Annotation of the results obtained in 2022
The objects of the project research are rare and endangered plants of Yakutia, during this reporting period we covered the works and performed experiments with the following plant species: Dracocephalum jacutense Peschkova, category "1", Lilium pilosiusculum (Freyn) Miscz, category "2 b". The following objects, which were not included in the 2022 work plan, were also included in the reporting period to expand the work: Artemisia martjanovii Krasch. ex Poljakov, category "3 e", Polygala sibirica L., category "3 c" - a rare species (Red Book of the RS(Y). The optimal protocols of seed material sterilization and conditions for in vitro cultivation of aseptic seedlings of the research objects Dracocephalum jacutense, Lilium pilosiusculum, Artemisia martjanovii, Polygala sibirica were developed and tested. Sterile seedlings from seeds of intact plants Dracocephalum jacutense, Lilium pilosiusculum, Artemisia martjanovii, Polygala sibirica were obtained. The method of seed stratification and sterilization for effective seedling development was perfected. The experiment with the use of suspension of graphene oxide (GOG) with different concentrations (0,5; 1; 1,5; 2; 2,5; 3 µg/l) for germination of seeds of the objects was carried out. The percentage of germination after treatment in all concentrations of OH suspension was much lower than in the control variant (control variant - seeds planted on nutrient medium MS without treatment with OH suspension). At the concentration of OH suspension from 2 to 3.5 µg/l, a 10-12% germination of seeds was observed. Selection of optimal type of seedlings explant grown in vitro for aseptic culture initiation and shoot induction of Dracocephalum jacutense, Lilium pilosiusculum, Artemisia martjanovii, Polygala sibirica was performed. The work on induction of Dracocephalum jacutense, Lilium pilosiusculum, Artemisia martjanovii, Polygala sibirica plants in vitro using explants of intact plants was carried out. MS medium with phytohormones NUC, BAP, 2,4-D, kinetin was used as a nutrient medium. The combination of 2,4-D 1 mg/l, NUC 1 mg/l and kinetin 1 mg/l was appropriate to obtain intensive callus formation. The growth dynamics of Polygala sibirica L. culture biomass under the influence of growth regulators was studied. Lilium pilosiusculum microshoots were obtained by induction of adventitious bud emergence by explant tissues. Active initiation of shoot growth on Lilium pilosiusculum bulb scales was obtained with a combination of cytokinin, BAP, and 2,4-D. Initiation and shoot growth were also observed on MS nutrient media with addition of BAP and GC. The optimal composition and concentration of growth regulators for indirect organogenesis of the objects under study from explants of sterile seedlings grown in vitro were tested and selected. Polygala sibirica, Artemisia Martjanovii microshoots were obtained by shoot growth initiation on callus tissue. Micropropagation of Polygala sibirica L. and Artemisia Martjanovii was obtained on MS nutrient medium variants with BAP growth regulator (0.5 µg/l, 1 µg/l). Intensive micropropagation is observed. Experiments on initiation of root growth and root formation of micropropagated Dracocephalum jacutense, Lilium pilosiusculum, Artemisia martjanovii, Polygala sibirica on the variants of nutrient medium MS with growth regulators NUC, IMC with a preliminarily influence on the introduced samples of micropropods by graphene oxide suspension (in concentration 0.5; 1.0; 1.5; 2.0; 2.5; 3.0 µg/L). The study of genetic stability is the penultimate process of microcloning, and this stage can provide convincing evidence of the preservation of genetic monoformism among Dr. jacutense Peschkova regenerate plants and intact plants, seedlings cultivated in vitro, or can reveal low genetic variability caused by different conditions of regenerate plants, hormone concentration and nutrient media. RAPD analysis for detection of genetic variability, is relevant for the study of poorly studied taxonomic groups, among which the model object Dracocephalum jacutense Peschkova. For genetic stability analysis, we isolated and preserved native genomic DNA from 5 dried samples. Genomic DNA was isolated from previously dried samples of Dracocephalum jacutense, using a commercial DNeasy Plant Mini Kit (Qiagen, Germany), according to the protocol and manufacturer's recommendations. Qualitative analysis of isolated genomic DNA was performed using spectrophotometry on a SpectroStar Omega instrument (BMG Labtech). Quantitative analysis (concentration) of isolated genomic DNA was performed by fluorimetry on a Qubit 2.0 instrument (Invitrogen™, USA). The following primers with 5'-3' sequences were chosen for validation of non-specific primers: (Pr2) AATCGGGCTG; (Pr18) AATCGGGCTG. DNA amplification was performed in a final volume of 25 μl. The PCR reaction was performed in a thermocycler (C1000 Bio-Rad, Bio-Rad Laboratories, Inc.) according to the developed protocol. The amplification products were separated by gel electrophoresis on a 1.5% agarose gel for 110 min at 70 V. The gel was visualized in a gel documentation system (GelDoc XR, Bio-Rad Laboratories, Inc.); DNA fragment sizes were assessed by comparison with a standard 3 bp marker ladder (Step100 Long "BiolabMix"). Based on the analysis, it was decided to use the Pr18 primer in further experiments. Using HPLC and HPLC combined with mass spectrometry in the four-step ion separation mode (MS/MS mode), we first studied the polyphenolic composition and compounds of other groups in intact and cultivated Dracocephalum jacutense Peschkova. The above-ground phytomass of plants and, separately, inflorescences, leaves, and stems were used for the analysis. A Shimadzu LC-20 Prominence HPLC high-pressure liquid chromatograph (Shimadzu, Japan) equipped with a UV-detector and a Shodex ODP-40 4E reverse-phase column was used to separate the multicomponent mixtures. The gradient elution program was as follows: 0.0 - 4 min, 100% CH3CN; 4 - 60 min, 100% - 25% CH3CN; 60 - 75 min, 25% - 0% CH3CN; control wash 75 - 120 min 0% CH3CN. All HPLC analysis was performed with a UV-VIS detector SPD-20A (Kanda-Nishikicho 1-chrome, Shimadzu, Chiyoda-ku, Tokyo, Japan) at wavelengths of 230 nm and 330 nm; temperature 40 °C. The injection volume was 1 μl. High precision mass spectrometric data were recorded on an amaZon SL mass spectrometer (manufactured by BRUKER DALTONIKS, Germany) equipped with an ESI source in negative and positive ion mode. MS was used in the scanning range m/z 100 -1700 for MS and MS /MS. The acquisition rate was 1 spectra/s for MS and 2 spectra/s for MS /MS. Data acquisition was controlled by the Windows software for BRUKER DALTONIKS. All experiments, respectively, were repeated three times. Ion separation and detection was performed in an ion trap. The four-step ion separation mode (MS/MS mode) was implemented. Samples of the substances under study were injected into the injector needle. Ionization was performed by electrospray. Each type of D. jacutense showed a significant difference in the composition of polyphenols as well as in the composition of other bioactive compounds. Structural identification of each compound was performed based on their exact mass and MS/MS fragmentation using HPLC-ESI ion trap-MS/MS. A total of 156 biologically active compounds were successfully characterized in D. jacutense extracts based on their exact MS, fragment ions, online database searches, and literature data. All identified compounds together with their molecular formulas, MS/MS data, and their comparative profile for D. jacutense are summarized in a table (in the publication). In plant extracts from the D. jacutense and introduced plant D. jacutense, 105 polyphenolic compounds and 51 compounds of other chemical groups were identified, a total of 156 compounds. Of these, polyphenols in extracts of wild plants accounted for 70.2 % (91 compounds), in extracts of introduced plants - 65 % (52 compounds). The similarity of wild and introduced D. jacutense extracts was noted for 54 compounds belonging to 12 groups of polyphenols and 12 groups of other compounds. Complete similarity in quantity and individual compounds for polyphenols was noted in the flavanones group. Benzenediol, pterocarpane, webonol, phenylpropanoic acid, dihydrochalcone, coumarin, coumarin glucoside, stearidonic and linolenic acids were found in other groups of compounds of both wild and introduced D. jacutense. The difference between extracts of wild and introduced D. jacutense was noted for 103 individual compounds, including 13 groups of polyphenols and 24 groups of other compounds. For the adaptation stage of plants regenerants the following devices were prepared: micro greenhouse with LED-lighting, maintaining a constant temperature, greenhouse with an artificial fog device. For the preparation of soil substrate for plants regenerants we buy peat with neutral acidity "Agrobalt N", acidic peat "Agrobalt B", vermiculite, natural biohumus is ordered from entrepreneurs. Necessary biofungicides and bacterial preparations, fertilizers are purchased. From the soil samples collected during the expedition (in 3-fold replication) from the places of natural growth of the research objects, the pH value was determined by the standard technique of determining soil pH in suspension. Dracocephalum jacutense grows in soils with pH 5.45+0.19, Lilium pilosiusculum - pH 5.9+0.23, Artemisia martjanovii grows in soils with pH 8.19+0.11, Polygala sibirica - pH 7.67+0.14. On the territory of the nursery of the clovergrass family of the Yakutsk Botanical Garden was allocated a plot on which beds are prepared with optimal for Dracocephalum jacutense with reduced acidity, for Polygala sibirica with the addition of limestone. For Lilium pilosiusculum regenerative plants, a site with reduced acidity is prepared; for Artemisia martjanovii regenerative plants, a site with a more alkaline index of the soil mixture is prepared. Stationary route expeditionary works to natural habitats of rare and endangered plant species of Yakutia are organized and carried out for the purpose of inspection, descriptions, collection of samples for works. Expeditionary works on the territory of Khangalassky District of the Republic of Sakha (Yakutia) are fulfilled during the period from June 15 to June 19, 2022. In the vicinity of the village. The following samples of Artemisia Martjanovii and Polygala sibirica were collected for herbarization, for phytochemical analysis, for genetic analysis, and soil samples. Geobotanical descriptions of phytocenoses of natural growth of these rare plant species were made. Intact plants Polygala sibirica and Artemisia Martjanovii were collected for cell-cultural work. Samples of intact plants were kept in temperature, light and humidity controlled conditions of an environmental chamber (MLR-352, Sanyo). Information on the work "Scientists and students of NEFU performed geobotanical works on the territory of Khangalassky district" is placed in the newspaper "Khangalas" (№42(12290) of 28.10.2022. Expeditionary works on the territory of Nyurbinsky district of the Republic of Sakha (Yakutia) were carried out from June 28 to July 07, 2022. In the vicinity of s. Lilium pilosiusculum samples were collected for herbarization, for phytochemical analysis, for genetic analysis, and soil samples in the vicinity of Zharkhan and Nyurba. Geobotanical descriptions of phytocenoses of natural growth of this rare plant species were made. Bulbs of Lilium pilosiusculum were collected for introduction of the plant into culture in vitro. Information "On the results of the expedition of the University" is placed in the newspaper "Nyurba (Ogni Nyurba)" (№40(12013) of 13.10.2022. Expeditionary works on the territory of Nyurbinsky district of the Republic of Sakha (Yakutia) were carried out from July 13 to July 18, 2022. Geobotanical description of phytocenosis of natural habitat Dracocephalum jacutense Peschkova was performed in the vicinity of Sangar settlement with collection of soil samples. We collected plant specimens for herbarization, for phytochemical analysis, for genetic analysis, and intact plants for use as explants for in vitro cell-cultural experiments. Information on Snakehead works is placed in the newspaper "Kobyaisky Vestnik" (№42(9275) of 28.10.2022. Information on expeditions and works on the project "SVFU scientists develop technologies of microclonal propagation of rare and endangered plant species" is presented at https://ulus.media/article/74857 from 13.10.2022. Information on the implementation of the project is available at https://www.s-vfu.ru/universitet/nauka/news/news_detail.php?ELEMENT_ID=193022 and https://www.s-vfu.ru/news/detail.php?SECTION_ID=15&ELEMENT_ID=193067. According to the results of the work 3 manuscripts were prepared, of which one manuscript is accepted for publication in the peer-reviewed Web of Science and Scopus journal (Q1) for 2022, one manuscript is accepted for publication in the peer-reviewed Web of Science and Scopus journal (Q3) for 2023.

 

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Annotation of the results obtained in 2023
When cultivated on MS with the addition of BAP (1 mg/l) from leaf and stem explants from an intact plant, callusogenesis of Dracocephalum jacutense was initiated in vitro under phytorack conditions at 25°C, humidity 60%, lighting intensity 5000 lux with a photoperiod of 18/6. When sterile seedlings of D. jacutense were transplanted to MS with the addition of IBA (1 mg/L), abundant root formation was observed on the 30th day; on MS with the addition of BAP (1 mg/L), development and abundant shoot formation were observed on the 15th day. Stably growing callus cultures of Polygala sibirica cells were obtained when cultured on MS with the addition of 2,4-D (1 mg/l), BAP (1 mg/l), and NAA (1 mg/l). At the same time, the increase in crude weight was greater by 1.9±0.2 times, dry weight by 5.5±0.1 times, than in the MS variant with the addition of 2,4-D and NAA in combination with kinetin. In vitro indirect morphogenesis of P. sibirica was initiated by culturing callus tissue on MS with addition of BAP (1 mg/L). In vitro rhizogenesis was initiated in propagated microplants of P. sibirica when cultured on MS with the addition of NAA (1 mg/l). Intensive in vitro shoot formation in Lilium pilosiusculum is observed when cultivated on MS with the addition of BAP (1 mg/L). When L. pilosiusculum microshoots were transplanted to MS with the addition of TDZ (5 mg/L), shoot formation with the appearance of loose callus mass was observed. When BAP (1 mg/l) and NAA (0.5 mg/l) were added to MS, shoot formation without leaves and development of 5 to 10 roots were observed. In the variant of cultivation on MS with the addition of BAP (1 mg/l) and NAA (1 mg/l), root system development with the formation of multiple young shoots in the amount of 10-20 pcs. was observed in microshoots of Lilia currevata. When L. pilosiusculum microplants are cultivated on MS with the addition of NAA (0.5 mg/l), initiation of root formation is observed, and thin and shortened roots are formed. Intensive root formation is observed when L. pilosiusculum microplants are cultivated on MS with the addition of NAA (1 mg/l). When microplants are cultivated on MS with the addition of IBA (1 mg/l), intensive root formation, shoot formation (up to 20–30 pcs.) and leaf growth are observed. The effect of a graphene oxide suspension on the development of microshoots of L. pilosiusculum is studied. At the same time, microshoots obtained on MS with the addition of IBA (1 mg/l) were transplanted into a nutrient medium with the same composition, but with the addition of a suspension of graphene oxide (0.5; 1; 1.5; 2; 2.5; 3 mg/l). With further cultivation of transplanted microshoots in phytorack conditions, the development of shoots and leaves is observed. Rooted microplants of L. pilosiusculum were transplanted into soil substrates to study the first stage of adaptation and kept in installed mini-greenhouses under phytostorage conditions with a photoperiod of 16/8. The highest frequency of in vitro induction of Artemisia martjanovii microshoot formation was observed when callus tissue pieces were cultured on MS supplemented with BAP (1 mg/L). The in vitro initiation of root formation in A. martjanovii microshoots was studied when cultured on MS with the addition of IBA (0.5/1 mg/L) and NUC (0.5/1 mg/L). Intensive root formation was observed in microshoots of A. martjanovii when cultivated on MS with the addition of NAA (1 mg/l). Growth of A. martjanovii microplants with rooting was scaled. Microplants have root length from 3 to 12 cm. Transplantation of rooted microplants of A. martjanovii into soil substrate was performed; the transplanted microplants are kept in installed mini-greenhouses under phytostorage conditions with a photoperiod of 16/8. A comparative analysis of genetic stability of in vitro samples of sterile seedlings, wild and introduced plants of Dracocephalum jacutense was carried out. The similarity coefficient of 0.666 (KJ) between samples of sterile seedlings and wild-growing Yakutian snakehead was revealed. The results of comparative analysis of genetic stability of microgrowths of Lilium pilosiusculum and Artemisia martjanovii on the basis of RAPD-analysis were obtained in comparison with wild plants and seedlings cultivated in vitro. According to the analysis data, a stable genetic stability between the obtained Lilium pilosiusculum microplants was revealed with a similarity coefficient value of 1 (KJ). Based on the results of a comparative analysis of the similarity coefficient for samples of Artemisia martjanovii, differences in DNA fragments were revealed in the variants of samples of sterile seedlings and microplants obtained on MS with the addition of NAA. Among the microplant samples of A. martjanovii, the highest value of 1 (KJ) was found among the microplant samples obtained on MS with the addition of NAA and BAP. Extracts of aboveground phytomass of Polygala sibirica were analyzed using HPLC-MS/MS with ion trap. 74 individual compounds have been tentatively identified, including 43 polyphenolic compounds and 31 compounds from other chemical groups. Based on the results obtained, we can conclude that Polygala sibirica from the Central Yakut population has very great potential for use in medicine. Of interest is the study of the complex of biologically active compounds of the polyphenol group, first discovered in the Polygalaceae family: Isoformononetin; Jaceosidine; Syringetin; Apigenin-7-O-glucoside; Herbacetin; Myricetin; Quercetin-3-O-hexoside; Afzelechin; Hesperitin; Eriocitrin; Delphinidin 3-O-glucoside; Malvidin 3-O-glucoside, etc. Phytochemical analysis of sterile Artemisia martjanovii seedlings obtained in vitro was performed using UPLC-ESI-MS. It was found that at least 4 polar compounds were present in the A. martjanovii extracts, which eluted from the chromatographic column within 1-4 minutes of analysis. The detected compounds are derivatives (phenylpropanoid esters) of cinnamic acid and chlorogenic acid. Semi-quantitative analysis of the major esters of cinnamic acid in extracts of samples of sterile A. martjanovii seedlings was carried out. Using UPLC-ESI-MS, structural identification of compounds was carried out in samples of roots, bulb scales and microplants of Lilium pilosiusculum. It was established that L. pilosiusculum extracts contain at least 5 polar compounds, which elute from the chromatographic column within 0.1–4 minutes of analysis. The detected compounds are derivatives of sucrose, incl. phenylpropanoid esters of sucrose. Semi-quantitative analysis of major sucrose esters in extracts of L. pilosiusculum samples was carried out. Rooted microplants of the research objects, after undergoing 2-3 weeks of adaptation in the soil substrate under mini-greenhouse conditions in the laboratory, were transferred for research in the botanical garden. For these works, a mini-greenhouse was built in the botanical garden. Regenerated plants are cared for with watering and fertilizing. The morphometric parameters of the observed regenerated plants are recorded weekly. The regenerated plants were transplanted into open ground; for the winter, the plantings were covered with lutrasil and mown grass. Expeditions to collect plant material for the project tasks were organized and carried out on the territory of Khangalassky Nyurba and Kobyaisky districts of RS(Ya). Information about the work performed with reference to the RSF grant was published in Ulus Media (09.17.2023) https://xn--80aaaaaqpp6as1cq2a.xn--p1ai/polyn-martyanova-istod-sibirskij-uchyonye-izuchayut-rasteniya-elanki /, newspaper “Nyurba (Lights of Nyurba)” (09/24/2023, No. 36(12060), p. 8), “Khangalas” (09.29.2023, no. 38(12384), p. 5), newspaper “Kobyaisky Herald” (20.10.2023, No. 41 (9325), P. 6). With the results of the study, members of the research team took part in 7 scientific and practical conferences at the regional, all-Russian and international levels with oral online reports, with correspondence and online participation. Based on the results of the work, 3 scientific articles were published in Russian and foreign journals, reviewed in the WoS and Scopus databases.

 

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