Species of Gentiana section Cruciata are highly similar in their morphological and anatomical characteristics, which complicates their taxonomic delimitation and phylogenetic interpretation. Molecular markers increasingly complement traditional morphology-based approaches, and the intergenic spacer of the 5S rRNA gene cluster (5S rDNA IGS) represents a promising candidate for such analyses. In this study, genetic variation among 10 species within section Cruciata was investigated using sequences of the 5S rDNA IGS region, and its potential utility for DNA barcoding and phylogenetic analysis was evaluated. Analysis of 38 cloned PCR products revealed a structural organization of the IGS region typical of other Gentiana species, including conserved regulatory elements, and a high level of nucleotide sequence similarity (91.2-99.5%). Interspecific polymorphism was comparable to intragenomic variation. Sequence alignment and clustering distinguished two groups of species that differed in their IGS nucleotide sequences. The resulting taxonomic grouping is generally consistent with previously reported phylogenetic relationships inferred from other molecular markers. The high similarity of IGS sequences among species of section Cruciata suggests their relatively recent divergence. Notably, G. cruciata, despite its migration from Asia to Europe, retained IGS region characteristics typical of the section.

The aim was to investigate biomass extracts of U. victoris strain UV-2 tissue culture for the content of lectins, to study their general characteristics and to optimize the method of ultrasonic extraction to obtain a soluble form of the lectin. Methods. Tissue culture method, direct hemagglutination test for detection of lectin activity (LA), determination of lectin carbohydrate specificity, ultrasonic extraction method. The results. Lectin activity was detected in the soluble (supernatant) and insoluble (sediment) fractions of the extract of biomass U. victoris tissue culture. In the soluble fraction LA was significantly less compared to that in the sediment, which indicated the presence of the membrane-bound form of the lectin. LA was characterized by pronounced species specificity: the reaction with mouse erythrocytes was the most intense. The study of carbohydrate specificity revealed a weak affinity for monosugars (galactose and galactosamine) and pronounced suppression of LA in the case of polysaccharides - hyaluronic acid, heparin and mucin. The possibilities of the ultrasound extraction method for separation the detected lectin from the the cell surface in the sediment fraction were investigated and the extraction procedure was optimized. The transition of LA into the soluble phase under the influence of ultrasound depended on the following parameters: the concentration and volume of the extract, as well as the time of exposure to ultrasound. It was established that the LA of the soluble phase doubled compared to the initial one with an extract concentration of 50 mg/ml and a volume of 20 ml under the action of ultrasound 40 kHz/70 W in the interval of 15-45 minutes. Longer ultrasound treatment had a negative effect on LA. Conclusions. Lectin activity of U. victoris tissue culture biomass extracts was discovered for the first time, which can become a promising source of lectin with a wide spectrum of pharmacological properties. The general characteristics of the lectin were given, and its species and carbohydrate specificity was revealed. The lectin was generally characterized and its species and carbohydrate specificity were established. Possibilities of ultrasound extraction were used and the procedure was optimized to obtain a soluble form of the lectin, necessary for its further fundamental and practical research.

У період з 26 по 29 вересня 2023 р. у м. Києві було проведено XVIII Міжнародну наукову конференцію «Фактори експериментальної еволюції організмів», присвячену 50-річчю заснування Інституту молекулярної біології і генетики НАН України (ІМБГ), 70-річчю відкриття подвійної спіралі ДНК та 180-річчю від дня народження Вольтера Флеммінга, а також асоційовані з конференцією VІІІ Симпозіум «Геном рослин» та Конференцію молодих вчених ІМБГ НАН України. Співорганізаторами цих наукових заходів були Українське товариство генетиків і селекціонерів ім. М.І. Вавилова (УТГіС), Інститут молекулярної біології і генетики НАН України (ІМБГ), ДУ «Інститут харчової біотехнології та геноміки НАН України» та Національний ботанічний сад ім. М.М. Гришка НАН України (НБС).

Aim.To study molecular genetic aspects of stress tolerance in the extremophile plant D. antar ctica by exploring ICE transcription factors involved in cold stress response, regulation of stomatal development and flowering time.Methods.The DaICE genes were assembled from SRA reads homologous to orthologous genes used as a reference.The predicted genes and proteins were analyzed with bioinformatics methods.Gene expression was analyzed using the RNA-seq data.Results.Two variants of the DaICE1 gene were found: a variant that encodes a full-length protein and a variant with a large deletion, which led to the loss of the N-terminus and part of the bHLH domain in the protein product.Two variants of CDS differed by four single-nucleotide polymorphisms, three of which were synonymous, with more dissimilar promoter sequences were found for the DaICE2.The organization of promoters and the intronexon structure of the genes were studied.For the predicted TF proteins, physico-chemical properties were determined, nuclear localization and the possibility of post-translational modifications were shown, phylogenetic analysis was carried out, conservative motifs were found, 3D structure was predicted.Gene expression was detected in most or all plant tissues and increased expression of DaICE1 variants was shown in the plants from natural conditions compared to laboratory ones.Conclusions.DaICE1 and DaICE2 are the candidate regulators of the CBF gene expression under cold stress in D. antarctica.

Keywords: Deschampsia antarctica E. Desv., plant tissue culture, phenolic compounds, flavonoids, tricin

Main objective of research to study the D. antarctica extracts antiviral activity which was grown in vitro and propagated by cloning. The D. antarctica aqueous ethanolic extracts was tested on in vitro models of MDСK – Madin-Darby Canine Kidney cells and PEK – Porcine Embryonic Kidney cells and influenza virus, А/FM/1/47(H1N1) strain and transmissible gastroenteritis virus – porcine coronavirus (TGEV). The antiviral activity of D. antarctica plant extracts (G/D9-1 genotype) on experimental models of influenza viruses and Coronavirus TGEV in vitro was conducted. D. antarctica plant extracts high antiviral activity on influenza viruses and Coronavirus TGEV in vitro was shown.

The intergenic spacer (IGS) of 5S ribosomal RNA genes (5S rDNA) (that are present in the genome of all living organisms) is characterized by a high variability, due to which it is a convenient and widely used object for clarifying the issues of genome evolution, population genetics, systematics, etc. The aim of this work was to study the IGS of 5S rDNA of the Antarctic pearlwort Colobanthus quitensis. The nucleotide sequence was determined by the methods of molecular genetic analysis, and molecular organization of the C. quitensis 5S rDNA IGS was studied. It was demonstrated that it contains the regulatory elements typical for other vascular plants. The existence of at least two classes of 5S rDNA repeats in the individual genome (that differ significantly in the length and nucleotide sequence of IGS) was established. In addition, based on differences in the length and nucleotide sequence, two subclasses of repeats with a long IGS and three subclasses with a short IGS were identified. A comparison of 5S rDNA IGS sequences in C. quitensis and Silene latifolia Poir. (a member of another section of the Caryophyllaceae family) demonstrated significant differences in the structure of a spacer region of 5S rRNA genes (except for its parts containing regulatory elements). In general, data obtained indicate a significant level of intragenomic polymorphism of C. quitensis 5S rDNA IGS.

This review presents the results of multi-year efforts for the development and integral investigation of the collection of Deschampsia antarctica e. Desv. plants originated from the maritime Antarctic. To create the collection, we have optimized the procedures and conditions for germination of seeds and generation of aseptic plants in vitro together with their subsequent clonal propagation, long-term in vitro culture, and acclimatization of plants to pot culture, and finally produced fertile plants able to set viable seeds. The collection of plants cultivated in vitro and in pots is consisted of clones of 22 different genotypes originated from eight island populations from the Argentine Islands - Kyiv Peninsula region. Some of these clones have been successfully cultured for more than ten years and were found to be genetically stable over this period. The collection was shown to be heterogeneous in a number of variables. The morphological and anatomical characteristics of the plants, as well as the data of karyological, molecular cytogenetic, molecular genetics, and biochemical analysis are presented. The created collection of D. antarctica plants can provide a valuable plant material with well-studied characteristics grown under controlled conditions to perform experimental research on the mechanisms for plant adaptation and resistance to various abiotic factors of Antarctic environment. Furthermore, development of the collection and its comprehensive characterization form the basis for further biotechnological developments focused on the use of this Antarctic plant as a source of biologically active substances, which can potentially be used for therapeutic and prophylactic purposes.

Keywords: Secale cereale L., Dasypyrum species, Agropyron cristatum L., subtelomeres, tandem repeats, fluorescence in situ hybridization

У період 3-5 жовтня 2022 року за участі організаторів -Українського товариства генетиків і селекціонерів ім.М.І.Вавилова (УТГіС), Інституту молекулярної біології і генетики НАН України, Національного дендрологічного парку «Софіївка» НАН України та Уманського національного університету садівництва -у форматі Zoom-конференції

Aim.To characterize a new hormone-independent strain К-27М of R. serpentina tissue culture.Methods.Plant tissue culture, biochemical and cytological analysis, statistical methods.Results.The strain is maintained on the specially designed simple-composition hormone-free 10C medium.The maximum biomass yield was at day 69 of subculture (693.5 g/l of live biomass, 43.0 g/l of dry biomass), the weight of cell biomass increased 15-18 times over a subculture period.The maximum content of indole alkaloids was observed from 88 to 108 days of subculture.The dry biomass contained 4.0% of total indole alkaloids, 1.64% of ajmaline-like alkaloids, 0.789% of ajmaline, 0.337% of vomilenine, and 0.006% of each yohimbine and reserpine.The strain is a heterogeneous mixoploid cell population with a modal class of 22 to 33 chromosomes (2n = 22), which characterized by significant cell and nuclear polymorphism.Conclusions.The K-27M strain of R. serpentina cultured tissue is a prospective producer of indole alkaloids.

Tissue culture of Rauwolfia serpentina is an alternative source of more than 20 indole alkaloids that have antiarrhythmic, hypotensive, psychotropic, and anti-inflammatory effects. The paper was aimed at determining the hypotensive activity of five fractions (containing different combinations of alkaloids) of the extracts from the biomass of R. serpentina tissue culture (K-27M line). To achieve this aim the following methods were used: in vitro plant tissue culture, HPLC analysis of indole alkaloids, assessment of the effects of fractions on the smooth muscle of the isolated rat thoracic aorta in vitro and on the blood pressure after intravenous administration in vivo. The five fractions of the extract from cell biomass of R. serpentina (K-27M line) were isolated from dried or live biomass and differed in both qualitative and quantitative compositions. The direction of action and the magnitude of the effect were found to depend on the percentage composition of alkaloids in the fraction. It was shown that fraction 1 containing ajmaline and acetylajmaline (total alkaloids content of 2.2%) and fraction 2 containing ajmaline, raucaffricine, and acetylajmaline (total alkaloids content of 6.4%) exerted vasoconstrictor effects on thoracic aorta muscles. Fraction 4 containing vomilenine, acetylajmaline, methylajmalicine, ajmalicine, and rauffloridine (total alkaloids content of 65%) and fraction 5 containing mainly acetylajmaline with lesser amounts of vomilenine, methylajmalicine, and ajmalicine (total alkaloids content of 47.4%) were found to have a relaxing activity in vitro. Intravenous administration of fractions 4 and 5 resulted in a short-time (15–30 s) hypotensia, with blood pressure drop by 25% to 30%. All the 5 fractions of the extracts from Rauwolfia serpentina K-27M line demonstrated either vasoconstrictor or relaxing effects depending on the alkaloids type and content.

The organization of promoters of DREB2B TF ortholog genes involved in the response to abiotic stresses was studied in D. antarctica E. Desv. extremophile plant and 12 more grass species with different cold and drought resistance. The evolutionary distances between the sequences of promoters were 0.621 on average, those between coding sequences (including introns) were 0.442, and the values of nucleotide diversity (π) for these regions were 0.410 and 0.274, respectively. Clustering of sequences corresponded in general to the accepted systematic division of the Poaceae family into subfamilies. Fifty-four cis -elements involved in the response to abiotic and biotic stresses, light, hormones (particularly, abscisic acid, auxin, methyl jasmonate, ethylene, gibberellin, and salicylic acid), and tissue-specific cis -elements were identified. Most of the cis -elements were associated with the response to abiotic stress that is consistent with data on DREB2B TF functions. Except for single peculiarities, representatives of different grass subfamilies and D. antarctica were similar in the total set of cis -elements in the DREB2B promoter that indicates the similarity of the regulation of expression of this gene and its potential functions in the response to stress in the studied species.

A laboratory prototype of enzyme biosensor based on pH-sensitive field-effect transistors has been created to determine the total content of indole alkaloids in Rauwolfia serpentina tissue culture. Enzyme acetylcholinesterase was immobilized on the surface of pH-sensitive field-effect transistors using glutaraldehyde for covalent crosslinking with bovine serum albumin. The biosensor was characterized by high sensitivity to the total content of indole alkaloids (minimum limit of determination 0.5 mu g/ml in the juice obtained from Rauwolfia serpentina tissue culture). The linear range of biosensor determination was from 2 to 15 mu g/ml of the total content of indole alkaloids. The analysis of indole alkaloids using a biosensor is easy and fast; unlike traditional methods, no expensive equipment and special sample preparation are required for analysis. The created biosensor could be further used to control the total content of indole alkaloids in modern biotechnological and pharmaceutical processes of the production of drugs and dietary supplements.

We used inter-simple sequence repeats and inter-retrotransposon amplified polymorphism markers to assess genetic variation in Deschampsia antarctica populations in the context of its uneven distribution in the northern and central maritime Antarctic. Genetic diversity and population structure and differentiation were assessed in nine populations from geographically isolated population groups of D. antarctica, including the South Shetland Islands, Anvers Island and the Argentine Islands regions. In total, 265 amplified DNA fragments were scored, of which 220 (83.0%) were polymorphic. The total sample showed low genetic diversity (unbiased expected heterozygosity = 0.081 and Shannon diversity index = 0.115) and high population differentiation (molecular variance among populations = 0.659). We also found a trend toward a decrease in genetic diversity and an increase in population differentiation toward the southern edge of the species range. Principal coordinates analysis of polymerase chain reaction data and Bayesian population structure analysis showed three main clusters, which included plants originating from three spatially isolated population groups. The unweighted pair group method with arithmetic mean clustering of populations based on Nei’s genetic distances was mainly in agreement with this pattern. Testing of isolation by distance using the Mantel test demonstrated a significant correlation between genetic and ln-transformed geographical distance (r = 0.703). The data obtained indicate that the geographically isolated D. antarctica populations in the maritime Antarctic might form genetic clusters within the total range.

Aim. The aim of the work was to determine the conditions for development of in vitro culture, induction of callus formation, and long-term tissue culture of Zingeria biebersteiniana (Claus) P. Smirn. Methods. In vitro clonal propagation, tissue culture techniques. Results. The seed germination rate was found to increase significantly after long-term cold stratification. The protocol for seed sterilization was developed, which yielded 57.3% of aseptic plants. Collections of in vitro and pot cultured plants were created. Experiments on the adaptation of in vitro propagated plants to pot culture conditions revealed a high level of their survival. The optimal medium for in vitro clonal propagation was MS, supplemented with 0.1 mg/L NAA; while the most effective media for induction of callus formation and for long-term tissue culture was B5 supplemented with 2 mg/L 2,4-D and 0.1 mg/L BAP. Conclusions. The protocols and conditions for seed germination, in vitro clonal propagation, induction of callus formation, as well as long-term tissue culture of Z. biebersteiniana have been developed. The developed techniques of in vitro culture can be used for conservation and restoration of genetic diversity of the species, as well as to obtain sufficient plant material for further studies.

Досліджено склад і антиоксидантні властивості екстрактів, вилучених із рослин Deschampsia antarc tica É. Desv., зібраних у регіоні Аргентинських островів — півострова Київ (Морська Антарктика).. Склад екстрактів вивчено методами високоефективної рідинної хроматографії та мас-спектрометрії з матрично- активованою лазерною десорбцією/іонізацією; антиоксидантні властивості досліджено методом Фоліна— Чокальтеу та DPPH-тесту. Встановлено, що основними класами речовин, які містяться в екстрактах, є поліфенольні сполуки, зокрема флавоноїди, гідроксикоричні та гідроксибензойні кислоти. Підтверджено, що флавоноїди переважно представлені глікозидами лютеоліну; склад та кількість фенольних сполук залежить від генотипу рослини, місця походження та умов навколишнього середовища. Виявлено, що загальний вміст поліфенолів у досліджених екстрактах знаходиться в діапазоні 4, 33—10, 93 мг/г сирої сировини; найбільшу кількість активних речовин містять екстракти, вилучені з рослин острова Україна, а найменшу — з острова Дарбу. Результати антиоксидантних досліджень показали, що екстракти виявляють високу антирадикальну активність і здатні за 60 хв інгібувати понад 50 % DPPH радикалів. Одержані дані щодо складу поліфенолів у рослинах Deschampsia antarctica É. Desv. Можуть слугувати маркером для моніторингу кліматичних змін.

Раувольфія зміїна (Rauwolfia serpentina Benth.) – тропічна чагарникова рослина, яка накопичує понад 50 різних алкалоїдів, що мають антиаритмічну та гiпотензивну дiю. Розробка в Інституті молекулярної біології і генетики НАН України технології одержання біомаси культури тканин Rauwolfia serpentina нaдає можливість розглядати її як перспективну рослинну сировину для виробництва лікарських препаратів і біологічно активних добавок. Однак застосування їх у клініці можливе лише після всебічних випробувань, у першу чергу вивчення гострої та підгострої токсичності. Мета дослідження – вивчити рівень гострої та підгострої токсичності екстракту біомаси високопродуктивної клітинної лінії К-27М Rauwolfia serpentina. Дослідження виконували на білих нелінійних мишах масою 25–35 г. Гостру токсичність екстракту вивчали шляхом внутрішньошлункового введення розчину тваринам, об’єм якого не перевищував 0,5 мл. Результати розраховували за методом В. Б. Прозоровського через 14 діб після одноразового введення. Підгостру токсичність проводили на трьох групах мишей з використанням системи рандомних номерів. Тварини групи 1 і 2 отримували 15 мг/кг та 45 мг/кг екстракту внутрішньошлунково у вигляді водного розчину, тварини групи 3 (контроль) отримували дистильовану воду. При вивченні гострої токсичності після введення 501–1000 мг/кг екстракту біомаси культури тканин раувольфії зміїної мишам спостерігали підвищену активність, судоми, каталепсію та загибель протягом 1 год. Клінічна картина гострого отруєння супроводжувалася загальмованістю, грумінгом, підвищенням тонусу хвоста, посиленням дихання, епізодами агресії. У разі введенні доз ≤ 398 мг/кг загибелі тварин не відбувалося. У мишей, які отримували 250–398 мг/кг екстракту, реєстрували загальмованість, грумінг, тремор протягом 30 хв з подальшим збільшенням рухової активності. Через 2 год тварини поверталися до нормального фізіологічного стану. У разі застосування екстракту в дозах 250 мг/кг і менше не виявили відхилень від фізіологічної норми. Оскільки при вивченні підгострої токсичності екстракту спостерігали летальність та/або виражені симптоми інтоксикації (атонія кишківника, метеоризм, кровонаповнення паренхіматозних органів), то згодом дози було знижено в 2 рази: у групі 1 з 15 мг/кг до 7,5 мг/кг; а в групі 2 з 45 мг/кг до 22,5 мг/кг. Надалі летальність тварин не виявляли. У тварин спостерігали зниження середньої маси тіла, виражене здуття живота, млявість, зниження рухової активності. У крові піддослідних мишей не відзначено достовірних змін кількості лейкоцитів, еритроцитів і тромбоцитів, однак виявлено значне збільшення відсотка нейтрофілів і еозинофілів у тварин групи 1 та збільшення відсотка базофілів у тварин групи 2. Встановлено, що величина LD50 екстракту раувольфії зміїної штаму К-27М становить (450 ± 35) мг/кг, що свідчить про його помірну токсичність (ІІІ клас). Результати гематологічного дослідження та біохімічних показників сироватки крові мишей можуть свідчити про запальні процеси в кишківнику та алергізуючу дію екстракту у вивчених дозах.

A laboratory prototype of an enzyme biosensor based on pH‑sensitive field-effect transistors has been developed to determine the total content of indole alkaloids in Rauwolfia serpentina Benth. Ex Kurz tissue culture. The biosensor was characterized by high sensitivity to th A laboratory prototype of an enzyme biosensor based on pH‑sensitive field effect transistors has been developed to determine the total content of indole alkaloids in Rauwolfia serpentina Benth. Ex Kurz tissue culture. The biosensor was characterized by high sensitivity to the total content of indole alkaloids (minimum limit of determination – 0.5 μg/ml of the total content of indole alkaloids contained in the juice obtained from tissue culture of Rauwolfia serpentina). The linear range of biosensor determination of the analyte was from 2 to 15 μg / ml of the total content of indole alkaloids. Analysis of indole alkaloids using a biosensor is simple and fast and does not require expensive equipment and special sample preparation for analysis, unlike traditional methods. The created biosensor can be further used to control the total content of indole alkaloids in modern biotechnological and pharmaceutical processes for the production of drugs and biologically active additives. e total content of indole alkaloids (minimum limit of determination – 0.5 μg/ml of the total content of indole alkaloids contained in the juice obtained from tissue culture of Rauwolfia serpentina). The linear range of biosensor determination of the analyte was from 2 to 15 μg / ml of the total content of indole alkaloids. Analysis of indole alkaloids using a biosensor is simple and fast and does not require expensive equipment and special sample preparation for analysis, unlike traditional methods. The created biosensor can be further used to control the total content of indole alkaloids in modern biotechnological and pharmaceutical processes for the production of drugs and biologically active additives.

Design of novel herbal drugs with a wide range of activity is an important object of pharmacology. Herbal remedies having antiarrhythmic, cardiotonic and vasodilatory effects are useful for treatment of heart arrhythmias. Vasodilating drugs are prescribed for medication of hypertension and migraine. Also they are a part of comprehensive treatment of various diseases such as disturbed peripheral blood circulation and atherosclerosis of arteries of extremities as well as problems with urination and potency. Search for novel herbal preparations with vasorelaxant activity in our view is a promising matter of current interest. This work is aiming in study of vasodilatory activity of methanol extract of strain K-27 tissue culture of Rauwolfia serpentina. Evaluation of vasodilatory activity extract of high-productive strain K-27 tissue culture of Rauwolfia serpentina has been carried out by the method of auxotonic mechanography of vascular smooth muscles. Strength and frequency of spontaneous contractions of portal vein as well as relaxation of aortic smooth muscles activated with phenylephrine were evaluation indicators of extract activity. In order to estimate working concentration of extract of strain K-27 tissue culture of Rauwolfia serpentina we have studied vascular activity at sequentially increasing concentrations of extract solution on the portal vein. Overlay of sequence of growing concentrations exhibited decrease in basal level of venous tone and dose-dependent suppression of phasic contractions with complete inhibition of spontaneous activity of portal vein at 1.44 mg/ml concentration and uprise of toxicity at higher concentrations. It has been shown dose-dependent relaxation of vascular preparations followed by development of α-adrenoceptor blocking effect and loss of sensibility to phenylephrine at the end of experiment. After discontinuing exposure with a substance the activating effect of phenylephrine remained not renewed during 30-50 minutes, at the same time contractile activity of aorta on the response to the other type activation (60 mM K+) took place. It was found that at the studied range of concentrations: 0.0288 μg/ml–28.8 μg/ml the extract of strain K-27 tissue culture of Rauwolfia serpentina has distinct vasodilatory effect. Also it was discovered α-adrenoceptor blocking effect of that extract, so it can find wide application in therapy as vasorelaxant and α-blocker, for instance in treatment of prostate diseases.