Gene delivery methods in plants
For
production of transgenic animals, DNA is usually microinjected into pronuclei
of
embryonic
cells at a very early stage after fertilization, or alternatively gene
targeting
of
embryo stem (ES) cells is employed. This is possible in animals due to the
availability
of specialized in vitro fertilization technology, which allows
manipulation of
ovule,
zygote or early embryo.
Such
techniques are not available in plants. In contrast to this in higher plants,
cells
or
protoplasts can be cultured and used for regeneration of whole plants.
Therefore,
these
protoplasts can be used for gene transfer followed by regeneration leading to
the
production of transgenic plants. Besides cultured cells and protoplasts, other
meristem
cells (immature embryos or organs), pollen or zygotes can also be used for
gene
transfer in plants. The enormous diversity of plant species and the
availability of
diverse
genotypes in a species, made it necessary to develop a variety of
techniques,
suiting different situations. These different methods of gene transfer in
plants
are discussed.
Target cells for gene transformation
The
first step in gene transfer technology is to select cells that are capable of
giving
rise
to whole transformed plants. Transformation without regeneration and
regeneration
without transformation are of limited value. In many species,
identification
of these cell types is difficult. This is unlike the situation in animals,
because
the plant cells are totipotent and can be stimulated to regenerate into whole
plants
in vitro via organogenesis or embryogenesis. However, in vitro plant
regeneration
imposes a degree of 'genome stress', especially if plants are
regenerated
via a callus phase. This may lead to chromosomal or genetic
abnormalities
in regenerated plants a phenomenon referred to as soma clonal
variation.
In
contrast to this, gene transfer into pollen (or possibly egg cells) may give
rise to
genetically
transformed gametes, which if used for fertilization (in vivo) may give
rise
to
transformed whole plants. Similarly, insertion of DNA into zygote (in vivo or
in
vitro)
followed by embryo rescue, may also be used to produce transgenic plants.
Another
alternative approach is the use of individual cells in embryos or meristems,
which
may be grown in vitro or may be allowed to develop normally for the
production
of transgenic plants.
Vectors for gene transfer
Most
vectors carry marker genes, which allow recognition of transformed cells (other
cells
die due to the action of an antibiotic or herbicide) and are described as
selectable
markers. Among these marker genes, the most common selectable
marker
is npt II, providing kanamycin resistance. Other common features of suitable
transformation
vector include the following: (i) multiple unique restriction sites (a
synthetic
polylinker); (ii) bacterial origins of replication (e.g. ColE1).
The
vectors having these properties may not necessarily have features, which
facilitate
their transfer to plant cells or integration into the plant nuclear genome.
Therefore,
Agrobacterium Ti plasmid is preferred over all other vectors, because of
wide
host range of this bacterial system and the capacity to transfer genes due to
the
presence
of T - DNA border sequences.
Gene delivery methods
To
achieve genetic transformation in plants, we need the construction of a vector
(genetic
vehicle) which transports the genes of interest, flanked by the necessary
controlling
sequences i.e. promoter and terminator, and deliver the genes into the
host
plant. The two kinds of gene transfer methods in plants are:
Vector-mediated or indirect gene transfer
Among
the various vectors used in plant transformation, the Ti plasmid of
Agrobacterium tumefaciens has been widely used. This bacterium
is known as
“natural
genetic engineer” of plants because these bacteria have natural ability to
transfer
T-DNA of their plasmids into plant genome upon infection of cells at the
wound
site and cause an unorganized growth of a cell mass known as crown gall. Ti
plasmids
are used as gene vectors for delivering useful foreign genes into target
plant
cells and tissues. The foreign gene is cloned in the T-DNA region of Ti-plasmid
in
place of unwanted sequences. To transform plants, leaf discs (in case of
dicots) or
embryogenic
callus (in case of monocots) are collected and infected with
Agrobacterium carrying recombinant disarmed Ti-plasmid vector. The
infected
tissue
is then cultured (co-cultivation) on shoot regeneration medium for 2-3 days
during
which time the transfer of T-DNA along with foreign genes takes place. After
this,
the transformed tissues (leaf discs/calli) are transferred onto selection cum
plant
regeneration
medium supplemented with usually lethal concentration of an antibiotic
to
selectively eliminate non-transformed tissues. After 3-5 weeks, the regenerated
shoots
(from leaf discs) are transferred to root-inducing medium, and after another 3-
4
weeks, complete plants are transferred to soil following the hardening
(acclimatization)
of regenerated plants. The molecular techniques like PCR and
southern
hybridization are used to detect the presence of foreign genes in the
transgenic
plants.
Structure and functions of Ti and Ri Plasmids
The
most commonly used vectors for gene transfer in higher plants are based on
tumour
inducing mechanism of the soil bacterium Agrobacterium tumefaciens,
which
is
the causal organism for crown gall disease, A closely related species A.
rhizogenes causes
hairy root disease. An understanding of the molecular basis of
these
diseases led to the utilization of these bacteria for developing gene transfer
systems.
It has been shown that the disease is caused due to the transfer of a DNA
segment
from the bacterium to the plant nuclear genome. The DNA segment, which
is
transferred is called T - DNA and is part of a large Ti (tumour inducing)
plasmid
found
in virulent strains of Agrobacterium tumefaciens. Similarly Ri (root
inducing)
megaplasmids
are found in the virulent strains of A. rhizogenes.
Most
Ti plasmids have four regions in common, (i) Region A, comprising T-DNA is
responsible
for tumour induction, so that mutations in this region lead to the
production
of tumours with altered morphology (shooty or rooty mutant galls).
Sequences
homologous to this region are always transferred to plant nuclear
genome,
so that the region is described as T-DNA (transferred DNA). (ii) Region B is
responsible
for replication. (iii) Region C is responsible for conjugation. (iv) Region D
is
responsible for virulence, so that mutation in this region abolishes virulence.
This
region
is therefore called virulence (v) region and plays a crucial role in the
transfer
of
T-DNA into the plant nuclear genome. The components of this Ti plasmid have
been
used for developing efficient plant transformation vectors.
The
T-DNA consists of the following regions: (i) An one region consisting of three
genes
(two genes tms and tms2 representing 'shooty locus' and one gene tmr
representing
'rooty locus') responsible for the biosynthesis of two phytohormones,
namely
indole acetic acid (an auxin) and isopentyladenosine 5'-monophosphate (a
cytokinin).
These genes encode the enzymes responsible for the synthesis of these
phytohormones,
so that the incorporation of these genes in plant nuclear genome
leads
to the synthesis of these phytohormones in the host plant. The phytohormones
in
their turn alter the developmental programme, leading to the formation of crown
gall
(ii) An os region responsible for the synthesis of unusual amino acid or sugar
derivatives,
which are collectively given the name opines. Opines are derived from a
variety
of compounds (e.g. arginine + pyruvate), that are found in plant cells. Two
most
common opines are octopine and nopaline. For the synthesis of octopine and
nopaline,
the corresponding enzymes octopine synthase and nopaline synthase are
coded
by T- DNA.
Depending
upon whether the Ti plasmid encodes octopine or nopaline, it is described
as
octopine-type Ti plasmid or nopalinetype Ti plasmid. Many organisms including
higher
plants are incapable of utilizing opines, which can be effectively utilized by
Agrobacterium.
Outside the T-DNA region, Ti plasmid carries genes that, catabolize
the
opines, which are utilized as a source of carbon and nitrogen. The T-DNA
regions
on all Ti and Ri plasmids are flanked by almost perfect 25bp direct repeat
sequences,
which are essential for T-DNA transfer, acting only in cis orientation. It
has
also been shown that any DNA sequence, flanked by these 25bp repeat
sequences
in the correct orientation, can be transferred to plant cells, an attribute
that
has been successfully utilized for Agrobacterium mediated gene transfer in
higher
plants leading to the production of transgenic plants.
Besides
25bp flanking border sequences (with T DNA), vir region is also essential for
T-DNA
transfer. While border sequences function in cis orientation with respect to T
-
DNA,
vir region is capable of functioning even in trans orientation. Consequently
physical
separation of T-DNA and vir region onto two different plasmids does not
affect
T-DNA transfer, provided both the plasmids are present in the same
Agrobacterium
cell. This property played an important role in designing the vectors
for
gene transfer in higher plants, as will be discussed later. The vir region
(approx
35
kbp) is organized into six operons, namely vir A, vir B, vir C, vir D, vir E,
and vir G,
of
which four operons (except vir A and vir G) are polycistronic. Genes vir A, B,
D,
and
G are absolutely required for virulence; the remaining two genes vir C and E
are
required
for tumour formation. The vir A locus is expressed constitutively under all
conditions.
The
vir G locus is expressed at low levels in vegetative cells, but is rapidly
induced to
higher
expression levels by exudates from wounded plant tissue. The vir A and vir G
gene
products regulate the expression of other vir loci. The vir A product (Vir A)
is
located
on the inner membrane of Agrobacterium cells and is probably a
chemoreceptor,
which senses the presence of phenolic compounds (found in
exudates
of wounded plant tissue), such as acetosyringone and β-hydroxyaceto
syringone.
Signal transduction proceeds via activation (possibly phosphorylation) of
Vir
G (product of gene vir G), which in its turn induces expression of other vir
genes.
Transformation techniques using Agrobacterium
Agrobacterium infection
(utilizing its plasmids as vectors) has been extensively
utilized
for transfer of foreign DNA into a number of dicotyledonous species. The only
important
species that have not responded well, are major seed legumes, even
though
transgenic soybean (Glycine max) plants have been obtained. The success in
this
approach for gene transfer has resulted from improvement in tissue culture
technology.
However, monocotyledons could not be successfully utilized for
Agrobacterium
mediated gene transfer except a solitary example of Asparagus. The
reasons
for this are not fully understood, because T -DNA transfer does occur at the
cellular
level. It is possible that the failure in monocots lies in the lack of wound
response
of monocotyledonous cells.
http://www.ejbiotechnology.info/content/vol1/issue3/full/1/figure1.html
Vectorless or direct gene transfer
In
the direct gene transfer methods, the foreign gene of interest is delivered
into the
host
plant cell without the help of a vector. The gene transfer system using
genetically
engineered vectors do not work out well particularly in monocot species.
Considering
the problem, direct gene transfer methods have been tried and the
methods
used for direct gene transfer in plants are:
Chemical mediated gene transfer
Direct
DNA uptake by protoplasts can be stimulated by chemicals like polyethylene
glycol
(PEG). This method was reported by Krens and his colleagues in l982. The
technique
is so efficient that virtually every protoplast system has proven
transformable.
PEG is also used to stimulate the uptake of liposomes and to improve
the
efficiency of electroporation. PEG at high concentration (15-25%) will
precipitate
ionic
macromolecules such as DNA and stimulate their uptake by endocytosis
without
any gross damage to protoplasts. This is followed by cell wall formation and
initiation
of cell division. These cells can now be plated at low density on selection
medium.
Initial studies using the above method were restricted to Petunia and
Nicotiana.
However,
other plant systems (rice, maize, etc.) were also successfully used later. In
these
methods, PEG was used in combination with pure Ti plasmid, or calcium
phosphate
precipitated Ti plasmid mixed with a carrier DNA. Transformation
frequencies
upto 1 in 100 have been achieved by this method. Nevertheless, there
are
serious problems in using this method for getting transgenic plants and all
these
problems
relate to plant regeneration from protoplasts.
Microinjection and Macroinjection
Plant
regeneration from transformed protoplasts, still remains a problem. Therefore
cultured
tissues, that encourage the continued development of immature structures,
provide
alternate cellular targets for transformation. These immature structures may
include
immature embryos, meristems, immature pollen, germinating pollen, isolated
ovules,
embryogenic suspension cultured cells, etc. The main disadvantage of this
technique
is the production of chimeric plants with only a part of the plant
transformed.
However, from this chimeric plant, transformed plants of single cell
origin
can be subsequently obtained. Utilizing this approach, transgenic chimeras
have
actually been obtained in oilseed rape (Brassica napus).
When
cells or protoplasts are used as targets in the technique of microinjection,
glass
micropipettes with 0.5-10μm diameter tip are used for transfer of
macromolecules
into the cytoplasm or the nucleus of a recipient cell or protoplast.
The
recipient cells are immobilized on a solid support (cover slip or slide, etc.)
or
artificially
bound to a substrate or held by a pipette under suction (as done in animal
systems).
Often a specially designed micromanipulator is employed for microinjecting
the
DNA. Although, this technique gives high rate of success, the process is slow,
expensive
and requires highly skilled and experienced personnel.
The
microinjection method was introduced by two groups of scientist led by
Crossway and
Reich in l986. Recently a method known as "holding pipette
method"
was
introduced. In this, the protoplasts are isolated from cell suspension culture
are
placed
on a depression slide, by its side with a microdroplet of DNA solution. Using
the
holding pipette, the protoplast has to be held and the DNA to be injected into
the
nucleus
using the injection pipette. After the micro injection the injected cells are
cultured
by hanging droplet culture method.
DNA
macroinjection employing needles with diameters greater than cell diameter has
also
been tried. In rye (Secale cereale), a marker gene was macroinjected
into the
stem
below the immature floral meristem, so as to reach the sporogenous tissue (De
la
Pena et al., 1987) leading to successful production of transgenic
plants.
Unfortunately,
this technique could not be successfully repeated with any other
cereal,
when tried in several laboratories. Therefore, doubt is expressed about the
validity
of earlier experiments conducted with rye (Potrykus, 1991).
Electroporation method
Electroporation
is another efficient method for the incorporation of foreign DNA into
protoplasts,
and thus for direct gene transfer into plants. This method was introduced
by
Fromm and his coworkers in 1986.
This
method is based on the use of short electrical impulses of high field strength.
These
impulses increase the permeability of protoplast membrane and facilitate entry
of
DNA molecules into the cells, if the DNA is in direct contact with the
membrane. In
view
of this, for delivery of DNA to protoplasts, electroporation is one of the
several
routine
techniques for efficient transformation. However, since regeneration from
protoplasts
is not always possible, cultured cells or tissue explants are often used.
Consequently,
it is important to test whether electroporation could transfer genes into
walled
cells. In most of these cases no proof of transformation was available.
The
electroporation pulse is generated by discharging a capacitor across the
electrodes
in a specially designed electroporation chamber. Either a high voltage (1.5
kV)
rectangular wave pulse of short duration or a low voltage (350V) pulse of long
duration
is used. The latter can be generated by a home made machine. Protoplasts
in
an ionic solution containing the vector DNA are suspended between the
electrodes,
electroporated and then plated as usual. Transformed colonies are
selected
as described earlier. Using electroporation method, successful transfer of
genes
was achieved with the protoplasts of tobacco, petunia, maize, rice, wheat and
sorghum.
In most of these cases cat gene associated with a suitable promoter
sequence
was transferred. Transformation frequencies can be further improved by (i)
using
field strength of 1.25kV/cm, (ii) adding PEG after adding DNA, (iii) heat
shocking
protoplasts at 45°C for 5 minutes before adding DNA and (iv) by using
linear
instead of circular DNA.
Microprojectiles or biolistics or particle gun for gene
transfer
In
1987, Klein and his colleagues evolved a method by which the delivery of
DNA
into
cells of intact plant organs or cultured cells is done by a process called
Projectile
Bombardment.
The micro-projectiles (small high density particles) are accelerated to
high
velocity by a particle gun apparatus. These particles with high kinetic energy
penetrate
the cells and membranes and carry foreign DNA inside of the bombarded
cells.
This method is otherwise called as "Biolistics Method". In recent
years, it has
been
shown that DNA delivery to plant cells is also possible, when heavy
microparticles
(tungsten or gold) coated with the DNA of interest are accelerated to a
very
high initial velocity (1,400 ft per, sec). These microprojectiles, normally
1-3pm in
diameter,
are carried by a 'macroprojectile' or the 'bullet' and are accelerated into
living
plant cells (target cells can be pollen, cultured cells, cells in
differentiated
tissues
and meristems) so that they can penetrate cell walls of intact tissue. The
acceleration
is achieved either by an explosive charge (cordite explosion) or by using
shock
waves initiated by a high voltage electric discharge. The design of two
particle
guns
used for acceleration of microprojectiles.
Gene gun
Transformed
plants using the above technique have been obtained in many cases
including
soybean, tobacco, maize, rice, wheat, etc.. Transient expression of genes
transferred
in cells by this method has also been observed in onion, maize, rice and
wheat.
There is no other gene transfer approach, which has met with so much of
enthusiasm.
Consequently considerable investment has been made in
experimentation
and manpower for development of this technique.
Sonication Method: This is a simple technique recently (l990) formulated by Xu
and
his
coworkers. In this method the explants (especially leaves) are excised and cut
into
segments, immmersed in sonication buffer containing plasmid DNA and Carrier
DNA
in a sterile glass petridish. Then the samples were sonicated with an
ultrasonic
pulse
generator at 0.5 c/cm2 acoustic intensity for 30 minutes. After 30 minutes, the
explants
were rinsed in buffer solution without DMSO and transferred to the culture
medium.
Transformation
This
method is used for introducing foreign DNA into bacterial cells e.g. E. Coli.
The
transformation
frequency (the fraction of cell population that can be transferred) is
very
good in this method. E.g. the uptake of plasmid DNA by E. coli is carried out
in
ice
cold CaCl2
(0-50C) followed by heat
shock treatment at 37-450C for about 90 sec.
The
transformation efficiency refers to the number of transformants per microgram
of
added
DNA. The CaCl2
breaks the cell wall at
certain regions and binds the DNA to
the
cell surface.
Conjuction
It
is a natural microbial recombination process and is used as a method for gene
transfer.
In conjuction, two live bacteria come together and the single stranded DNA
is
transferred via cytoplasmic bridges from the donor bacteria to the recipient
bacteria.
Liposome mediated gene transfer or Lipofection
Liposomes
are small lipid bags, in which large number of plasmids are enclosed.
They
can be induced to fuse with protoplasts using devices like PEG, and therefore
have
been used for gene transfer. The technique, offers following advantages: (i)
protection
of DNA/RNA from nuclease digestion, (ii) low cell toxicity, (iii) stability and
storage
of nucleic acids due to encapsulation in liposomes, (iv) high degree of
reproducibility
and (v) applicability to a wide range of cell types.
In
this technique, DNA enters the protoplasts due to endocytosis of liposomes,
involving
the following steps: (i) adhesion of the liposomes to the protoplast surface,
(ii)
fusion of liposomes at the site of adhesion and (iii) release of plasmids
inside the
cell.
The technique has been successfully used to deliver DNA into the protoplasts of
a
number of plant species (e.g. tobacco, petunia, carrot, etc.).
Gene transformation using pollen or pollen tube
There
has been a hope that DNA can be taken up by the germinating pollen and can
either
integrate into sperm nuclei or reach the zygote through the pollen tube
pathway.
Both these approaches have been tried and interesting phenotypic
alterations
suggesting gene transfer have been obtained. In no case, however,
unequivocal
proof of gene transfer has been available. In a number of experiments,
when
marker genes were used for transfer, only negative results were obtained.
Several
problems exist in this method and these include the presence of cell wall,
nucleases,
heterochromatic state of acceptor DNA, callose plugs in pollen tube, etc.
Transgenic
plants have never been recovered using this approach and this method,
though
very attractive, seems to have little potential for gene transfer.
Calcium phosphate precipitation method for gene transfer
Foreign
DNA can also be carried with the Ca ++ ions, to be released inside the cell
due
to the precipitation of calcium in the form of calcium phosphate. In the past,
this
method
was considered to be very important for gene transfer in plants.
Incubation of dry seeds, embryos, tissues or cells in DNA
Incubation
of dry seeds, embryos, tissues or cells in known DNA (viral or non viral
having
defined marker genes) has been tried in many cases and expression of
defined
genes has been witnessed. However, in no case proof of integrative
transformation
could be available. In all these cases, plant cell walls not only work as
efficient
barriers, but are also efficient traps for DNA molecules. It would be very
surprising
if DNA can cross cell walls efficiently without permeabilizing them either by
PEG,
or by electroporation or by any other device.
Selection of transformed cells from untransformed cells
The
selection of transformed plant cells from untransformed cells is an important
step
in
the plant genetic engineering. For this, a marker gene (e.g. for antibiotic
resistance)
is introduced into the plant along with the transgene followed by the
selection
of an appropriate selection medium (containing the antibiotic). The
segregation
and stability of the transgene integration and expression in the
subsequent
generations can be studied by genetic and molecular analyses
(Northern,
Southern, Western blot, PCR).
Though
several methods have been described for gene transfer using naked DNA,
the
recovery of genetic recombinants, otherwise called as "transgenic
plants"
appears
to be a rare phenomenon. A concerted effort
· To
accurately identify genes which can be shown to influence agronomically
important
characters
· To
apply the technique to clone the isolated genes
· To
anneal them to appropriate vectors and
· To
evaluate their expression in agronomically important crop varieties will
solve
the deficiencies in the conventional breeding procedures.
The
last five years have seen successful outcomes and transgenic plants have been
produced
in some crop species by using both vector mediated and direct gene
transfer
techniques. However, all the programmes were not successful because of
lack
of proofs for the integrative gene transfer. Considering the needs to have
integrative
gene transfer, Potrykus points out that all the successful gene
transfers
should
have the following proofs.
1.
Serious controls for treatments and analysis.
2.
A tight correlation between treatment and predicted results.
3.
A tight correlation between physical (Southern blot, in situ hybridization)
and
phenotypic data.
4.
Complete Southern Analysis to show the hybrid fragments of host DNA
and
foreign DNA, and the absence or presence of contaminating fragments.
5.
Data that allow discrimination between false positives and correct
transformants
in the evaluation of the phenotypic evidence.
6.
Correlation of the physical and phenotypic evidence with transmission to
sexual
offspring, as well as genetic and molecular analysis of offspring
populations.
Questions
1. The vectors used in genetic engineering
should possess
a).
Multiple unique restriction sites b). Bacterial origins of replication
c).
Marker genes, which allow
recognition
of transformed cells
d).
All the above
2. Agrobacterium
Ti plasmid is preferred over all other vectors because ……
a).
Wide host range b). Capacity to transfer genes due to
the
presence of T - DNA border
sequences
c). Both a and b d). None of the above
3. The
natural genetic engineer is ….……
a). Agrobacterium tumefaciens b). Bacilius subtilis
c).
E. coli d). None of the above
4.
Agrobacterium tumefaciens causes ….……
a). Crown gall b).
Hairy root
c). Root rot d).
None of the above
5. Agrobacterium rhizogenes causes ….……
a).
Crown gall b). Hairy root
c). Root rot d).
None of the above
6.
Ti plasmids have ……… regions in common.
a). 4 b).
5
c). 3
d). None of the above
6.
Region which is responsible for tumour induction in Ti plasmids is …..
a). Region A b).
Region B
c).
Region C d). Region D
7.
T-DNA region of Ti plasmids is …..
a). Region A b).
Region B
c).
Region C d). Region D
8.
Region B is responsible for …..
a).
Tumour induction b). Replication
c).
Conjugation d). Virulence
9.
Region A is responsible for …..
a). Tumour induction b). Replication
c).
Conjugation d). Virulence
10.
Region C is responsible for …..
a).
Tumour induction b). Replication
c). Conjugation d).
Virulence
11.
Region D is responsible for …..
a).
Tumour induction b). Replication
c).
Conjugation d). Virulence
12.
The gene responsible for shooty locus in T-DNA region is …..
a).
tms b). tms2
c).
tmr d). tms and tms2
13.
The gene responsible for rooty locus in T-DNA region is …..
a).
tms b). tms2
c). tmr d).
None of the above
14.
The chemical mediated (PEG) transfer is proposed by……..
a). Krens b).
c).
d). None of the above
15.
The optimum concentration of PEG for DNA transfer is...…..
a). 15-25% b).
30-40%
c).
10% d). None of the above
16.
The microinjection method was introduced by...…..
a). Crossway and
Reich b). Fromm
c).
Klein d). Xu
17.
The electroporation method was introduced by...…..
a).
Crossway and Reich b). Fromm
c).
Klein d). Xu
18.
The particle gun for gene transfer was introduced by...…..
a).
Crossway and Reich b). Fromm
c). Klein d).
Xu
19.
The sonication method was introduced by...…..
a).
Crossway and Reich b). Fromm
c).
Klein d). Xu
Additional reading…
http://www.biotechnology4u.com/plant_biotechnology_gene_transfermethods_plants.
html
http://depts.washington.edu/agro/
http://www.ejbiotechnology.info/content/vol1/issue3/full/1/bip/
http://arabidopsis.info/students/agrobacterium/
