Features of the clonal micropropagation of plants of the genus sequoia with a high content of biologically active substances

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Abstract

Introduction. Sequoia sempervirens (D.Don) Endl. are the tallest long–lived relict plants capable of accumulating unique secondary metabolites that can be used in phytopharmacognosy. To preserve the biodiversity of rare plant species, it is advisable to apply biotechnology methods and create in vitro genetic banks, including by the method of clonal micropropagation. Stress-resistant and highly productive plants can be created using methods of cellular biotechnology, in particular, in vitro cell selection.

The purpose of the study. development of technology for in vitro production of highly productive microclones of relict gymnosperms of the genus Sequoia, study of the formation and localization of secondary metabolites and their biological activity.

Material and methods. The object of the study was Sequoia sempervirens (D.Don) Endl. plants. Aseptic culture was obtained from shoots of the first year of vegetation of intact plants. Explants were cultured on MS nutrient medium with different hormonal composition. The localization of phenolic compounds was studied in leaves, stems, apical buds of intact sequoia plants and in microclones, as well as in callus tissue. Histochemical methods were used for this: the material was stained with 0.08% Fast Blue reagent raster for the sum of phenolic compounds, and a reaction with vanillin reagent in hydrochloric acid vapor was used to study the localization of flavans (catechins and proanthocyanidins). The quantitative content of different classes of polyphenols was determined using spectrophotometric methods. The cytotoxic properties of the extracts were studied using an MTT test.

Results. Microclone cultures of evergreen sequoia (Sequoia sempervirens (D.Don) Endl.) were obtained in vitro. The dependence of culture growth in vitro on the hormonal composition of the nutrient medium and the endogenous content of polyphenols in the primary explant has been studied. The accumulation and localization of secondary compounds in in vitro Sequoia sempervirens microclones has been shown for the first time. Secondary metabolites are predominantly localized in epidermal, parenchymal and conductive tissues in both intact plants and microclones in vitro. The localization of secondary compounds in in vitro cultures as producers of substances with high biological activity is characterized. Studies on the cytotoxicity of extracts of sequoia cultures in vitro demonstrate high rates in relation to the HeLa cervical cancer cell line and the human glioblastoma A172 line.

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About the authors

S. M. Zaitseva

Russian State Agrarian University – Moscow Agricultural Academy named after K.A. Timiryazeva

Author for correspondence.
Email: smzaytseva@yandex.ru
ORCID iD: 0000-0001-9137-3774
SPIN-code: 5553-8033

Ph.D. (Biol.), Associate Professor, Department of Biotechnology

Russian Federation, Timiryazevskaya st., 49, Moscow, 127434

E. L. Bolotina

Russian State Agrarian University – Moscow Agricultural Academy named after K.A. Timiryazeva

Email: lizavetarodbol@yandex.ru
ORCID iD: 0009-0007-9006-6044
SPIN-code: 2791-6818

Post-graduate Student, Department of Biotechnology

Russian Federation, Timiryazevskaya st., 49, Moscow, 127434

Е. А. Kalashnikova

Russian State Agrarian University – Moscow Agricultural Academy named after K.A. Timiryazeva

Email: kalash0407@mail.ru
ORCID iD: 0000-0002-2655-1789
SPIN-code: 6776-2635

Dr.Sc. (Biol.), Professor, Department of Biotechnology

Russian Federation, Timiryazevskaya st., 49, Moscow, 127434

R. N. Kirakosyan

Russian State Agrarian University – Moscow Agricultural Academy named after K.A. Timiryazeva

Email: mia41291@mail.ru
ORCID iD: 0000-0002-5244-4311
SPIN-code: 5260-8784

Ph.D. (Biol.), Associate Professor, Department of Biotechnology

Russian Federation, Timiryazevskaya st., 49, Moscow, 127434

A. A. Balakina

Federal Research Center of Problems of Chemical Physics and Medicinal Chemistry RAS

Email: balakina@icp.ac.ru
ORCID iD: 0000-0002-5952-9211
SPIN-code: 2217-3493

Ph.D. (Biol.), Chief Research Scaientist, Laboratory of Molecular Biology

Russian Federation, Ac. Semenov avenue 1, Chernogolovka, Moscow region, 142432

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2. Fig. 1. Localization of phenolic compounds in leafs and stems (а, б) integumentary and (в, г) conducting tissues of Sequoia used as explants for initiating in vitro cultures. (reaction to flavans with vanillin reagent (а-в) and the total content of phenolic compounds with Fast Blue reagent (г))

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3. Fig. 2. Cuttings of evergreen sequoia used as explants for cultivation in vitro (a), activation of growth processes in meristems (б, в), callus tissue (г) and single morphogenesis on callus tissue of the fourth passage, cultured on MS nutrient medium (IAA – 0.5 mg/l, 2,4-D – 0.5 mg/l, 6-BAP – 0.5 mg/l) at 16-hour photoperiod (г, д)

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4. Fig. 3. Microclones on MS nutrient media with different hormonal composition (a, б – medium without hormones; в, г –  medium with the addition of hormones IAA – 0.5 mg/l, 2,4-D – 2 mg/l, 6-BAP – 0.5 mg/l) with a 16-hour photoperiod. Adaptations of micro-plants in ex vitro conditions (д)

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5. Fig. 4. Localization of phenolic compounds in the stem (а) and needles (б) of the sequoia microclone and in the cell walls of leaf parenchyma needles (в). Reaction to the total content of phenolic compounds with the Fast Blue reagent (а, б) and to flavans with the vanillin reagent and (в)

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6. Fig. 5. Localization of phenolic compounds (в–д) in the callus "cushion" of the morphogenesis zone during the formation of sequoia microclones (а, б). Formation of conductive structures in the callus "cushion" in the morphogenesis zone (в–д)

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7. Fig. 6. Localization of secondary metabolites in morphogenic callus of sequoia (а). Reaction to the amount of soluble phenolic compounds with the Fast blue reagent (б, в) and the vanillin reagent for the localization of flavans (г, д)

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8. Fig. 7. Accumulation of polyphenols in primary explants, microclones and callus tissue of sequoia

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9. Fig. 8. Effect of sequoia extract on the viability of normal and tumor cells. HepG2 – cell line of human hepatocellular carcinoma; HeLa – line of “immortal” cervical cancer cells; FetMSC  – mesenchymal stem cells from the bone marrow of a 5-6 week human embryo; A172 – line of human glioblastoma

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