Murashige & Skoog Research Articles

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.

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.

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.

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

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 regeneration of black pepper (Piper nigrum L.)

This investigation was done to determine how various plant growth regulators such as benzyl adenine (BA) and indole-3-butyric acid (IBA) affected the in vitro plant regeneration of the black pepper plant. The largest number of shoots was produced when the shoot tips and nodal segments of black pepper were utilized as explants and inoculated in Murashige Skoog (MS) media supplemented with 1.5 mg/L BA. The treatment with 2.0 mg/L BA at 8 weeks after induction (WAI) produced the most leaves (3.2). In the 2.0 mg/L treatment of IBA, the greatest number of roots was regenerated. The treatment 2.0 mg/L BA + 2.0 mg/L IBA produced the highest results for shoot induction, shoot length, and overall leaf production when the effects of both hormones were combined. The same treatment also produced the highest proportion of root induction and longest roots. Regenerated plantlets survive at a 46.67% rate in a shaded building and at a 57.14% rate in the open air under bright sunlight. In order to produce black pepper on a big scale, an effective approach for in vitro regeneration of black pepper has been established. Bangladesh J. Agri. 2023, 48(2): 75-85

Development of regenerated plants of interspecific wheat hybrids using immature embryos as explants

Nowadays particular attention in winter wheat breeding is paid to the introduction of biotechnological methods. The culture of immature embryos in vitro is an alternative way to produce hybrids that often do not develop fully in vivo. The purpose of the current study was to estimate the ability of immature embryos of interspecific winter wheat hybrids for callus induction, morphogenesis, and plant regeneration on artificial nutrient media, as well as to select optimal conditions for obtaining full-fledged plants with all the botanical characteristics of the species. The research material included 5 hybrid combinations of interspecific crosses between durum (Triticum durum DESF.) and common (Triticum aestivum L.) winter wheat. The material was collected during one field season (2022). Immature embryos were used as explants. Culture media were according to Murashige-Skoog (MS), and they differed in the content of hormones and organic substances. There has been found that the hybrid combinations were characterized by a variety of morphogenic responses. The manifestation of callusogenesis was not high enough and averaged 22–32 % depending on the nutrient medium. Rhizogenesis averaged from 1–6.7 %, and ended with the formation of roots. The process of formation of embryoid- and hemmorizogenesis was best demonstrated by the hybrid combination No.1, and on two media. All processes of morphogenesis excepting ‘rhizogenesis’ varied greatly among hybrid combinations. The largest number of regenerated plants was obtained from hybrid combination No.1 (61 pcs., 54.5 %). After vernalization and acclimatization, 38 % of the plants survived from the number of planted embryos. The largest number of heads (79 pcs.) was formed according to hybrid combination No.1, on average 1.46 heads per plant. However, not all heads were fertile. The results of the correlation analysis made it possible to identify indicators that closely correlated with the amount of plant regeneration, namely, the high yield of embryoid- and hemmorizogenic structures ensured a high frequency of plant regeneration (r = 0.769).

Tolerance Level of Butterfly Pea (Clitoria ternatea L.) to Stress Acidity Through Tissue Culture Technique

Butterfly pea (Clitoria ternatea L.) a high-quality legume that is rich in protein and grows on various soil types with a pH range of 5.5-8.9. This experiment was conducted to get the level of tolerance of butterfly pea plants to stress acidity at different levels through tissue culture technique. The study was designed using a complete randomized design with 6 treatments with the different levels of AlCl3 addition using Murashige Skoog (MS) media with 20 replications (P0 (0 ppm AlCl3), P1 (100 ppm AlCl3), P2 (200 ppm AlCl3), P3 (300 ppm AlCl3), P4 (400 ppm AlCl3), and P5 (500 ppm AlCl3)). Data were analyzed using analysis of variance (ANOVA), and if there was a significant difference, data were further analyzed using Duncan’s multiple range test. The variables observed were acidity media changes, plant height gain, number of leaves, number of branches, number of tillers, percentage of leaves withering, and leaf color. The results showed that the butterfly pea plant has mechanism of adaptation to acid stress on the parameters of plant height gain and number of leaves at the end of the observation. However, the level of plant tolerance on the parameters of the number of branches and the number of tillers was ≤ 300 ppm (pH 3.73).

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%.

In vitro culture introduction and microclonal reproduction of triploid Aspen (Populus Tremula L.)

In response to global warming issues, the entire world community seeks to decarbonize its energy and economy. One of the ways to reduce concentrated greenhouse gases in the atmosphere is to enlarge forest areas and encourage forest regeneration after fires. The given paper describes sterilization methods of primary explants. Studies have shown that 0.1% diacid solution supplemented with Tween 20 is the most effective at producing aseptic viable cultures of triploid aspen (Populus tremula L.) in vitro. This method provides the necessary amount of viable sterile cultures for their subsequent micro-multiplication (clonal), sufficient for the further mass production of planting material. Murashige & Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) had a more intensive formation of adventive buds compared to the medium with kinetin. Higher BAP concentration resulted in more regenerants from one explant with their height shortened. Woody Plant Medium (WPM) with different concentrations of 6-benzylaminopurine and indolyl-acetic acid (IAA) is more effective for working with triploid aspen. Effective reproduction at triploid aspen cultivation was shown at 0.5 mg/l concentration of BAP. The research results can be successfully used in vitro culture introducing of triploid aspen, experiments on micro-reproduction of other tree crops to get high-quality planting material.

In Vitro Propagation of Several Valuable Selections of Robinia pseudoacacia L. as a Fast and Sustainable Source for Wood Production

There is an increasing trend in forest production towards planting rapid-growing trees as attractive, environmentally friendly energy sources. This study aimed to establish an alternative to the traditional propagation of a number of selections of Robinia pseudoacacia L. by developing an in vitro culture protocol. This study’s topic is of great importance, and it reflects an ongoing concern at the University of Oradea’s Faculty of Environmental Protection’s sustainable research program. The explants from four forms (called S1, S2, S3, and S4), selected for their phenotypic characteristics, were inoculated on four culture media (Murashige–Skoog (MS), Anderson, Chée–Pool, and Driver and Kuniyuki Woody (DKW)) with the same phytohormonal balance. DKW medium proved to be the better support of morphogenic activity, and it was further tested under different phytohormonal balances. Different results were observed depending on the hormone content in the DKW environment. In the presence of 0.5 mg/L benzylaminopurine (BAP) and 0.04 mg/L aminoisobutyric acid (AIB), 91.5% of the explants developed an average of 4.45 ± 0.18 shoots, whereas the average upper shoot height (3.82 cm) was recorded on DKW medium with 0.5 mg/L BAP and 0.04 mg/L α-naphthaleneacetic acid (NAA). Auxin, 0.05 mg/L AIB, promoted root production (5.27 ± 0.15 roots/explant), while 0.1 mg/L NAA promoted root length. In conclusion, the S4 selection produced the greatest outcomes of all environmental variables in terms of both the number of shoots and their heights.

The growth of paprika (Capsicum annuum L.) from cotyledon on various media in vitro

Micro propagation of paprika is still facing problems on suitable media composition. This research aimed to determine the growth of paprika (Capsicum annuum L.) from cotyledon on various media in vitro. The research was arranged as completely randomized design with 10 treatments, four replications, used Murashige-Skoog (MS) fortified with: 1). 3 benzylamino purine (BAP) + 0.3 indoleacetic acid (IAA) + 0.2 silver nitrate (AgNO3); 2). 3 BAP + 0.3 IAA + 0.3 gibberellin (GA) + 0.2 AgNO3; 3). 3 BAP + 0.3 GA + 0.3 naphthaleneacetic acid (NAA) + 0.2 AgNO3; 4). 3 BAP + 1.5 NAA + 3 AgNO3 + 1 glutamine; 5). 3 BAP + 0.5 NAA + 3 AgNO3 + 1 glutamine; 6). 4 BAP + 1.5 NAA + 3.5 AgNO3 + 1 glutamine; 7). 5 BAP + 1 IAA; 8). 10 BAP + 2 GA; 9). 5 thidiazuron + 2 GA; 10). 7.5 kinetin + 2 GA. All units are in mg/L. The treatment effects were observed on callus and shoot growth, analyzed by ANOVA and the mean differences were tested using Tukey Test. All treatments induced callus but only 10), 9) and 7) regenerated shoots of 3.75, 1.50 and 1,38 successivelly. Generally, addition of 7.5 mg/L kinetin + 2 mg/L gibberellin to Murashige-Skoog basal media was the best to induce shoot.

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.

Efecto de diferentes tipos de sustratos y auxinas en el establecimiento ex vitro de segmentos nodales de arándano Var. Biloxi

Micropropagation of Vaccinium spp. using Murashige Skoog (MS) and benzylaminopurine (BAP) has been successfully developed. This work aimed to determine the effect of different types of substrates and auxins (naftalen acetic acid NAA, indol acetic acid IAA y, indol butyric acid IBA) on ex vitro establishment and rooting of nodal segments of V. corymbosum Var. Biloxi to create a donor bank of blueberry plants. The influence of five different substrates and three types of auxins using five concentrations was assessed for a better nodal segment ex vitro establishment. The 40 % coconut fiber substrate, 20 % pomine + 40 % peat improved ex vitro establishment of V. corymbosum Var. Biloxi nodal segments. Otherwise, using 100 ppm of NAA the ex vitro establishment of V. corymbosum Var. Biloxi nodal segments increased. These results will facilitate the conformation of a donor bank of youth plants of V. corymbosum, increasing possibilities of in vitro establishment for massive micropropagation. Keywords: acclimatization, eriaceae, hormones, Vaccinum.

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.

Clonal micropropagation of rocket (Eruca sativa Mill.)

Purpose. To optimize the method of clonal micropropagation of rocket (Eruca sativa Mill.). Methods. Seeds of rocket cultivars 'Znahar' and 'Lybid' were used for introduction into in vitro culture. Solutions of sodium hypochlorite (35%), ethanol (70%) and HgCl2 (0.2%) were used for sterilization. A 5% solution of chloramine served as a control. Sterile material was planted on liquid and solid agar nutrient media according to Murashige-Skoog (MS) and Hamborg-Eveleg (B5). A solid nutrient medium MS supplemented with 0.5 mg/l BAP was used as a control at all stages of reproduction. 6–benzylaminopurine (BAP), benzylaminopurine (BA), kinetin, IAA, NAA and IBA were added to media for reproduction and rooting. The control was IAA (0.5 mg/l). Results. Sterilization of seeds with HgCl2 resulted in obtaining 93 and 90% sterile material; however, no viable seeds were found. The highest indicators of material sterility in the studied rocket varieties were noted for the use of sodium hypochlorite solution: in 'Znahar' 93% and 'Lybid' 89%. In addition, viability in this treatment was at the level of 90 and 85%, respectively. Higher indicators of both the number of shoots (8 and 6) and their height (8 and 3 cm) of both studied varieties were obtained on the MS medium (medium B5 – 5 and 5 shoots, and 5 and 4 cm, respectively). The most intensive shoot formation occurred with the use of kinetin. In particular, on the MS medium, variety 'Znahar' formed 19 shoots, 'Lybid' 17 shoots; on B5 – 16 and 13 shoots, respectively. At a concentration of 0.8 mg/l, the length of the shoots of the studied varieties was with IAA 10 and 8 cm, with NAA 15 and 13 cm and with IBA 18 and 16 cm, respectively. The same tendency was observed when the concentration increased to 1.2 mg/l. The number of lateral roots varied from 3 to 7 in the control and from 4 to 11 in the experimental treatments. Most of the roots were formed at an IBA concentration of 1.2 mg/l. In the case of adding 0.8 mg/l of NAA, 7 roots were formed in 'Znahar' and 5 in 'Lybid'; when increasing the concentration to 1.2 mg/l the number was 10 and 9, respectively. Conclusions. The worst indicators of viable seed material were obtained after sterilization with a HgCl2 solution (93 and 90% of sterile and 0% of viable seeds) and the best with sodium hypochlorite solution (93 and 89% of sterile and 90 and 85% of viable seeds). On the liquid nutrient media according to different prescriptions, the smallest indicators of the height of shoots and their number were obtained. Also, in these treatments, the vitrification of plants, their slow growth and insignificant shoot formation were observed. The longest root system was formed in the studied rocket varieties with adding IBA to the nutrient medium. At the same time, under such conditions, regardless of the IBA concentration, plants formed too long root system that can be injured during planting; therefore, it is more appropriate to use IAA and NAA or their combination.

Characterization of the physico-chemical properties of the natural habitat and in vitro culture effects on the biochemistry, proliferation and morphology of Lemna minuta

In this study, the ecological conditions of the natural habitat of Lemna minuta Kunth in Morocco were investigated, and the impact of five synthetic growth media (Murashige-Skoog (MS), Schenk-Hildebrand (SH), Hoagland medium (HM), 10X Algal Assay Procedure (AAP), and Swedish Standard Institute medium (SIS)) on the morphophysiological and biochemical parameters was analysed. The morphophysiological parameters included root length, frond surface area, and fresh weight, while the biochemical parameters included photosynthetic pigments, carbohydrates, and protein content. The study was conducted in vitro in two phases: an uncontrolled aeration system (Phase I) and a controlled aeration system (Phase II).The results showed that the pH, conductivity, salinity, and ammonium levels in the natural habitat were within the optimal range for duckweed growth. The measured orthophosphate concentrations were higher compared to previous observations, while the recorded chemical oxygen demand values were low. The study also revealed a significant effect of the culture medium composition on the morphophysiological and biochemical parameters of the duckweed. The fresh weight biomass, relative growth rate in fronds, relative growth rate in surface area, root length, protein content, carbohydrates, chlorophyll (a), chlorophyll (b), total chlorophyll, carotenoids, and the chlorophyll (a/b) ratio were all affected by the culture medium.The most accurate regression models described the growth index GI(F) based on time and in vitro culture conditions in both phases. In Phase I, the best models for MS, SIS, AAP, and SH media were linear, weighted quadratic, cubic, and weighted cubic, respectively. In Phase II, the best models for all growth media were linear. The time coefficients (in days) for Phase II were 0.321, 0.547, 1.232, 1.470, and 0.306 for AAP, HM, MS, SH, and SIS, respectively.Comparing the morphophysiological and biochemical parameters of fronds from different media and analysing the regression model results showed that the SH and MS media were the best among the tested media for the in vitro culture of L. minuta in controlled aeration conditions. However, further research is needed to develop new synthetic media that best promote the growth and maintenance of this duckweed in long-term culture.

The role of growth regulators in the ontogenesis of plants of potato cancer differentiators in vitro culture

Goal. To study impact of growing regulators on morphometric indexes and potato cultivar-differentiators productivity in culture in vitro.
 Methods. The studies conducted in the Laboratory of Biotechnology for Agricultural Crops of Ukrainian Plant Quarantine Research Station Institute of Plant Protection of NAAS. Murashige-Skoog (MS) nutrient medium was used with the addition of different concentrations of Epin-maxi growth regulators (MS — control; MS + Epin-maxi r., (2,4-epibrassinolide, 0.012 g/l); MS + Epin-maxi, 0.025 g/l) and Poteitin (MS — control; MS + Poteitin w.s., (2,6-dimethylpyridine-1-oxide with succinic acid, 0.15 mg/l); MS + Poteitin, 0.3 mg/l) on different varieties of potato cancer of different ripeness groups (Schedryk — early, Slovyanka — medium-ripe). Plants grew in terms of cultivation room during 16-hour photoperiod a light intensity of 2000—2500 lux, temperature 22—25°C and humidity of 60—80%. The grafting conducted in quantity 25 plants with three times experiment repeating.
 Results. The morphogenesis optimal indexes received during their put into the nutritious medium MS + Epin-maxi 0.025 g/l and MS + Poteytin 0.3 mg/l. The plants’ height of variety Schedryk increased on 21st day of growing on 4.9—25.2 mm, in control (65.2 mm) and internodes number increased 0.4—0.9 pcs in control (5.2 pcs.). The plant’s height increased of variety Slovyanka in variants with stimulators was more than on 12.1—28.4 mm and the internodes number increased 0.5—1.1 pcs. The root-forming increase determined in all experiments variants: roots quantity — on 2.6—3.4 pcs., their length was 25.6—29.8 mm, the average microtubers weight was in 1.4—1.6 times higher, the mictotubers weight increase on 1 plant in 1.6—1.9 times, the quantity of microtubers on one plants in 1.6—1.8 times in comparison with control.
 Conclusions. The optimal indexes of morphometric parameters and potato cultivar-differentiators productivity on culture in vitro received in variants of research with Epin-maxi 0.025 g/l and Poteytin 0.3 mg/l. These stimulators, when added to the nutrient medium, contributed to an increase in plant height, the number of internodes, root formation (number of roots and their length), average mass of microtubers, mass and number of microtubers per plant.

The effect of 2,4-D, BA and Thidiazuron on somatic embryo induction of liberica coffee of Tungkal Composite from Jambi

The Tungkal Composite of liberica coffee rejuvenation program at Jambi Province is urgently needed due to the latest status of the plants are old and showing diseased attacked. To support the rejuvenation, the provision of seedlings could be done by tissue culture approach through somatic embryogenesis. This research aimed to study the effect of 2,4-D, BA and TDZ on callus formation and embryo induction from liberica coffee leaf explants. Explants used were leaf sections with or without veins. Explants were grown on Murashige Skoog (MS) basal medium with 2,4-D + BA and 2,4-D + TDZ. A completely randomized design was employed in the trial, and each experimental unit was replicated three times. Results showed that callus could be formed on both types of explants, but leaf explants with veins could produce callus faster. The use of BA in medium could induce callus formation faster than TDZ. The higher the concentration of 2,4-D, BA and TDZ, the slower callus initiation. The addition of TDZ in the medium resulted in mostly callus, while callus from medium with BA showed a friable structure. Globular somatic embryos were formed in all treatments with varying amounts. TDZ can induce more globular somatic embryo than BA.

Season, basal media and plant growth regulators effect in wood plant in vitro propagation: a comprehensive review

Wood plant in-vitro propagation is currently widely used and is not limited to the preparation of superior seeds but has been widely utilized for the production of phytopharmaceuticals and the production of a valuable and specific tree for the benefit of man. Studies focused on the influence of explant collection seasons, selection of basic media, and plant growth hormone. The results of the study showed the magnitude of the variables that must be considered in the propagation of tissue cultures even in the same species and types of cultures. The period of January and March is the best time for explant retrieval because it provides the best explant response in bud breakage, lowest browning, and best bud induction. The basic media of MS (Murashige & Skoog), as well as its various modifications and the basic medium of WPM (Wood Plant Medium), tend to be widely chosen as the basic media at various levels of subcultures in many woody plant species. The application of growth regulators shows a tendency to use BAP (benzyl amino purine) and NAA (naphthalene acetic acid) whose application is separately or in combination with a certain concentration according to the species and subculture phase. Other plant growth regulators (PGRs) that are also quite widely used are the IBA (indole butyric acid), IAA (indole acetic acid), and G3 (gibberellic acid) groups which are used in the multiplication and rooting stages. The information from the study can be used as basic information at the stage of optimizing tissue culture activities in different species, explanatory, and regeneration types. The success of the optimization stage will be used as an improvement to the previous woody plant tissue culture protocol so that optimization of success is achieved. In vitro propagation is always associated with the number of resources used, so efforts to improve in vitro protocols are a very important activity to evaluate that will lead to increased effectiveness to reduce resource inputs, and be able to realize low-cost wood plant tissue culture propagation.