Murashige-Skoog Medium Research Articles

Клональное микроразмножение сортов земляники садовой (Fragaria х ananassa Duch.) нейтральнодневного типа плодоношения

Abstract. The article presents the results of the evaluation of the efficiency of clonal propagation of commercially promising day-neutral varieties of garden strawberry. Day-neutral strawberry varieties usually form fewer rosettes on a plant during the growing season than regular varieties, and therefore vegetative propagation is ineffective for these varieties. The aim of the study is to increase the efficiency of propagation of day-neutral varieties of garden strawberry (Fragaria x ananassa Duch.), characterized by weak runnerforming ability, by optimizing the process of microclonal propagation in vitro and subsequent adaptation of regenerated plants to ex vitro conditions. The article indicates the features of microclonal propagation technology of neutral-day strawberry varieties Vivara, Sweet Eve and Evis Delight. The studies were conducted in the laboratory of the Federal State Budget Scientific Institution NCFSСHVW in 2022-2023. MurashigeSkoog (MS) agar medium with the addition of plant growth regulator 6-BAP was used as the main medium (at the introduction stage – 6-BAP – 0.2 mg/l, at the micropropagation stage – 0.75 mg/l, hormone-free MS medium – control). As a result of the studies, it was found that the variety of garden strawberry Sweet Eve is characterized by a higher shoot-forming ability. At a 6-BAP concentration of 0.75 mg/l in the medium, an average of 10.5 new shoots are formed from one shoot in the third passage. Evis Delight variety showed a low shoot-forming capacity of up to 3.8 shoots/exp. After the fourth passage, even on a hormone-free medium, Sweet Eve variety showed an aftereffect of 6-BAP, with the formation of an average of 8.8 shoots from one explant. Such a strong aftereffect of 6-BAP was not noted in Vivara and Eve’s Delight varieties. Rooting began after the fifth passage on a hormone-free medium. The root formation efficiency was 95- 100%. At this stage, longer shoots up to 5.9 cm and roots up to 4.8 cm are formed in Vivara variety. Evis Delight variety stood out in terms of the number of leaves (an average of 9.1 pcs.) and roots (7.5 pcs.). The use of the biopreparation Agrinos 1 at the adaptation stage had a positive effect on the formation of the vegetative mass and root system of strawberry plants. The application of the preparation at a concentration of 5 ml/l promotes the growth of the root system, the length of which is 1.6-2.2 times greater than the length of the roots in the control variant. The number of leaves increases by 1.1-1.3 times, and the average height of the bushes is 1.5-1.7 times greater than the control values. Key words: clonal micropropagation, day-neutral strawberry, 6-benzylaminopurine, multiplication factor, microshoots

Activity of gemmogenesis in regenerated plants of hazelnut cv. ‘Akademyk Yablokov’ and antibiotic therapy for elimination of bacterial contamination in vitro

Background. The problem of obtaining certified planting material with biotechnological methods is important for expanding commercial and homestead plantations of hazelnut cultivars in Belarus. Materials and methods. Regenerated plants of cv. ‘Akademyk Yablokov’, representing the genus Corylus L., were a model object for studying in vitro morphogenesis and effectiveness of antibiotics against bacterial contamination, so that a protocol could be developed to obtain healthy planting material of hazelnut cultivars. The plants produced during this study were included in the duplicate ex situ collection of nut crops preserved in vitro in the active growth state. Results. Single exposure to the antibiotic kanamycin monosulfate at a concentration of 100 mg/L during antibiotic therapy in the stage of in vitro micropropagation eliminated bacterial infection in 83.3% of regenerated plants, and twofold exposure in 100%. Further cultivation revealed its phytotoxic aftereffect manifested in the form of necrosis on most of the regenerated plants and a decrease in the activity of gemmogenesis and growth. Neither single nor twofold exposure to cefotaxime sodium salt at a concentration of 90 mg/L caused elimination of bacterial infection, but gemmogenesis and regenerated plant growth retained their activity during subsequent cultivation on antibiotic-free media. The best development parameters were observed on a modified Murashige–Skoog medium with 6 mg/L 6-BA, 0.01 mg/L IBA, and 0.1 mg/L GA3 (average number of shoots: 2.2; number of microcuttings: 2.3), and a modified DKW medium with 6 mg/L 6-BA, 0.01 mg/L IBA, and 0.1 mg/L GA3 (average number of shoots: 2.05; microcuttings: 2.9). The use of zeatin as a cytokinin to stimulate adventitious morphogenesis or activate the growth of axillary meristems at a concentration of 5 or 6 mg/L was not as effective as 6-BA.

Efficient Production of Some Bioactive Depsides and Simple Phenolic Acids by Microshoots of Aronia × Prunifolia (Purple Aronia) Agitated Cultures as the Result of Feeding Strategy with Four Different Biogenetic Precursors

A precursor feeding strategy was used for the first time in agitated microshoot cultures of Aronia × prunifolia. This strategy involved the addition of biogenetic precursors of simple phenolic acids (phenylalanine, cinnamic acid, and benzoic acid) and depsides (caffeic acid) into the culture media, with an assessment of its effect on the production of these bioactive compounds. The in vitro cultures were maintained in Murashige–Skoog medium (1 mg/L BAP and 1 mg/L NAA). Precursors at five concentrations (0.1, 0.5, 1.0, 5.0, and 10.0 mmol/L) were fed into the medium at the time of culture initiation (point “0”) and independently on the 10th day of growth cycles. The contents of 23 compounds were determined in methanolic extracts of biomass collected after 20 days of growth cycles using an HPLC method. All extracts contained the same four depsides (chlorogenic, neochlorogenic, rosmarinic, and cryptochlorogenic acids) and the same four simple phenolic acids (protocatechuic, vanillic, caffeic, and syringic acids). Chlorogenic and neochlorogenic acids were the predominant compounds in all extracts (max. 388.39 and 263.54 mg/100 g d.w.). The maximal total contents of all compounds were confirmed after feeding with cinnamic acid (5 mmol/L, point “0”) and caffeic acid (10 mmol/L, point “0”), which caused a 2.68-fold and 2.49-fold increase in the contents of the estimated compounds vs. control cultures (603.03 and 558.48 mg/100 g d.w., respectively). The obtained results documented the efficacy of the precursor feeding strategy in enhancing the production of bioactive compounds in agitated cultures of A. × prunifolia and suggest a potential practical application value.

In vitro germination and seedling formation of Plantago tomentosa Lam. (Plantaginaceae): influence of concentrations of the MS medium

Plantago tomentosa Lam. (Plantaginaceae) is an herbaceous plant native to Brazil. It is widely used in folk medicine. The species has potential for use in the pharmaceutical and food industry due to its possible bioactive properties and the presence of mucilage in the seeds. The objective of this work was to investigate the influence of different concentrations of Murashige-Skoog (MS) medium on germination and seedling formation in vitro of P. tomentosa. The seeds were collected in Lajeado, disinfected, and inoculated in bottles with MS medium added to 7 g L-1 of agar and 30 g L-1 of sucrose, in four treatments: 25%, 50%, 75%, and 100% of the medium concentration. The experimental design was completely randomized, consisting of 17 replications of ten seeds for each treatment. The evaluation of germination and formed seeds was carried out every two days to determine the variables of germination percentage (GP), mean germination time (MGT), germination speed index (GSI), seedling formation percentage (SFP), mean seedling formation time (MSFT) and seedling formation speed index (SFSI). No significant difference was found between treatments for all the assessed variables. It is concluded that the use of a concentration of 25% of the MS medium is viable, as it promoted a high percentage of germination and seedling formation and in a time interval similar to that of the other concentrations, with the advantage of using fewer reagents.

Induction of Somatic Embryogenesis of Pontianak Siamese Orange Cotyledon Cultures on Murashige Skoog Media with the Addition of 2,4-D and Kinetin

Conventional citrus propagation has weaknesses, one of which is susceptible to CVPD(Citrus Vein Phloem Degeneration) and other diseases which are the main cause of the decline in population and production of pontianak siam orange plants in West Kalimantan. One alternative to in vitro propagation of citrus seedlings is the induction of somatic embryogenesis. The purpose of the study was to obtain the best combination of 2,4-D and kinetin concentrations for callus induction and somatic embryo induction from Pontianak siam orange cotyledon explants, observing the developmental phase (stage) of embryos formed. The implementation of the study used a factorial completely randomized design (CRD) consisting of 2 factors. The first factor is the concentration of 2,4-D consisting of 4 concentration levels, namely 0; 0.5; 1; 1.5; 2 mg/L. The second factor is kinetin concentration consisting of 4 concentration levels, namely 0; 0.5; 1; 1.5; 2 mg/L.The observation parameters include the time of callus emergence (hst), the percentage of explants forming callus (%), the texture and color of the callus, observing the stage of the embryo found. The results showed that 72% of treatments were able to induce callus from pontianak siam orange cotyledons with an average callus appearance at 7 hst. The callus formed is compact and crumbly with varied callus colors, namely white, yellowish, greenish and brownish. The best treatment that produces embryogenic callus is the combination of D0.5K0.5; D1K1; D0.5K1.5; D1.5K1.5 and D2K2 with the characteristics of crumbly callus and yellowish and brownish colors. The results of microscopic observations found that the somatic embryo phase formed was the pro-embryo phase.

Changes in Metabolism and Content of Chlorophyll in Common Duckweed (Lemna minor L.) Caused by Environmental Contamination with Fluorides.

The impact of fluorine on plants remains poorly understood. We examined duckweed growth in extracts of soil contaminated with fluorine leached from chicken manure. Additionally, fluorine levels were analyzed in fresh manure, outdoor-stored manure, and soil samples at varying distances from the manure pile. Fresh manure contained 37-48 mg F- × kg-1, while soil extracts contained 2.1 to 4.9 mg F- × kg-1. We evaluated the physiological effects of fluorine on duckweed cultured on soil extracts or in 50% Murashige-Skoog (MS) medium supplemented with fluorine concentrations matching those in soil samples (2.1 to 4.9 mg F- × L-1), as well as at 0, 4, and 210 mg × L-1. Duckweed exposed to fluorine displayed similar toxicity symptoms whether in soil extracts or supplemented medium. Fluoride at concentrations of 2.1 to 4.9 mg F- × L-1 reduced the intact chlorophyll content, binding the porphyrin ring at position 32 without affecting Mg2+. This reaction resulted in chlorophyll a absorption peak shifted towards shorter wavelengths and formation of a new band of the F--chlorophyll a complex at λ = 421 nm. Moreover, plants exposed to low concentrations of fluorine exhibited increased activities of aminolevulinic acid dehydratase and chlorophyllase, whereas the activities of both enzymes sharply declined when the fluoride concentration exceeded 4.9 mg × L-1. Consequently, fluorine damages chlorophyll a, disrupts the activity of chlorophyll-metabolizing enzymes, and diminishes the plant growth rate, even when the effects of these disruptions are too subtle to be discerned by the naked human eye.

The homeodomain of the <i>Raphanus sativus</i> WOX4 binds to the promoter of the <i>LOG3</i> cytokinin biosynthesis gene

BACKGROUND: The WOX4 transcription factor plays a crucial role in maintaining the organisation of cambium meristem during secondary growth, but its direct targets are unknown. AIM: The aim of our work were to study the effect of WOX4 overexpression on the root development and gene expression in radish (Raphanus sativus L.), a root crop related to Arabidopsis thaliana, and to search for direct targets of the WOX4 in radish. MATERIALS AND METHODS: Radish line 19 of the St. Petersburg State University radish genetic collection was used. Plants were grown on Murashige–Skoog medium and then in soil at 23оС and 16 h of daylight. Total DNA was extracted from radish seedlings using the CTAB method. The PCR-amplified full-length RsWOX4-2 gene, gene fragments or homeobox sequence were cloned into the vectors for overexpression (pB7WG2D), RNA interference (pH7GWIWG2) and yeast one-hybrid assay (pDEST22), respectively, using the Gateway system. The vectors for overexpression and RNA interference of RsWOX4-2 were transformed into Escherichia coli DH10B and then into Agrobacteium rhizogenes Arqua chemically competent cells. Radish seedlings were transformed with A. rhizogenes containing vectors for overexpression and RNA interference of RsWOX4-2, and GUS-overexpressing A. rhizogenes was used as a control. Total RNA from transgenic radish roots was extracted with Trizol reagent. RNA reverse transcription was performed using dT-18 primers and RevertAid reverse transcriptase. qPCR was performed using the Eva Green reagent kit on a CFX96 thermocycler with fluorescence detection system. Results were processed using the 2–ΔΔCT method. Yeast transformation was performed using the competent Saccharomyces cerevisiae cells of Y2H Gold strain. For the yeast one-hybrid assay, the obtained yeast colonies transformed with plasmids containing TF homeodomain sequence and promoter regions of genes were grown on DDO and TDO selective media with different concentrations of 3-amino-1,2,4-triazole. Statistical processing based on Student’s t-test and graphing were performed using the ggplot2 package for the R programming language (v.4.0.2). RESULTS: Overexpression of the RsWOX4-2 gene affects the structure of the radish root stele and alters the number of vessels and cambium cells. Overexpression and RNA interference of the RsWOX4-2 causes changes in the expression levels of putative target genes with the WOX family transcription factor conserved binding sites in their promoters. Using the yeast one-hybrid assay, we have shown that the DNA-binding homeodomain of RsWOX4-2 interacts with the TAATCC site in the promoter of the RsLOG3 gene, which encodes the enzyme for cytokinin biosynthesis. CONCLUSIONS: We have demonstrated the effect of RsWOX4-2 overexpression on radish root stele and gene expression and identified the RsLOG3 as the putative direct target of the WOX4 transcription factor in radish.

Combination of thidiazuron and basal media type on optimizing in vitro growth of Grammatophyllum stapeliiflorum orchids

Grammatophyllum stapeliiflorum, recognized as the melancholic orchid, represents one of the elusive orchid species facing the brink of extinction, thus exacerbating its rarity. Tissue culture technology becomes imperative for the propagation of this orchid species. This study aims to scrutinize the influence of different concentrations of the synthetic growth regulator thidiazuron (TDZ) on distinct basal media types, Murashige Skoog (MS) and Vacin and Went (VW), on the in vitro growth of G. stapeliiflorum orchids. Employing a complete randomized factorial design, the study entails two research factors: TDZ concentration with 4 treatment levels, A0 = 0 mg/L, A1 = 0.25 mg/L, A2 = 0.50 mg/L, and A3 = 0.75 mg/L, and the second factor being the media type, B1 = ½ MS and B2 = ½ VW. Findings indicate significant effects of MS and VW media on the percentage of viable explants, browning, globular forms, and shoots. Interaction between TDZ concentration and media type didn't yield significant effects on each experimental parameter. Treatment with 0.25 and 0.50 mg/L TDZ on ½ VW media demonstrated optimal results for explant growth percentage, browning, and explants in the globular phase, at 97.33%, 2.67%, and 2.67%, respectively. Moreover, treatment with 0.75 mg/L TDZ on ½ VW media resulted in the highest percentage of explant coloration. The combination of TDZ and media has shown the potential to enhance the production efficiency and conservation of this rare orchid species through tissue culture technology.

ВВЕДЕНИЕ В КУЛЬТУРУ IN VITRO И МИКРОКЛОНАЛЬНОЕ РАЗМНОЖЕНИЕ БОЛЬШЕГОЛОВНИКА СЕРПУХОВИДНОГО STEMMACANTHA SERRATULOIDES (GEORGI) M. DITTRICH

Stemmacantha serratuloides (Georgi) M. Dittrich is a perennial herbaceous medicinal plant, a source of a number of phytoecdysteroids with adaptogenic, anabolic and tonic effects. Due to the rarity of this plant species and the strong dependence of its seed productivity on weather conditions, the relevance of research aimed at its biotechnological cultivation and reproduction is high. The aim of this study was to introduce S. serratuloides plants from the Cis-Ural population of the Republic of Bashkortostan into in vitro culture and to develop an effective protocol for micropropagation of this species. It was shown that an effective method of preparing seeds for in vitro culture is their successive sterilization with 96% ethanol (1 min) and 20% bleach (20 min), as well as me- chanical scarification, without a preliminary stratification step. When using 1 mg/l of 2,4-dichlorophenoxyacetic acid and 1.5 mg/l of 6-benzylaminopurine on Murashige-Skoog medium, no regeneration of shoots on explants of cotyledons and hypocotyls occurred. Shoot regeneration was induced from root explants using Murashige-Skoog medium supplemented with 6-benzylaminopurine and indoleacetic acid at concentrations of 1 mg/l and 1.5 mg/l, respectively. Rooting of shoots was carried out on MS medium containing 0.25 mg/L IAA. This micropropagation technology can be used for rapid propagation of plants for various biotechnological purposes, for example, for further transformation by Agrobacterium rhizogenes and creating of hairy root cultures.

ИНТЕНСИВНОСТЬ МОРФОГЕНЕЗА РЕГЕНЕРАНТОВ КАРТОФЕЛЯ ПРИ ИСПОЛЬЗОВАНИИ ОСВЕЩЕНИЯ РАЗЛИЧНОГО СПЕКТРАЛЬНОГО СОСТАВА

The aim of research is to establish the optimal parameters of LED lighting for in vitro cultivation of meristem plants of different potato varieties in order to optimize the elements of the original potato seed production technology. Research was conducted in 2023 in the laboratory of agricultural plant biotechnology of the Samara Research Institute of Agriculture. The object of research was meristem plants of six potato varieties. The explants were grown on a Murashige-Skoog nutrient medium for 40 days using various sources of artificial LED lighting. The photoperiod duration was 16 hours, the air temperature was 22–24 °C, the relative humidity was 70–75 %. After 10, 20, 30 and 40 days of cultivation, the biometric para¬meters of the regenerants were measured – plant length and the number of internodes. When growing meristematic potato plants in vitro, lighting of different spectral composition had a positive effect on the growth and development of regenerants at different periods of their development. At the initial stages of vegetation, the maximum parameters of growth and development of regenerants were revealed when using white and combined lighting. By the end of the vegetation period, plants in the variant with red-violet spectrum lighting were characterized by a significantly higher length and number of internodes. A pronounced variety-specific response of meristematic plants to different lighting conditions was shown. During the entire exposure period, an increase in the share of the influence of the interaction of genotypic and environmental factors on the variability of plant length was noted. The interaction of factors was also decisive for the number of internodes in the first 30 days of vegetation. In the last decade of vegetation, the maximum contribution to the variability of the trait was made by the lighting factor. For practical use in ori¬ginal seed production of potatoes, individual selection of LED lighting parameters for each variety is recommended with a gradual increase in the red-violet spectrum after the tenth day of exposure.

Phenylethanoid glycosides accumulation and antiradical activity of fractionated extracts of Plantago ovata Forssk. callus cultures lines

The main phenylethanoid glycosides in the Plantago genus are acteoside (verbascoside) and plantamajoside, compounds with broad biological effects. This is a report on Plantago ovata callus induction, proliferation and establishment as well as the content of those phenylethanoids in that cell biomass. In the experimental studies, callus initiated from various seedling explants (roots, hypocotyls and leaves) was cultured on MS (Murashige-Skoog) media augmented with 2,4-D (2,4-dichloroacetic acid) and KIN (kinetin) or NAA (α-naphthaleneacetic acid) and BAP (6-benzylaminopurine). Callus proliferating on MS without NH4NO3 (ammonium nitrate) supplemented with 2,4-D (1.0 mg/l) and KIN (0.5 mg/l or 1.0 mg/l) turned out to be a good growth system for biomass production—mean increase of fresh weigh calculated on three following passages was 9.1 ± 1.8. The phytochemical analyses and antiradical DPPH (1,1-diphenyl-2-picryl-hydrazyl) tests revealed that the antioxidant activity is due to the presence of phenylethanoid glycosides. The quantitative screening of the callus extract by TLC (thin-layer chromatography) video densitometric method showed the highest content of acteoside (9.58 ± 0.75 mg/g dry weight) in root-derived and plantamajoside (8.15 ± 0.81 mg/g d.w.) in hypocotyl-derived callus biomass. In in vitro redifferentiated cultures of P. ovata, compounds with a demonstrated therapeutic effect, can be obtained in a manner that is completely independent of cultivation or harvesting from the wild.Graphical abstract

The effectiveness of flow-through substrateless hydroponics in the adaptation of strawberry microplants

A new approach to the adaptation of test-tube strawberry microplants under flow-through hydroponics conditions was tested. The study was carried out on strawberry cultivars “Asia”, “Florence”, “Kimberly”. Murashige-Skoog medium was used for in vitro cultivation. During propagation, 1.0 mg•L-1 6-BAP was added to the medium, during elongation - 0.05 mg•L-1 6-BAP, and during rooting - 1.0 mg•L-1 IBA. Plants were grown in vitro in a light room at a temperature of 23±1°C, a 16-hour photoperiod and a light intensity of 5-6 klx. Flow hydroponics was used to adapt microplants. Plastic containers with dimensions of 0.6 x 0.4 x 0.04 m (volume 9 l), specially prepared for working in a hydroponic system, were taken. They were covered on top with non-woven polymer material, perforated with planting holes with a diameter of 3-4 mm according to a 4 x 4 cm pattern (150 pcs/box), into which plants were planted. This reduced labor costs for adaptation and ensured the efficiency of the operation to 86- 100% within 1 month.

Callus Induction Followed by Regeneration and Hairy Root Induction in Common Buckwheat.

This section describes a set of methods for callus induction followed by the successful regeneration of whole plants and obtaining a culture of transgenic hairy roots from buckwheat plants (Fagopyrum esculentum Moench.). Callus induction and regeneration are key steps for many biotechnological, genetic, and breeding approaches, such as genetic modification, production of biologically active compounds, and propagation of valuable germplasm. Induction of hairy roots using Agrobacterium rhizogenes is also an important tool for functional gene research and plant genome modification. While many efforts were invested into the development of the corresponding protocols, they are not equally efficient for different cultivars. Here, we have tested and optimized the protocols of callus induction, regeneration, and transformation using A. rhizogenes for a set of cultivars of F. esculentum, including wild ancestor of cultivated buckwheat F. esculentum ssp. ancestrale and a self-pollinated accession KK8. The optimal medium for callus induction is Murashige-Skoog basal medium with 3% sucrose which includes hormones 2,4-dichlorophenoxyacetic acid 2mg/L and kinetin 2mg/L; for shoot initiation 6-benzylaminopurine 2mg/L, kinetin 0.2mg/L, and indole-3-acetic acid 0.2mg/L; for shoot multiplication 6-benzylaminopurine 3mg/L and indole-3-acetic acid 0.2mg/L; and for root initiation half-strength Murashige-Skoog medium with 1.5% sucrose and indole-3-butyric acid 1mg/L. A. rhizogenes R1000 strain proved to be the most efficient in inducing hairy roots in buckwheat and T-DNA transfer from binary vectors. Seedling explants cut at the root area and immersed in agrobacterium suspension, as well as prickling the cotyledonary area with agrobacteria dipped syringe needle, are the most labor-effective methods of infection, allowing to initiate hairy root growth in 100% of explants.

Obtaining a primary suspension cell culture of Dracocephalum palmatum Stephan ex Willd

Dracocephalum palmatum Steph. grows on the southern slopes of the Oymyakon plateau in Yakutia (Northeast of Russian Federation) in conditions of harsh continental climate with continuous permafrost. The aboveground phytomass of the plant contains various complexes of secondary metabolites including polyphenolic compounds. It is a potential source of secondary metabolites needed for practical use in the pharmaceutical industry. The aim of the study is to obtain a primary suspension cell culture of Dracocephalum palmatum, growing in the conditions of the Cold Pole — Oymyakon. The work includes optimization of the nutrient medium for introducing calluses into a suspension culture, analysis of the dynamics of biomass growth of the obtained suspension culture, and morphological characteristics of the cells of the suspension culture. The callus cell cultures of Dracocephalum palmatum, cultivated on Murashige-Skoog (MS) medium with the addition of 0.5 mg/L α- naphthylacetic acid (NAA) and 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), were most successfully transitioned into suspension culture. The maximum biomass growth of cell suspension culture was observed when cultivated in liquid MS medium with the addition of 2,4-D (0.5 mg/L), 6- benzylaminopurine (0.5 mg/L), and NAA (0.5 mg/L). The primary cell suspension culture of Dracocephalum palmatum, cultivated for 22 days, had an increase in wet weight of 9,2084 g, dry weight — 0,34135 g, and contained dedifferentiated aggregates of parenchyma-like cells and single round-shaped cells. Samples of the obtained cell suspension culture of Dracocephalum palmatum will be used for the analysis of secondary metabolites and for the development of optimal cultivation conditions in a bioreactor.

IN VITRO PRODUCTION OF VIRUS-FREE CARNATION (DIANTHUS CARIOPHYLLUS L.) PLANTING MATERIAL

The methods of culture of apical meristems and direct and indirect morphogenesis in vitro were used for production of virus-free planting material of carnation. A scheme for obtaining aseptic material has been developed, which consists of stepwise treatment of explants: Thimerosal - 2 min, 70% ethyl alcohol - 0.5 min and 0.08% AgNO 3 - 1 min, which reduces the level of contamination by fungal infection. Expounded the results of studies of callusogenesis and direct and indirect morphogenesis in the culture of in vitro explants of Dutch carnation, their dependence on the content of growth regulators in the nutrient medium. It was established that there were almost no significant differences in the course of callusogenesis processes within carnation varieties . At the same time, the frequency of callusogenesis was 100%. Under the conditions of indirect morphogenesis realization, it is necessary to take into account the age of callus tissues. The growth and intensive shoot formation of carnations was noted on the Murashige-Skoog nutrient medium supplemented with BAP at a concentration of 0.5 mg/l. The best medium for rooting was the MS medium with half the concentration of macro- and microsalts with the addition of 0.5 mg/l of NAA, which is recommended by us for rooting regenerating carnation plants of various varieties. Peat : perlite in a 1:1 ratio was used as a substrate for the adaptation of regenerating plants . Survival of carnation plants to conditions in vivo for the variety "Raffino Linde" was 90%, while for the variety "Tiya" - 83%, respectively.

Effect of culture filtrates of Fusarium fungi on oat callus cultures

The effect of three concentrations (30, 40 and 50%) of the culture filtrate (CF) of the genus Fusarium fungi (F. sporotrichioides, F. poae, F. equiseti, F. oxysporum) on the growth and development parameters of oat callus culture was evaluated. CF at the callus proliferation stage was used to select somaclonal cell lines with mycotoxin tolerance traits. Prior to this, callusogenesis induction was carried out on the Murashige-Skoog (MS) medium with 3 mg/l 2,4-D and 2 mg/l UIC in the culture of immature embryos of the Tyumensky Golozerny, Tubinsky, Zolotoy Pochatok and Talisman varieties. The material for the experiment was selected from the ears of the plants grown in the summer period of 2016–2018 on the experimental fields in the Krasnoyarsk forest-steppe. Callus size was recorded when transplanted onto the proliferation media (MS + 1.5 mg/l 2,4-D), control and CF-containing media. After 30 days of cultivation, callus growth, signs of necrosis and organogenesis were noted. At the end of the experiment, the number of the regenerants formed was counted. The presence of CF in the proliferation medium already at a concentration of 40% provided a decrease in the proliferative activity and increased the frequency of necrosis by at least 50%. Similar results were obtained at the CF level of 50%. On the media with F. poae CF, the reduction of callus viability reached 60–70%. The calluses that remained viable under these conditions had a frequency of regenerant formation and organogenesis 2–3 times higher than the samples that were not influenced by the selective factor. This is particularly pronounced when F. sporotrichioides CF is added. However, this effect was not observed when F. oxysporum CF was applied. This may be due to the differences in the composition of the mycotoxin complex of this mushroom species from the others used in the study. For further work on the technology of creating oat forms with resistance to mycotoxins of the Fusarium genus fungi, it is assumed to use a level of selective pressure not lower than 40%.

The effectiveness of growth regulators and light color spectrum on callus growth of Amorphophallus muelleri Blume. var. Madiun1

Introduction: Plant Growth Regulators (PGRs) play a role in regulating organogenesis and morphogenesis in shoots, roots, and callus formation. Color spectrum of light is one of the quality light factors that affects plant physiological processes. This study aimed to determine the effect of cytokinin and auxin on Murashige-Skoog (MS) medium and light on callus induction and proliferation of porang (Amorphophallus muelleri Blume.) var. Madiun1. Methods: This study used completely randomized design, with the first factor was PGRs (combination auxin and cytokinin) and the second was color spectrum of light (white light, blue light, and its combination) during incubation. Variables observed were emergence time, color, texture, structure and calli growth, also shoots emerging from calli. Results: The results showed an interaction between PGRs with a combination of light color spectrum on callus growth. The fastest callus growth occurred in combination 5.0 mg.l-1 6-benzyl amino purine (BAP) with 0.2 mg.l-1 naphthalene acetic acid (NAA) which was incubated in a combination of white and blue light for 16 hour irradiation. The combination 5.0 mg.l-1 BAP with 0.2 mg.l-1 NAA was able to induce callus emergence time, and the shoots appearing were faster, whereas combination of white and blue light was able to accelerate callus emergence from bulbil and adventitious shoots emergence. Conclusion: The combination of white and blue light color spectrum for 16 hours irradiation can accelerate callus emergence from bulbil and adventitious shoots emerging from calli, and interaction with combination of 5.0 mg.l-1 BAP and 0.2 mg.l-1 NAA can accelerate porang’s callus growth.

РАЗМНОЖЕНИЕ ПОДВОЯ ВИШНИ АИ 70 В КУЛЬТУРЕ IN VITRO

The article presents the results of evaluating the effectiveness of clonal micropropagation of the clone rootstock of cherry AI 70 (FSBSI NCFSCHVW breeding). In the course of the research, the influence of the chelated forms of iron Fe-EDTA and Fe-EDDHA, as well as the concentration of 6 BAP (0.75 and 1.0 mg/l) on the efficiency of shoot formation was evaluated. We took the Murashige-Skoog medium as the basis. The greatest number of shoots in the rootstock AI 70 is formed on MS medium containing Fe-EDDHA (100 mg/l) and 6 BAP 1 mg/l. At the first passage, the multiplication factor is 5.2 shoots/exp., at the fourth passage the number of shoots increases to 12.3 pieces/exp. On average, 8.8 new shoots are formed for each passage. In the control variant on MS medium with Fe-EDTA, an average of 6.6 shoots/exp.

Rhizogenesis of rocket (Eruca sativa Mill.) and wall-rocket Diplotaxis tenuifolia (L.) DC.

Purpose. To test the efficiency of the plant growth regulating substances at different concentrations for stimulating the rhizogenesis of rocket (E. sativa) and wall-rocket (D. tenuifolia). Methods. The experiment was carried out at the Biotechnology Laboratory of the Institute of Bioenergy Crops and Sugar Beet of the National Academy of Agrarian Sciences in 2020–2022. Rocket varieties ‘Znahar’, ‘Lybid’, ‘Zlat’, ‘Silvetta’ and wall-rocket variety ‘Liudmyla’ were used in the study. Seeds were examined for their quality indicators, sterilized with a solution of 35% laundry detergent and ethanol and seeded in vitro. Sterile seedlings were transferred to the Murashige-Skoog (MS) medium supplemented with benzylaminopurine (BAP) for propagation. Naphthylacetic acid (NAA), indolyl-3-acetic acid (IAA), indolyl-3-butyric acid (IBA) and gibberellic acid (GA) were added to the nutrient medium according to the MS prescription in different concentrations. Results. The introduction of IBA (0.8 mg/l) and GA into the nutrient medium, regardless of the concentration, resulted in the longest root system in all treatments. The studied modification made it possible to obtain root length on the 9th day of cultivation from 7 to 64 cm and on the 14th day from 10 to 70 cm. On the 14th day of cultivation, in the plants grown under the concentration of GA >1.0 mg/l, the root not only the elongated and browned, but reached 70 cm in length. For the introduction of IBA into the medium previously supplemented with GA, on the 9th day of cultivation, negative effect was noted in all treatments, namely the elongation of internodes and central root. On the 14th day, vitrification of shoots and significant elongation of both central and lateral roots occurred in all treatments. The central root reached 70 cm in length, which is not advisable during the adaptation period. The results of the experiment indicate that on the 21st day, the longest root system (12 cm and 42 cm) was obtained in the rocket variety ‘Zlat’ (0.1 mg/l GA and 1.5 mg/l GA, respectively) and the shortest root length (6 cm and 21 cm) in the wall-rocket variety ‘Liudmyla’ (0.1 mg/l GA and 1.5 mg/l GA, respectively). Importantly, the root length of ‘Zlat’ variety on the 21st day of cultivation, compared to the 14th day, at a concentration of 0.1 mg/l increased by only 3 cm, while in the 'Liudmyla' variety by 2 cm. Considering this, it is impractical to carry out cultivation up to 21 days. The lowest root length was obtained with the use of IBA and GA: rhizogenesis occurred more slowly than in previous treatments, and on the 14th day the root length was in the range from 3 to 20 cm. Conclusions. The longest root system in all studied treatments was obtained in variety ‘Zlat’, while shortest in ‘Liudmyla’. The optimal concentrations of NAA + IBA + GA for the cultivation of the studied rocket and wall-rocket varieties are 0.3 and 0.5 mg/l.

In vitro direct organogenesis of the medicinal single-mountain local prioritized vulnerable Greek endemic Achillea occulta under different medium variants

Achillea species are of high medicinal value based on their use in traditional medicine and their content in secondary metabolites with potential application in dermatology and cosmetic industry. Achillea occulta is a single-mountain local prioritized vulnerable Greek endemic encountering propagation problems through seeds and root division. In vitro propagation can be used for the rapid and mass production of clonal plants complying with phytochemical and sanitary quality criteria. In this study, several culture medium variants were tested including the effect of different indole-3-butyric acid (IBA) concentrations with 6-benzyladenine (BA), different cytokinin types, agar concentrations, and zeatin (ZT) sterilization methods on in vitro shoot proliferation, as well as the effect of different auxin types and concentrations on in vitro rooting and ex vitro survival rate of shoot-tip explants. The results showed BA was the most preferable cytokinin type as evidenced by all proliferation attributes and autoclaved ZT provided longer shoots. Therefore, the two more promising and cost-effective treatments for proliferation were 2.2 μM BA + 0.25 μM IBA or 4.4 μM BA + 0.5 μM IBA after 21 days of culture on modified Murashige-Skoog (MS) medium (x2 Fe) enriched with 20 g L-1 sucrose and 6-7 g L-1 Agar (90-93.8% shoot formation, 2.7-2.9 shoots 0.4-0.6 cm long, 2.5-2.6 proliferation rates). Explants rooted better after 21 days of culture on plain MS medium with 2 μM IBA or 1 μM α-naphthalene acetic acid (NAA) (13.3-20% rooting, 1-1.5 roots 1.8-2 cm long). NAA gave more roots, IBA longer roots, while both auxins exhibited highest rooting (13.33-20%) and ex vitro survival rates (50-66.7%).