direct gene transfer. The spontaneous uptake of DNA
by pant cell is quite low, therefore different chemical and physical treatments
are employed to facilitate the entry of DNA into plant cell. The gene
constructs to be delivered into plant cell either present on vector or uncloned
condition. Various method of direct gene transfer are as followes:-
(1) Chemical
method:Certain
chemicals like polyethylene glycol (PEG), polyvinyl alcohol and calcium
phosphate enhance the uptake of DNA by plant protoplast. In PEG mediated DNA
delivery plant protoplasts are suspended in a transformation medium rich in Mg++
ion. Linearised plasmid DNA containing the gene construct is added, to the
protoplast suspention following which 20% PEG is added and pH adjusted to about
8. The protoplasts may be given a 5min heat shock at 45°c following the
transfer to ice just before the additional DNA, since it increases the
frequency of transformation by several orders of magnitude. Use of carrier DNA
thus promotes transformation but it is neither desirable nor necessary. After a
period of incubation the PEG concentration is reduced while the Ca++
ion is enhanced. This enhances the transformation frequency.
(2) lectroporation:Induction
of DNA into cells by exposing them for very brief period of high voltage
electrical pulses which is thought to induce transient pores in the
plasmalemma, is called electroporation. There are basically two system of
electroporation – (a) low voltage long pulse method, (b) high voltage short
pulse approach. Plant protoplasts are suspended in a suitable ionic solution
containing linearised recombinant plasmid DNA. The electroporation mixture is
then exposed to the choosen voltage pulse combination for the desired number of
cycles. Protoplasts are then cultured to obtain cell colonies plants. They are
then cultured to obtain cell colonies and plants. The optimal voltage and time
will depend on the plant sp., the source of protoplast and the resistance of
medium. Generally low voltage, long pulse produce high rates of transient
transformation while high voltage short pulse gives high rates of stable
transformation.
(3) Particle-gun
method:In
this method one of two mm tungsten or gold particles, coated with DNA to be
used for transformation, are accelerated to velocities which enable their
entry, into plant cells or nuclei. Particle acceleration is achieved by using a
device called particle gun or gene gun. This method was 1st used by
Klein et al (1988). It can be used to transform shoot apical meristem, leaf
blades, immature and mature embryos, mature pollen, root and shoot sections
etc. Meristematic cell show higher transformation frequency than non dividing
cells. This gene transfer method can be applicable to all plants and it is
independent of regeneration ability of the sp.
(4) Lipofection:Introduction
of DNA into the cell via liposome is known as lipofection. It is generally used
in animal cell culture.
(5) Microinjection:In
case of microinjection the DNA solution is injected directly inside the cell
using capillary glass micropipette, with the help of micromanipulation of a
microinjection assembly. It is easier to use protoplast than cells since cell
wall interfere with the process of microinjection. The protoplast are usually
immobilized in agarose or on glass coated with polylysine or by holding them
under suction by a micropipette. The process of microinjection is technically
demanding and time consuming, a maximum of 40-50 protoplast can be microinjected
in one hour. Successful transformation by this technique has been achieved in
tobacco, alfalfa etc.
(6) Fiber
mediated DNA delivery: In this approach DNA is delivered into
the cell cytoplasm and nucleus by silicon carbide fiber of 0.6mm diameter and
10-80 mm length. In one study suspention culture cells were mixed with plasmid
DNA having gus gene and the silicon carbide fiber all suspended in the medium.
The mixture was vortexed and the cells were assayed for transient gus
expression. The procedure and mechanism is similar to microinjection. The
method was successful in both maize and tobacco suspention culture cells. It is
widely applicable most rapid and in expensive method of DNA delivery.
(7) Laser
induced DNA delivery:
Laser has been successfully used for high frequency transfection in
animal cells. However this technology was failed to produce transformation in
plant cell.
(8) Pollen
transformation:Some reports have claimed gene transfer by soaking
pollen grains in DNA solution prior to their use for pollination. However this
report could not be substantiated by other workers using cloned genes. The
method is highly attractive in view of its simplicity and general
applicability, but so far there is no definite evidence for a transgene being
transferred by pollen soaking in the DNA solution.
(9) DNA delivery
via growing pollen tubes: In this approach stigma of a flower is
cut off sometime after pollination and DNA solution is applied on to the cut
surface. The time of stigma excision will depend on the rate of pollen tube
growth. This method is simple, easy and very promising, provided consistent
result and stable transformation are achieved. This would necessitate a much
better understanding of the mechanism of DNA transfer into the zygote and the
factors affecting it.
(10)Macroinjection:The
injection of plasmid DNA into the lumen of developing inflorescence using a
hypodermic syringe is called macroinjection. It is hypothesized that the DNA is
taken up by microspores during some specific stage of their development. This
approach is very simple and easy, however the consisting and frequency of
transformation is very low.
(11) Direct DNA
uptake by mature zygotic embryos:When dry isolated embryos of
wheat, barley, rye, etc. are imbibed in a DNA solution, they take up the DNA
and show the expression of marker gene. Dry seeds whose seed cores have been
removed by grinding, also take up DNA when imbibed in a DNA solution. Imbibed
seeds or embryos are germinated on appropriate selective media to isolate the
transformed embryos.
.jpg)
Comments
Post a Comment