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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