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physical parameters to be experimentally selected are
as follows: helium pressure required to accelerate
DNA-carrying microparticles; vacuum pressure in a
chamber with target cells; distance separating a target
tissue from the source of particles and the dissecting
filter; the size and number of microparticles used in a
shot; the number of shots at a target tissue; etc. The
chemical parameters include the availability and concentration
of reagents for DNA precipitation on metal
particles, namely, inorganic salts of calcium and magnesium,
and organic components (PEG, glycerol,
ethanol, spermidine). This group of parameters also
includes the pH value, concentration of integrated
DNA, and the nature of the metal for microparticles
(tungsten, gold, platinum). The biological parameters
are primarily associated with the degree of competence
of transformed cells for regeneration of morphogenic
structures and subsequent regeneration of
plants, with the degree of plasmolysis of explant cells
induced by pre- and postosmotic treatment (cultivation
on a medium with an increased osmotic pressure),
with the nature of integrated DNA and its structure
(double-stranded or single-stranded circular
DNA, linear double-stranded or single-stranded plasmid
DNA). A very important factor for the efficiency
of bioballistic transformation is the state of the plant
cell population at the moment of attack, i.e., the ratio
of cells at the stage of DNA synthesis and cell division.
However, despite a seemingly difficult choice of conditions
for optimal bioballistic transformation, such
conditions have already been determined for a large
number of plant species [9].
The main disadvantage of bioballistic transformation,
in addition to expensive equipment and the
necessity of selecting conditions, is the integration of a
large number of copies of the transgene sequence into
the recipient plant genome, which can lead to transgene
silencing (Table). However, it was demonstrated
for corn that the number of transformation events with
single-copy integration can be increased using, for
example, low DNA quantities for bombardment, but
this can result in a reduction in transformation efficiency
[10]. It should be noted that in comparison to
agrobacterial transformation, bioballistics is characterized
by a lower transformation efficiency (Table), a
high probability of integration of sequences of the vector
construct, and by the loss of integrity of the expression
cassette [8].
Protoplast transformation (direct transfer of DNA
into protoplasts) is based on the use of protoplasts of
mesophyll cells (plant mesophyll cells deprived of the
cell wall as a result of treatment with a specific set of
cellulolytic enzymes) [11]. T-DNA transfer occurs as
a result of alteration of the osmotic pressure inside
plant cells during their treatment with PEG [12]. A
schematic diagram for generating transgenic plants
with protoplast transformation is presented in Fig. 2
for stable tobacco transformants.
The advantages of this method are the possibility of
delivering large DNA fragments and using simple vectors
(in the linear form also) (Table), and the lack of
agrobacterial contamination during production of
transgenic plants. However, protoplast transformation
is a rather laborious method with a low transformation
efficiency as compared, for instance, to agrobacterial
transformation, and the efficiency of transformation
depends on the plant genotype (table). All these factors
limit the application of protoplast transformation.
Electroporation is based on integration of the vector
T-DNA sequence into protoplasts with the use of a
special device, an electroporator. As a result of physical
treatment, T-DNA is transferred into plant cells.
The procedures for selecting transformed cells and
producing plant transformants follow the same principle
as in the case of protoplast transformation [11].
Like in the case of direct transfer of DNA into protoplasts,
the efficiency of electroporation, as a method
for generating transgenic plants, to a large extent
depends on the plant genotype. The method itself is
characterized by a low transformation efficiency and
requires specific expensive equipment (table), which
limits its application.
Plastid transformation is based on the transfer of
genes into chloroplast DNA. A schematic diagram of
plastid transformation is presented in Fig. 3. Only ballistic
transformation is used for transformation of plastids.
The procedures for selecting transformed cells
and producing transformed plants follow the same
principle as in the case of agrobacterial transformation
of plants. The vector DNA is transferred into the plastid
genome via homologous recombination (Fig. 3).
The vector for plastid transformation includes
sequences homologous to the sequences of the plastid
DNA genes. This facilitates the integration of the
sequences without disturbing the integrity and functions
of the plastid genome genes.
The first successful transformation of plastids was
performed for the unicellular green alga Chlamydomonas
reinhardtii and later for tobacco plants [13]. The
spectrum of plants used for plastid transformation was
restricted for a long time to representatives of one or
two species. However, technologies for efficient transformation
and generation of transplastomic plants,
including potato [14], lettuce [15], tomatoes [16], and
Arabidopsis [17], are being actively developed at
present.
The advantages and disadvantages of this method
are described in detail in review [13] and are summarized
in the table. Important advantages are a high target
protein yield and the presence of a chaperone
machine for correct folding of proteins (e.g., human
serum albumin or interferons); in addition, since
chloroplasts are prokaryotic in nature, they can
express native bacterial genes. Another unquestionable
merit of transplastomic plants is their biosafety.
Since the plant pollen does not contain plastids, dispersion
of transgenes in the environment is impossi-
RUSSIAN JOURNAL OF GENETICS Vol. 48 No. 11 2012
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