1070 VYACHESLAVOVA et al. 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
EXPRESSION OF HETEROLOGOUS GENES IN PLANT SYSTEMS 1071 Creation of an expression vector Isolation of tobacco mesophyll protoplasts Direct transformation of tobacco protoplasts with PEG K T Formation of cell colonies on a selective medium Callus formation and morphogenesis Rooting on a selective medium Molecular genetic analysis T 0 T K Transplantation into soil, seed production T 1 Segregation analysis T 2 Analysis of stable transgene heritability in generations T Morphological and biochemical 3 K T characteristics Fig. 2. A schematic diagram of generation of transgenic plants with the use of protoplast transformation. K, control; T, transgene. RUSSIAN JOURNAL OF GENETICS Vol. 48 No. 11 2012
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