A. A. Zagorskaya

Comparative analysis of HBV M-antigen production in leaves of individual transgenic carrot plants

76 The advantages of plants as alternative systems for expression of heterologous proteins are illustrated by numerous examples of accumulation in plant tissues of complex functionally active immunoglobulins, secretory granules, collagen, hemoglobin, and cytokines [1]. Transgenic plants accumulating antigens against various pathogens can be used as edible vaccines, including those against hepatitis B virus [2].

[T-DNA-induced mutations in transgenic plants].

The review surveys experimental data on changes of individual traits in genetically modified (transgenic) plants. The attention is focused on mutations induced by T-DNA insertions upon Agrobacterium-induced transformation of dicotyledonous plants. The character of mutation appearance in transgenic plants is examined. The prospects of mutations induced by T-DNA insertions are considered.

Transgenic tobacco plants producing human interleukin-18.

Studies conducted over the last decade have shown that genetically modified plants are potentially less expensive and safer sources of recombinant proteins than the commonly used expression systems based on bacteria, yeast, and cultured mammalian and insect cells. World’s leading biotech companies have already created transgenic plants producing hormones, cytokines, growth factors, and enzymes. The recombinant proteins from plants were found to be similar in biological activity to their counterparts derived from other expression systems [1]. Transgenic plants that produce epitopes of human and animal pathogens are promising for oral immunization [2]. Vaccines against hepatitis B, rabies, Norwalk virus, and diarrhea, obtained from plants, have now passed first clinical trials [3, 4]. However, when subunit vaccines are used, immunological tolerance may develop to the pathogen [5] (for example, no immune response is observed to antigens consumed every day in food). To improve the immunization efficacy of “edible vaccines” and to prevent the development of immunological tolerance, researchers actively seek proteins that can act as adjuvants [6]. Interleukin-18 (IL-18) was found to be a promising adjuvant in laboratory animals subjected to intranasal vaccination [7]. It was possible to avoid the induction of immunological tolerance to ovalbumin in newborn mice by combining this antigen with oral administration of IL-18; their combination elicited the systemic and mucosal immune responses to ovalbumin in the mice [8]. In this context, it is of interest to study the possibility of supplementing edible vaccines with IL18 as an adjuvant. We believe that transgenic plants may be convenient vehicles for delivery of IL-18 by the oral route to gastrointestinal mucosae. It is also of interest to study whether pure recombinant IL-18 can be obtained by affine chromatography.

Inheritance of Altered Flower Morphology and Kanamycin-Resistance in Transgenic Tobacco Plants

Inheritance of altered flower morphology and resistance to antibiotic kanamycin was studied in the first and second generations (T1and T2, respectively) of self-pollinated transgenic tobacco plants. In most transformants, kanamycin resistance was closely linked to mutant phenotype. T-DNA-induced mutations were shown to be dominant.

Abnormalities of meiotic division caused by T-DNA tagged mutation in tobacco (Nicotiana tabacum L.).

Mutations obtained by insertional mutagenesis have been useful tools for investigation of the complex and multistep meiotic process (Peirson et al., 1997; Bai et al., 1999). The mutations make it possible to clone the gene of interest and to identify its products. The transposable element system has been used for transposon tagging of genes in maize (Hake et al., 1989; McCardy et al., 1989), and in Arabidopsis (Feldman, 1991; Aarts et al., 1993). A T-DNA—induced meiotic mutation has been found among a series of transgenic tobacco plants (Deineko et al., 1999). The characteristic features of the plants are male sterility and modified structure of the flower. DN (deformed nuclei) is a monoinsertional dominant mutation with an unusual cytological phenotype. The mutation causes defects of the position and shape of the nuclei in the first and the second meiotic divisions, cytomixis at meiosis 1, nuclear deformation, block of the spindles or their misorientation at meiosis 2.

Auxin: Regulation and its modulation pathways

Indole-3-acetic acid (IAA), or auxin, is a key regulator of almost all processes of plant growth and development. In general, the auxin biosynthesis, metabolism, and transport systems form a global auxin machinery, which is controlled by a great number of specific genes. The regulation of this auxin system is implemented at least in four functional levels, i.e. biosynthesis, metabolism, transport of auxin, and response to auxin signaling. Not all the genes involved in the operation and regulation of auxin machinery are known to this day, and there are still many uncertainties in our ideas about the functioning of this key plant growth system. The list of genes associated with auxin action expands rapidly. However, only a few of them are likely to be used as viable targets for directed crop improvement in genetic transformation programs. This review reflects our current understanding of the internal regulation mechanisms of the plant auxin system and reveals the most attractive gene targets for use in genetic engineering.

Auxins: Biosynthesis, metabolism, and transport

Indole-3-acetic acid (IAA) is the key plant hormone of the auxin class, which controls all processes of plant growth and development. Information on IAA biosynthesis, metabolism, and transport is of great importance for understanding nearly all morphogenetic processes in plant development. Nevertheless, our knowledge about the organization of this auxin machine, which is of significance to all plants, is by no means complete. At present, six independent pathways of IAA biosynthesis are known, each of which has its own regulation and genetic base. Auxin metabolism and active transport are no less intricate systems with internal and external regulation. A lot of genes that determine the behavior of the auxin machine are still unknown, but those revealed can be employed in genetic engineering to obtain new plant forms with improved properties. The review presents modern concepts of the biosynthesis, metabolism, and active transport of auxin with regard to the genes underlying these processes.

Migration of DNA-Containing Organelles between Tobacco Microsporocytes during Cytomixis

Ultrastructural analysis of intercellular migration of DNA-containing organelles (nuclei, mitochondria, and plastids) in tobacco microsporogenesis during cytomixis was conducted. It was demonstrated for the first time that the migrating part of the nucleus is covered with ribosomes and can contain the accumulation of nuclear pores. The possibility of mitochondrial migration between the plant cells was proven for the first time. It was demonstrated that mitochondria extremely rarely pass into neighboring cells, and their movement occurs through one cytomictic channel. In turn, plastids can generate the accumulations around cytomictic channels and actively migrate between the cells, even through small size cytomictic channels. It was established that plastids can pass into another cell through one or several cytomictic channels, and several plastids can also simultaneously migrate through one channel. The consequences of migration of DNA-containing organelles in the cells producing the pollen are discussed.

Suspension-cultured plant cells as a platform for obtaining recombinant proteins

Production of recombinant proteins in suspension cultures of genetically modified plant cells is a promising and rapidly developing area of plant biotechnology. In the present review article, advantages related to using plant systems for expression of recombinant proteins are considered. Here, the main focus is covering the literature on optimization of cultivation conditions of suspension-cultured plant cells to obtain a maximal yield of target proteins. In particular, certain examples of successful use of such cells to produce pharmaceuticals were described.

Identification of the NtFZY gene family in Tobacco (Nicotiana tabacum) involved in the tryptophan-dependent auxin biosynthesis pathway

140 The main endogenous auxin—indolee33acetic acid (IAA)—is involved in almost all processes of plant growth and development. This is one of the key plant hormones [1]. The biochemical pathways of IAA biosynthesis and, accordingly, the genes that control these processes are still poorly understood. To date, five different biochemical pathways of IAA synthesis (both tryptophanndependent and tryptophannindee pendent) are known [2, 3]. However, none of these pathways has been studied comprehensively in terms of the enzymes and intermediates of biosynthesis and the genes that control them. Today, nothing is known about the genes that ensure the biosynthesis of auxin in the important model object tobacco (Nicotiana tabacum L.). Therefore, the identification and characc terization of the tobacco NtFZY gene family, which ensures the tryptamine pathway of auxin biosynthesis, is of considerable theoretical and practical interest. The novelty and priority of this work is that the genes involved in the biosynthesis of auxin, ortholoo gous to the YUCCA genes of Arabidopsis thaliana, were identified in N. tabacum for the first time. Despite the fact that tobacco is widely used in various biochemical and genetic engineering studies, its genetics remains poorly studied. The NtFZY1–NtFZY4 genes, identii fied by us, are the first identified tobacco genes that are involved in the tryptamine pathway of auxin biosynn thesis and encode flavin monooxygenaseelike proteins functioning at the initial stages of the tryptophann dependent pathway of auxin biosynthesis. Identificaa tion of YUCCA genes in tobacco is promising not only in terms of broadening the possibilities of a detailed study of the tryptamine pathway of IAA biosynthesis in plants in general. These genes can later be used to cree ate new genotypes of different agronomically imporr tant plants with altered auxin dynamics. It should be noted that not all IAA biosynthesis pathways are present in all plant families [4]. Two bioo synthetic pathways, in which CYP79B and YUCCA genes play the key role, were most thoroughly studied in A. thaliana. The CYP79B gene is involved in the indole–acetaldoxime pathway, whereas the YUCCA gene functions in the tryptamine pathway [3]. The FZY gene encodes a monooxygenaseelike protein, is an ortholog of the YUCCA gene of A. thaliana and was detected in petunia [5]. This fact suggests that the YUCCA pathway of the synthesis of IAA is represented in the Solanaceae family. Another confirmation of this assumption was the discovery of the YUCCA pathway and FZY orthologs in the …