Sergey Victorovich Evsyukov

Bacterium Rhizobium rhizogenes has the unique ability to cause hairy root disease. Symptoms of this disease occur when the bacterial genes rolA, rolB, rolC, and rolD, contained in plasmid T-DNA, are expressed after integration into the plant genome. A biotechnological method for obtaining fast-growing in vitro cultures of hairy roots capable of synthesizing secondary metabolites is based on this feature. Also, the ability of the bacterium to mediate horizontal gene transfer may have led to the appearance of rol-gene homologs in plant genomes, which by now have been found in a wide range of species. The variability of rol-genes may play an important role in the evolution of the whole mechanism of natural transformation, as it can potentially affect the physiological properties of transformed plants. The sequence analysis revealed a significant overall degree of variability in rol-genes between bacterial strains. But at the same time, a part of rol-gene sequences remained conserved in both bacterial and plant genomes. A detailed study demonstrated that in all of the considered bacterial rol-genes, as well as in plant genes potentially capable of expression in full-length form, the variability was represented by either nucleotide substitutions or insertions and deletions of multiples of three, which did not permit reading frame displacement.

Cryopreservation is one way to preserve rare, endangered species. However, during the cryopreservation process, plant cells undergo considerable stress, which may lead to cell death. In our work, orthodox Stipa seeds of six rare species were cryopreserved: S. sareptana, S. ucrainica, S. tirsa, S. dasyphylla, S. adoxa, and S. pulcherríma. Short-term cryopreservation (14 days) stimulated germination of all Stipa species studied. Prolonged cryopreservation (70 days and more) decreased the germination of all Stipa seeds except S. sareptana. The decrease in germination progressed over time as a result of the cumulative stress of cryopreservation rather than the initial stress. To stimulate germination, seeds were stratified and treated with GA₃, KNO₃, NaOH, and H₂O₂. After four years of seed cryopreservation, it was possible to obtain seedlings of all the Stipa species studied with 30 days of stratification and 180 days of germination. After five years of cryopreservation and seed treatment with 30% NaOH for one hour, the best germination was obtained in S. adoxa and S. pulcherrima. After treatment with 5% H₂O₂ for 20 min, the best germination was obtained in S. sareptana, S. ucrainica, and S. dasyphylla. S. sareptana seeds germinated in all the aforementioned experiments. S. sareptana has a non-deep physiological dormancy and is the most widespread and drought-tolerant Stipa species studied. The best habitat adaptation and stress tolerance correlated with this species’cryotolerance. S. sareptana was recommended for further cryopreservation, while storage protocols for the other Stipa species studied need further improvements.

Due to population growth, instability of climatic conditions, and reduction of the areas of natural ecosystems, it becomes necessary to involve modern biotechnological approaches to obtain highly productive plant material. This statement applies both to the creation of plant varieties and the production of new pharmaceutical raw materials. Genetic transformation of valuable medicinal plants using Agrobacterium rhizogenes ensures the production of stable and rapidly growing hairy roots cultures that have a number of advantages compared with cell culture and, above all, can synthesize root-specific substances at the level of the roots of the intact plant. In this regard, special attention should be paid to the collection of hairy roots of the Institute of Plant Physiology RAS, Russian Academy of Sciences, the founder of which was Dr. Kuzovkina I.N. Currently, the collection contains 38 hairy roots lines of valuable medicinal and forage plants. The review discusses the prospects of creating a hairy roots collection as a basis for fundamental research and commercial purposes.

In this study the transgenic lines (TLs) of tobacco (Nicotianatabacum L.), which overexpress the heterologous gene encoding the bacterial enzyme choline oxidase were evaluated. The goal of our work is to study the effect of choline oxidase gene expression on the sensitivity of plant tissues to the action of NaCl. The regenerative capacity, rhizogenesis, the amount of photosynthetic pigments and osmotically active compounds (proline and glycine betaine) were assessed by in vitro cell culture methods using biochemical and morphological parameters. Transgenic lines with confirmed expression were characterized by high regeneration capacity from callus in the presence of 200 mmol NaCl, partial retention of viability at 400 mmol NaCl. These data correlated with the implicit response of regenerants and whole plants to the harmful effects of salinity. They turned out to be less sensitive to the presence of 200 mmol NaCl in the cultivation medium, in contrast to the WT plants.