The genus Taxus (yew) is a source of a number of high-value medicinal substances, particularly, paclitaxel (taxol)—a complex diterpenoid compound with a powerful antitumor action (trade name of Taxol®). Paclitaxel is one of the most efficient drugs in chemotherapy owing to its specific ability to suppress proliferation of tumor cells via stabilization of their microtubules. The world-wide demand for taxol is 800–1000 kg a year and these figures annually rise by 20%. The growing need for paclitaxel and its derivatives and the shortage of plant resources necessary for their production made compounds of the taxane group one of the most important objects for development of biotechnological methods of their production. Out of all the possible ways of taxol production (isolation from wild or plantation trees, total chemical synthesis or semisynthesis, use of yew cell cultures, techniques of metabolic engineering, and use of yew endophytic fungi), the most promising is industrial cultivation of Taxus spp. cell cultures. This review examines the papers dealing with investigation of secondary metabolism in dedifferentiated cells in vitro of various yew species and feasibility of industrial use of cell cultures for production of taxoids. We revealed a number of specificity of Taxus spp. cell cultures: (1) from a cytophysiological aspect—difficult initiation of cell cultures, their low growth characteristics, specific media and culturing conditions; (2) from a phytochemical aspect—distinction from intact plants in qualitative composition and content of secondary metabolites accounted for by specificity of cell culture as a biological system; predominant formation of С14-hydroxylated rather than of С13-hydroxylated taxoids; an opportunity for elevation of the content of taxoids—including commercially valuable ones (paclitaxel and baccatin III) with the aid of different tools (elicitation, stress exposures, two-phase cultivation and some others); (3) from a biotechnological aspect—possibility of industrial cultivation of yew cell cultures; existence of several successful industries (Germany and the Republic of Korea).

Taxane diterpenoids (taxoids) are found only in representatives of the Taxaceae family (different yew species); however, the unique structure and therapeutic properties of taxoids, which are in demand in medicine, have made these compounds one of the most studied secondary metabolites of higher plants. In this work, for the first time, a detailed study is performed into the structural diversity of polyesters of 14-hydroxylated taxoids in Taxus canadensis a yew species, for intact plants of which nonpolar 14-hydroxylated taxoids have not been previously described. At the first stage of the work, it was shown using chromato-mass spectrometry that polyesters of 14-hydroxylated taxoids (yunnanxan, taxuyunnanin C, sinenxan B, sinenxan C) are the dominant diterpenoid secondary metabolites in T. canadensis callus cell culture. Based on these results, as well as the similarity of the metabolism of cultivated in vitro plant cells and root cells in planta, it was suggested that polyesters of 14-hydroxylated taxoids will preferentially accumulate in the roots in intact T. canadensis plants. The validity of this hypothesis was confirmed using liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). According to chromato-mass-spectrometric screening in T. canadensis needles, polyesters of 14-hydroxylated taxoids are actually found only in trace amounts, while they are one of the major (quantitatively) diterpenoids in the roots. One of the main 14-hydroxylated taxoids of T. canadensis roots, yunnanxan, was isolated preparatively and uniquely identified using NMR spectroscopy and high-resolution mass spectrometry. This work is the first report on the presence of polyesters of 14-hydroxylated taxoids in intact Canadian yew plants.

The work aimed on studying the effect of synthetic phytohormones on growth and biosynthetic parameters of a suspension culture of Tribulus terrestris L., and the influence of cytokinins on these characteristics. In addition to the previously obtained results about alternative action of two types of auxins, 2,4-D and α-NAA (the first of them activates cell growth in vitro but inhibits the formation of steroidal glycosides, while the second causes cytodifferentiation, leading to a slowdown and arrest of culture growth, but activates the formation of secondary compounds), it was shown that the type of cytokinin and its concentration can modulate its action. It was found that, after transferring a culture from a “growth” medium (containing 2,4-D) to a medium with α-NAA and a different composition of cytokinins (BAP or kinetin in various concentrations), it retains the ability to actively grow for at least three growing cycles. Then, with a suboptimal combination of phytohormones, the cell culture stops growing and dies. However, with a certain composition of growth regulators in a nutrient medium (1 or 2 mg/L α-NAA + 1 mg/L kinetin), after a long lag phase (12–18 days), culture growth resumes and its long-term cultivation is subsequently possible. Suspension on a medium with 2 mg/L α-NAA and 1 mg/L kinetin T. terrestris were maintained for 70 growing cycles without deterioration of growth characteristics (the maximum level of accumulation of dry biomass Mmax was 12–16 g/L, growth index I was 13–18, specific growth rate μ was 0.18–0.28 days–1). In this case, the content of steroidal glycosides in cells did not fall below 0.2–0.3% of dry biomass. Using the UPLC-ESI-MS method, the presence in the cell biomass of at least four steroidal glycosides of the furostanol type—derivatives of hydroxy-diosgenin and tigogenin/neotigogenin—was shown. The results obtained confirm the general patterns of development of cell populations in vitro and are important for optimizing the growth and biosynthetic characteristics of plant cell cultures.

The study of the mechanisms of adaptogenic action of furostanol glycosides (FG) on plant cells was carried out in vitro and a comparison was made of the effects of hypothermia and hyperosmotic stress on a suspension cell culture of Medicago sativa L. It has been shown that the cell population of M. sativa in vitro possesses functional specificities that determine different sensitivity to the action of these abiotic stressors, which was expressed in different levels of cell viability: high (85%) under hypothermia and low (25%) under hyperosmotic action. Hypothermia stimulated the rate of generation of superoxide anion ( $${\text{O}}_{2}^{{\bullet - }}$$ ), and it was accompanied by a high constitutive activity of antioxidant enzymes (guaiacol-peroxidase, ascorbate peroxidase and glutathione peroxidase), the level of which reflects the compensatory potential of cells. Preservation of a high level of cell viability under the action of hypothermia, despite a 40% increase in the rate of generation of $${\text{O}}_{2}^{{\bullet - }}$$ , indicates that the formed reactive oxygen species did not cause damage to lipid structures and macromolecules in cells. Exogenous treatment with FG with this type of stress effect promoted an increase in the activity of antioxidant enzymes but did not have a noticeable effect on the initially high level of cell viability. In conditions of hyperosmotic stress, preliminary exposure to FG led to a threefold increase in cell survival (from 25 to 73%) and a 30% increase in the activity of soluble peroxidase in comparison with its level under the influence of only a stressor. Exposure to FG also caused an increase in activity antioxidant enzymes, a decrease in the level of lipid peroxidation and an increase in the activity of enzymes of the malate dehydrogenase (MDH) complex. However, in contrast to hypothermia, the observed changes caused a significant increase in the viability of M. sativa cells in vitro. An increase in the formation of osmolytes in the reactions NAD/NAD·H-MDH was indicated by the increased concentration of osmotic (mannitol), which causes the initial degree of cell plasmolysis, in comparison with the control. The article discusses specificities of M. sativa cell culture influencing the specificity of its stress-resistance, possible mechanisms of cell protection in vitro in hypothermia and hyperosmotic stress and the role of FG in these processes.

Rhizogenic, callus, and suspension cultures in vitro were obtained for Digitalis lanata and their growth, cytophysiological and biochemical characteristics were investigated. The obtained cultures were characterized by good growth characteristics (growth indexes I in the range of 5–13). Suspension cell culture had a specific growth rate μ within 0.2–0.3 days –1 and it was characterized by a two-phase growth curve (growth retardation during the exponential phase). In the obtained cultures, a study of the qualitative and quantitative composition of secondary metabolites by UPLC-ESI-MS and HPLC-ESI-MS showed the absence of cardiac glycosides. At the same time, phenylethanoids and steroidal glycosides of the furostanol type were found in all studied cultures. The total content of phenylethanoids in callus and suspension cultures was approximately 0.5% of the dry biomass. Based on the results of mass spectrometry, ten phenylethanoid structures, including digiciliside A, digiciliside B, maxoside, purpureaside E, and their methyl derivatives and isomers, and also seven furostanol glycosides with aglycones tigogenin and gitogenin were identified. It has been shown that the composition of secondary metabolites depends on the degree of cell differentiation: furostanol glycosides were prevalent in a rhizogenic culture consisting mainly of differentiated cells, while the diversity of phenylethanoids significantly increases in callus and suspension cell cultures consisting of dedifferentiated cells. The results of the study confirm the hypothesis put forward in our previous works about the specificity of secondary metabolism and its high intensity in plant cell cultures.

Secondary metabolism (the formation of substances of specialized metabolism) is one of the distinctive specificities of the plant organism. Data on complete sequences of plant genomes indicate that from 15 to 25% of all genes are involved in this process (the biosynthesis of enzymes, transporters and transfactors serving it), while many aspects of secondary metabolism, including its physiological functions, are still unclear. The use of plant cell and organ cultures is a promising approach to solving fundamental and applied problems in specialized metabolism. The mechanisms of formation of secondary metabolites in plant systems in vitro with different levels of cell differentiation can differ significantly from those in an intact plant, which, when comparing these biological systems, can be effectively used to study the principles of regulation, organization and functioning of secondary metabolism. To date, more than 100 000 compounds of specialized metabolism (alkaloids, isoprenoids, phenolic compounds and a number of "minor" groups of secondary metabolites) are known, which, as a rule, are biologically active substances. This fact determines their practical significance and wide application in medicine: today, about a third of all medicinal substances are of plant origin. An acute shortage of medicinal plant materials makes the use of plant cell and organ cultures a very promising source of biologically active substances. Cardiac glycosides are one of the most important groups of secondary metabolites, which have been used in medicine for the treatment of cardiac diseases for more than two centuries. Interest in them is now growing significantly due to the discovery of new properties, including antiviral and antitumor activity. The review analyzes the main results of works devoted to the study of the formation of cardiac glycosides in plant cell and organ cultures producing these compounds.

The effect of exogenous furostanol glycosides (FG) on the activity of redox enzymes was investigated in suspension cell culture of alfalfa (Medicago sativa L.). It was shown that 60-min-long treatment of alfalfa cells with FG at a concentration of 10–5 M triggered formation of ROS and activated enzymes of the antioxidant complex: superoxide dismutase and guaiacol-dependent peroxidase where the highest activity was associated with ion-bound fraction. Application of inhibitor of NADPH oxidase diphenyliodonium chloride showed that ROS are generated in the presence of FG by NADPH oxidase of plasma membrane. It was found that treatment of alfalfa in vitro cells with FG elevated activity of the key enzyme of pentose phosphate pathway: glucose-6-phosphate dehydrogenase (G-6-P DH) and glutathione reductase. A relationship was revealed between the operation of G-6-P DH, NADPH oxidase, and glutathione reductase. It is assumed that, owing to suppression of one of the main consumers of NADPH (NADPH oxidase), a rise in the activity of glutathione reductase may eliminate the inhibition of G-6-P DH. Under hyperosmotic stress, FG improved the viability of alfalfa cells in vitro to 70%, whereas it was only 24% in control material. At the same time, viability reached 90% in reference cells without treatment. Such an effect of FG became apparent as a result of elevation in activity of aldehyde dehydrogenase, reduction in lipid peroxidation (by 24%), and activation of antioxidant enzymes. Adaptation mechanisms operating on the level of redox systems are discussed.

Callus and suspension cell cultures have been obtained from aseptic seedlings of Siberian bloat fruit (Phlojodicarpus sibiricus), a producer of coumarins. P. sibiricus callus cultures were characterized by white-yellow coloration, a combination of loose and dense cell aggregates, and satisfactory growth. The dry biomass growth index for cultures of leaf, hypocotyle or root origin was 7-9, 10-12 and 11-13, respectively. Suspension cell cultures were initiated from calluses of leaf and hypocotyle origin; these cultures were also white-yellow and consisted mainly of cell aggregates of the meristem-like and parenchyma-like types with different aggregation degrees depending on the origin. Cell viability during the growth cycle was at the level of 70-80%. In contrast to the original callus cultures, the suspension culture of leaf callus origin had the highest growth characteristics (growth index of about 10). Preliminary phytochemical screening by UPLC ESI MS showed the presence of khellactone-group coumarins in the biomass of P. sibiricus primary calluses (1st-3rd growth cycle) and suspension cultures derived from them. Sibirian bloat fruit, Phlojodicarpus sibiricus, callusogenesis, suspension cell culture, coumarins The works on obtaining and growing P. sibiricus cells, as well as studying their growth characteristics were financed by the Russian Scientific Foundation grant no. 74-00097. The study of phytochemical characteristics of P. sibiricus cell cultures was supported by the state assignment of the Ministry of Education and Science of Russia (FSRG-2020-0019).

The immobilization of the microalgae (MA) of cells on biosorbents based on polyethyleneimine and cellulose-containing plant components is studied. Polyethyleneimine-based polycationic polymers have a high level of affinity for the surface structures of the MA and ensure that the cells are strongly attached to the surface of the sorbent. The introduction of plant fillers in the composition of such polymers allows us to increase their biodegradation rate and reduce costs. In the present study, six insoluble porous polymeric materials obtained by the cryopolymerization of a mixture of polyethyleneimine and various plant components using diethylene glycol diglycidyl ether were synthesized. The lyophilized biomass of plant cell cultures of Ajuga turkestanica and Polyscias fruticosa , as well as the cell-structured material (CSM) obtained from their biomass, is used as a bio-filler. Studying the kinetics and evaluating the efficiency of the immobilization of cells of the model MA culture showed the high sorption ability of the obtained materials: after 24 h of cultivation, the efficiency of immobilization was 72–77%. Assessment of the level of the photosynthetic activity of the MA cells using the method of pulse-simulated fluorimetry demonstrated that the studied biosorbents did not have any toxic effect on the cells and did not affect their functional activity. Novel biosorbents can be effectively used to collect the biomass of microorganisms from natural aquatic environments and industrial cultivators.

The role of furostanol glycosides (FGs) in the intensification of main metabolic processes in cultured yam (Dioscorea deltoidea Wall.) cells was shown. The effects of FGs were associated with changes in the complicated network of intracellular enzymatic reactions. This was evidenced by an increase in the activities of the enzymes from the glucose glycolytic oxidation pathway (hexokinase and fructose-1,6-difosphate aldolase) and the malate dehydrogenase complex (oxidoreductive-NADH/NAD-malate dehydrogenase and malic enzyme-decarboxylating NADP-malate dehydrogenase) as well as the stimulation of mitochondrial respiration accompanied by changes in activity of the alternative respiratory pathway. At the lag-phase of the growth cycle, the cell treatment with FGs was capable to guide glucose conversion through the glycolytic pathway. This was witnessed by a decrease in the activity of glucose-6-phosphate dehydrogenase and increase in that of hexokinase. Inhibition of NADPH oxidase afforded by diphenyliodonium chloride resulted in a decrease in activities of the key enzymes of glycolysis and the pentose phosphate cycle of glucose oxidation. Simultaneously, the aldolase activity increased and raised the content of its product glyceraldehyde 3-phosphate. The effect may explain the earlier-observed activation of the aldehyde-utilizing enzymes diminishing the initial POL level. The modulating action of FGs on activities of the enzymes of sugar glycolytic oxidation was shown. This was expressed as the activation of the tested enzymes at the lag- and exponential phases of the cellular growth and as the absence of the FG effect at the stationary phase. The short-term exposure of yam cells to FGs intensified the total mitochondrial respiration. An inhibitory analysis revealed the dominance of the cytochrome respiration pathway at the lag-phase, while its contribution became considerably lower at the later growth phases. FGs did not affect the rate of this respiration path but increased the potential activity of alternative oxidase (AO) during the lag- and exponential phases of the growth cycle. The FG-induced activation of AO, which may be intended to maintain a balance between carbohydrate metabolism and the rate of electron transport, is discussed.

In the framework of the present study, callus and suspension cultures of Phlojodicarpus sibiricus cells were obtained. Their morphological, physiological and biosynthetic characteristics were investigated.