doi: 10.15389/agrobiology.2021.3.602eng

UDC: 581.19:581.143.6

The authors express their gratitude to the staff of LLC «Sintegal» (Tomsk) for providing with meta-chloro-benzhydryl urea preparation and to the employee of the Department of Plant Physiology, Biotechnology and Bioinformatics of TSU Yu.V. Medvedeva for maintaining the collection of Saussurea orgaadayi plant cell culture.



I.F. Golovatskaya , A.E. Reznichenko, N.I. Laptev

National Research Tomsk State University, 36, pr. Lenina, Tomsk, 634050 Russia, e-mail (corresponding author ✉),,

Golovatskaya I.F.
Laptev N.I.
Reznichenko A.E.

Received December 6, 2020

Meta-chloro-benzhydryl urea (m-СBU) is an inducer of the human monooxygenase system, its key enzymes belong to the cytochrome P-450 superfamily (CYP). Currently, there is no information about the role of m-СBU in the plant vital activity regulation; however, the participation of CYP in the metabolism of secondary metabolites, for example, flavonoids (Fl), and most phytohormones have been shown. Saussurea orgaadayi V. Khan. and Krasnob. is a poorly studied plant species. Its cell culture, in accordance with our data, doubles the total amount of endogenous Fl during the transition from exponential growth to the stationary phase. The present study, for the first time shows statistically significant (p < 0.05) differences in the growth responses of the callus culture of the S. orgaadayi to m-СBU in different concentrations. It was revealed that the change in the growth index in terms of fresh and dry weight is related to a change in the volume and shape of cells, as well as the occurrence frequency of different groups of cells. Here, for the first time, the dynamics of the content of Fl, accompanying changes in the growth of culture under the influence of m-СBU was assessed. The aim of this work was to determine the role of meta-chloro-benzhydryl urea in the accumulation of flavonoids and the variability of cytomorphological characteristics of S. orgaadayicallus culture (cell linear dimensions, volume and shape, the frequency of cells of different sizes, growth index for the fresh and dry biomass). A callus culture derived from cotyledon explants of sterile seedling was repeatedly passaged on a modified Murashige-Skoog (MS) agar nutrient medium supplemented with sucrose, vitamins and growth regulators 0.8 mg/l 2,4-D and 0.5 mg/l 6-BAP. The culture was grown in the dark at a temperature of 22-24 °С in MS medium added with growth regulators and 0.01, 0.1, 1, 10, or 100 μM m-СBU (Sintegal, LLC, Russia). In the control, m-СBU was not added. After 30 days of subculture, 2/3 of the material was used to determine the wet and dry biomass followed by the isolation of flavonoids, and 1/3 of the material was fixed in Clark’s solution for 2 days. To prepare micropreparations, the cell culture was macerated in a 3 N hydrochloric acid solution with constant shaking until a homogeneous cell suspension was obtained. Cytophotometric analysis was performed using light microscopy (video camera Moticam 2300, Motic, Spain) with software. The sizes of 100 cells were measured for each variant, their shape was estimated, and the volume was calculated. To calculate the growth index (GI), the initial (beginning of subcultivation, M0) and the final fresh or dry weight of calli (on day 30 of subculture, M30) were determined and expressed as a percentage of the control: GI = (M30 – M0)/M0. The total amount of Fl in the callus culture was quantified based on the colored Fl complexation with aluminum chloride followed by measurement of the optical density (a UV-1650 spectrophotometer, Shimadzu Corp., Japan). As a result of the studies, a dose-dependent effect of m-СBU on cell growth was established due to their division (0.1 μM) and stretching (1-100 μM), which was accompanied by a 2.1-3.5-fold and 1.5-2.9-fold increase in the GIf and GId of callus culture, respectively (p < 0.05). At a concentration of 0.1 μM, the number of small meristematic cells increased by 16 % compared to the control. At the same time, the average volume of large cells decreased by 31 % as compared to the control, which indicates inhibition of cell elongation processes. With the increase in m-СBU concentration, the frequency of cells of two groups increased, by 55 and 30 % for medium-sized cells at 1 and 10 μM, respectively, and by 50 and 57 % for large-sized cells at 1 and 100 μM, respectively. The volume of cells also increased compared to the control, by 61 % at 10 μM for large cells and by 18 % at 100 μM of small cells. m-СBU reduced the total amount of endogenous flavonoids by 80-95 % (p < 0.05) upon activation of growth processes in S. orgaadayi cells in vitro. The content of the total Fl decreased maximally at 0.01 and 0.1 μM m-СBU and did not differ significantly from the control at 100 μM m-СBU. The maximum 3.5-fold increase in GIf in the medium with 1 μM m-СBU occurred simultaneously with an 83 % decrease in the amount of Fl. m-СBU can be used in plant biotechnology as a cell growth modulator in callus cultures to reduce the content of growth-inhibiting metabolites. To activate cell division, the most preferable dose is 0.1 µM m-СBU, while to change the content of Fl, which doubles the biomass of the culture, 100 µM m-СBU should be used.

Keywords: Saussurea orgaadayi, cell culture, meta-chloro-benzhydryl urea, morphogenesis, flavonoids.



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