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1068 VYACHESLAVOVA et al. Agrobacterium vir genes ChvE Sugars Ti plasmid T-DNA T-DNA VirD2 VirA VirD1/VirD2 VirE2 T-DNA complex VirG Phenols wounding integrated T-DNA integrated T-DNA nucleus plant cell Fig. 1. A diagram of agrobacterial transformation of a plant cell. tion of a protein product are selected from a large number of primary transformants. Several factors are known that influence the efficiency of agrobacterial transformation of plants. These are plant genotype, the explants used, the Agrobacterium strain, composition of nutrient media, conditions and duration of the interaction of Agrobacterium and plant explants (inoculation and cocultivation stages), etc. [7]. The method of Agrobacterium-mediated transformation of plants has a number of advantages, including a low number of copies of integrated sequences, the possibility of transferring large-size sequences, the possibility of expression of genes encoding multimeric proteins (e.g., immunoglobulins), and a high level of stable expression (Table). However, despite the apparent simplicity of the method, its main limitation is the dependence of transformation efficiency on plant genotype. In connection with this, the method often requires optimization for an individual plant species or vector construct. Taking into consideration the use of transgenic plants obtained by agrobacterial transformation as highly efficient producers of target proteins, a drawback of this method is an insufficiently high yield of a target protein relative to the total soluble protein. Another problem is silencing of target genes in transgenic plants. Bioballistic transformation of plants is based on the physical process of T-DNA delivery. T-DNA contained in a plant vector is precipitated onto special metal particles (gold, tungsten), and these particles are transferred into the plant genome with the use of a gene gun (bombardment of plant tissue). Thus, the genes contained in the T-DNA region are integrated into the plant genome and expressed in the transformed plant cells. The procedures for selecting transformed cells and producing from them primary plant transformants follow the same principle as in the case of plant transformation with A. tumefaciens. At present, transgenic plants belonging to different families of the plant kingdom have been obtained with this method [8]. Since the time this method was developed in 1990, numerous bioballistic devices have also appeared. Different principles of operation and specific design features of these devices necessitate optimization of the conditions of ballistic transformation for every device and every plant species [8]. The efficiency of bioballistic transformation depends on different factors, which are divided into three groups: physical, chemical, and biological. The RUSSIAN JOURNAL OF GENETICS Vol. 48 No. 11 2012

EXPRESSION OF HETEROLOGOUS GENES IN PLANT SYSTEMS 1069 Comparative characteristics of stable and transient expression of genes in plants Stable expression Transient expression Characteristic agrobacterial transformation Nuclear transformation bioballistic transformation protoplast transformation Plastid transformation bioballistic transformation agroinfiltration viral infection Dependence on genotype Dependent Less dependent Dependent Less dependent Dependent Dependent Host plant range Wide Wide Limited Limited Limited Limited Vectors Complex Simple Simple Complex Complex Complex Possibility to use linear DNA No Yes Yes Yes No No Transfer of genes with a size of over 1000 Kb Possible Possible Possible Impossible Possible Possible Copy number (number of insertions) Low copy number Multiple T-DNA integrations and rearrangements Low copy number Single copy? Without integration Without integration Posttranslational modifications of protein products Possible Possible Possible Impossible Possible Possible Expression level Medium Low Low High** High High Transformation efficiency High* Medium Low Very low High High Specific expensive equipment Not required Required Not required Required Not required Not required Complexity of procedures Simple Simple Complex Complex Simple Simple Biosafety Relative*** Relative*** Relative*** High**** High***** High***** * High transformation efficiency for some species, but special protocols are required. ** Usually from several percent of the total soluble protein to the physiological maximum >50% of the total soluble protein. *** Special approaches are required to prevent the release of a transgene into the environment. **** Maternal inheritance of plastids, the pollen is free of plastids. ***** Transgenic plants are not generated. Most recombinant proteins can be produced under controlled conditions. RUSSIAN JOURNAL OF GENETICS Vol. 48 No. 11 2012