Callus Cells Research Articles

An Attempt to Restore the Fertility of Miscanthus × giganteus

Miscanthus × giganteus is a popular industrial plant with great potential in ecological agriculture. It forms numerous rhizomes that are important in the sequestration of carbon dioxide. The plant can be a source of lignin and cellulose, biomass for renewable energy production, and can be used in small garden architecture, or to strengthen the banks of landslides. Breeding this species is difficult, as it is a sterile allotriploid with 57 chromosomes. The aim of the study was to obtain fertile plants of this species by treating its callus and regenerants with chromosome doubling agents such as colchicine, oryzalin, trifluralin, and caffeine at variable concentrations and durations. Callus cells naturally showed large variations in the number of chromosomes but only euploid cells regenerated plants. Treatment of the regenerants with 1252 µM colchicine for 18 h allowed for obtaining two hexaploid shoots; however, they died before flowering. Colchicine and oryzalin stimulated the formation of mixoploid shoots. The investigated substances, except for caffeine, were highly toxic to plants. M. × giganteus plants with 114 chromosomes may die because such a high number of chromosomes may be unfavorable for cells of this species.

ОРГАНОГЕНЕЗ IN VITRO КАК РЕАЛИЗАЦИЯ СВОЙСТВА ПЛЮРИПОТЕНТНОСТИ СТВОЛОВЫХ КЛЕТОК МОРФОГЕННОГО КАЛЛУСА

The article provides the brief review of the literature and own works devoted to the peculiarities of organogenesis as the morphogenesis pathway in calli in the conditions of in vitro culture. Particular attention is paid to the issues of realization of the morphogenic potency of callus cells due to the pluripotency property. A comparison of organogenesis in vitro with similar events in organogenesis in planta confirms the validity of the principle of universality of plant morphogenesis processes in cultural and natural conditions [Батыгина, 2014]. The used terminology is analyzed. Such controversial issue as the property of stemness of callus cells is touched upon. The prospects of using organogenesis in vitro as a model for studying the most complex biological phenomenon of organogenesis in planta are discussed.

Polyesters of 14-Hydroxylated Taxoids Found for the First Time in <i>Taxus canadensis</i> Intact Plants

Taxane diterpenoids (taxoids) are found only in representatives of the Taxaceae family (different yew species); however, the unique structure and therapeutic properties of taxoids, which are in demand in medicine, have made these compounds one of the most studied secondary metabolites of higher plants. In this work, for the first time, a detailed study is performed into the structural diversity of polyesters of 14-hydroxylated taxoids in Taxus canadensis a yew species, for intact plants of which nonpolar 14-hydroxylated taxoids have not been previously described. At the first stage of the work, it was shown using chromato-mass spectrometry that polyesters of 14-hydroxylated taxoids (yunnanxan, taxuyunnanin C, sinenxan B, sinenxan C) are the dominant diterpenoid secondary metabolites in T. canadensis callus cell culture. Based on these results, as well as the similarity of the metabolism of cultivated in vitro plant cells and root cells in planta, it was suggested that polyesters of 14-hydroxylated taxoids will preferentially accumulate in the roots in intact T. canadensis plants. The validity of this hypothesis was confirmed using liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). According to chromato-mass-spectrometric screening in T. canadensis needles, polyesters of 14-hydroxylated taxoids are actually found only in trace amounts, while they are one of the major (quantitatively) diterpenoids in the roots. One of the main 14-hydroxylated taxoids of T. canadensis roots, yunnanxan, was isolated preparatively and uniquely identified using NMR spectroscopy and high-resolution mass spectrometry. This work is the first report on the presence of polyesters of 14-hydroxylated taxoids in intact Canadian yew plants.

Cellular and metabolic characteristics of peach anther-derived callus

Callus is an important material for horticulture research, especially for biotechnological applications, and can be used for the production of high-value metabolites. In this paper, anthers of peach (Prunus persica L.) varieties ‘Zaojiubao’, ‘Yanhong’, and ‘Xueyulu’ were used to induce callus lines, and the tissue morphology, subcellular structure, and metabolite profiles were characterized. Peach anther-derived calli were successfully induced in MS+1.0 mg/l ZT+1.0 mg/l 2,4-D + 0.5 mg/l IAA medium, and were stably subcultured in MS+1.0 mg/l BA+0.2 mg/l IBA+0.1 mg/l GA3 medium. SSR analysis confirmed the callus lines originated from somatic cells. Furthermore, microscopic observation of callus cells revealed some interesting cytological features, such as abundant mitochondria, which may be associated with vigorous growth and strong resistance to senescence, and suggests their potential for protoplast-related experiments such as transient expression. The metabolites in anther-derived calli were comparatively analyzed. Naringenin-7-O-glucoside (prunin), which has medicinal value as an anti-inflammatory and antiviral compound, was highly enriched in the callus lines, and cyanidin-3-O-glucoside caused the red coloration of peach anther calli. Additionally, chlorogenic acid, and amino acid derivatives such as aspartic acid and leucine, as well as other organic acids, lipids, and alkaloids, were detected in the callus lines. This study showed a series of peach anther-derived callus lines have great potential for cell biology and transformation research, especially for the production of high value components, which can be used for medicinal purposes, health products, and food additives.

Overexpression of the <i>Ib-rolB/C</i> gene perturbs biosynthesis of caffeoylquinic acid derivatives in transgenic calli of sweet potato

Ipomoea batatas is a root crop widely cultivated in South America, Africa, and Asia. This is also a source of caffeoylquinic acid derivatives (CQAs) with potential health-promoting benefits. Sweet potato genome carries two separate cellular T-DNA (cT-DNA) regions (IbT-DNA1 and IbT-DNA2). Especially, IbT-DNA2 contains five ORFs homologous to the Agrobacterium rhizogenes T-DNA, namely ORF13, ORF14, ORF17n, ORF18/ORF17n, and RolB/RolC proteins [1]. Unfortunately, presently there is insufficient information on IbT-DNA2 genes function in the physiological processes of sweet potatoes.
 In this study, expressional levels of the IbT-DNA2 genes and the effect of Ib-rolB/C overexpression were examined using I. batatas cell culture. We discovered that I. batatas cT-DNA genes were not expressed in callus, and abiotic stresses and chemical elicitors affected their transcriptional levels weakly. Additionally, two Ib-rolBC-transgenic cell lines have been established though Agrobacterium-mediated transformation of I. batatas callus cells. Overexpression of Ib-rolB/C gene reduced biomass accumulation of transgenic cell lines by 1.21.6 times and increased the CQAs content by 1.51.9-fold. To justify the metabolic fluctuations, the study also looked into the expression patterns of the major biosynthetic genes, namely bPAL, IbC4H, Ib4CL, IbHCT, and IbHQT. The obtained data demonstrated that the overexpression of the Ib-rolB/C reduced the IbPAL transcript but considerable increase in the transcript levels of the IbHQT. We propose that this result was obtained through as-yet-uncharacterized signaling pathways activated by RolB/RolC.

Morphological, Anatomical, Physiological and Biochemical Changes during Adventitious Roots Formation of Bougainvillea buttiana ‘Miss Manila’

Bougainvillea, a widely used ornamental plant, is mainly propagated by cuttings and tissue culture. Still, large-scale production of Bougainvillea is often difficult because of rooting issues. Therefore, based on an early establishment of the regeneration system for tissue culture in Bougainvillea by our research team, we further studied its rooting mechanism. It was observed that the morphology and anatomical structure of Bougainvillea buttiana ‘Miss Manila’ contained endogenous hormones, such as indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA3), and zeatin-riboside (ZR), including peroxidase (POD), polyphenol oxidase (PPO), and IAA-oxidase (IAAO) activities. Following the culture, Days 0–15 consisted of the induction and initiation stages, while Days 15–25 included the expression stages. No latent root primordium was found in the Bougainvillea plantlet, which belonged to the induced rooting type. The root primordium was derived from callus cells generated by divisions of parenchyma cells in the basic tissues. It was found that the changes in the POD, PPO, and IAAO activities were closely related to the formation of adventitious roots (AR), in which the highest rooting values occurred during the transition from the initiation stage to the expression stage, whereas the endogenous IAA and ABA contents had negative and positive correlations during the induction, initiation, and expression stages.; The values of GA3 and ZR also peaked during the transition from the initiation to the expression stage. ZR and GA3 were found to promote adventitious root formation, while ABA inhibited it. The IAA/ABA, ABA/ GA3, and IAA/ZR ratios also shifted at the onset of the expression stage of AR, indicating these values were closely related to their occurrence. Overall, this study provides the basis for further research considering AR formation in Bougainvillea, and the propagation of various Bougainvillea varieties.

Visualizing PVS2 component permeation into Oryza sativa callus cells by coherent raman scattering microscopy

Visualizing PVS2 component permeation into Oryza sativa callus cells by coherent raman scattering microscopy

COMPARATIVE ANALYSIS OF THE EFFICIENCY OF THE METHODS
 OF REGENERATION FROM RAPESEED EXPLANTS OF MAGNAT AND
 LEADER VARIETIES

A comparative analysis of the effectiveness of organogenesis from the callus cells of hypocotyl segment explants in rapeseed varieties Leader and Magnat regionalized in Belarus was carried out. It was established that the Leader, compared to the Magnat variety, is more suitable for organogenesis from plant explants in vitro. It was shown that for the Leader variety the regeneration efficiency is up to 10 plants adapted ex vitro per explant, when using a method, according to which hypocotyl explants are first exposed to auxin (2,4-dichlorophenoxyacetic acid, 1 mg/l), and then to cytokinin (6-benzylaminopurine, 5–8 mg/l).

Diverse forms of xylem-Like cells and strand formation in Xylogenic Eucalyptus bosistoana callus culture

In vitro xylem induction system is a basic tool in physiological, biochemical, and molecular studies of secondary cell wall formation, lignin biosynthesis and deposition associated with tracheary element formation. Eucalyptus bosistoana is a Class 1 durable hardwood tree species, selected by the New Zealand Dryland Forest Initiative for good quality wood and high adaptability to the NZ growing conditions. Xylogenic E. bosistoana callus culture was established and up to 40% of the callus cells were xylem-like cells (XLCs) which may have differentiated from small, cytoplasmically dense or compact dividing, and exhibited increased lignin contents during culture. The eucalyptus XLCs showed diverse sizes, patterns of secondary cell wall thickenings similar to the xylem cells in the young shoots and organized development including cell–cell connections of the XLCs to form xylem strands. This is the first report of the organized development of XLCs in E. bosistoana callus culture.

Engineered magnetic plant biobots for nerve agent removal

Biohybrid micro/nanorobots that integrate biological entities with artificial nanomaterials have shown great potential in the field of biotechnology. However, commonly used physical hybridization approaches can lead to blockages and damage to biological interfaces, impeding the optimal exploitation of natural abilities. Here, we show that magnetically propelled plant biobots (MPBs), employing tomato-callus cultivation engineering in the presence of Fe3O4 nanoparticles (NPs), are capable of active movement and directional guidance under a transversal rotating magnetic field. The Fe3O4 NPs were transported through the cell growth media and then taken up into the plant tissue cells (PTCs), imparting the plant biobot with magnetic function. Moreover, Fe ions support the growth of callus cells, resulting in nanoparticle incorporation and enabling faster growth and structurally compact texture. The magnetic plant biobots demonstrated rapid and efficient removal of chlorpyrifos (approximately 80%), a hazardous nerve gas agent that causes severe acute toxicity, and recovery using an external magnetic field. The eco-friendly plant biobots described here demonstrate their potential in biomedical and environmental applications.

Influence of Plant Growth Regulators and Artificial Light on the Growth and Accumulation of Inulin of Dedifferentiated Chicory (Cichorium intybus L.) Callus Cells.

Chicory (Chicorium intybus L.) is a perennial herb of the family Asteraceae, widely distributed in Asia and Europe, commonly used industrially as a raw material for extracting inulin because of a high content of inulin and biologically active compounds. Light conditions and plant growth regulators (PGRs) are two of many factors that affect the growth and inulin content of chicory callus. The aim of this work is to study the effect of PGRs and light conditions on proliferation and accumulation of inulin of chicory callus in vitro. In this study, we used semi-solid MS medium supplemented with different auxins (including Indole-3-acetic acid (IAA), naphthylacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D)) at a concentration of 5.5–9.5 mg/L in combination with 2.0 mg/L 6 benzylaminopurine (BA) to determine induction and proliferation of callus. The increasing value of callus fresh weight was used to assess the growth of the callus in treatments. The results showed that a steady increase in callus fresh weight and inulin content in callus cells was obtained when they were cultured on MS medium supplemented with a combination of 2.0 mg/L BA with 7.5 mg/L IAA in lighting conditions with radiation equalized by the flux density of photosynthetic photons and ratios of radiation levels in the region of FR—far red > R—red. Increasing demand for organic inulin sources in production practice can be met by our finding.

Transcriptional regulation of cell proliferation competence-associated Arabidopsis genes, CDKA;1, RID1 and SRD2, by phytohormones in tissue culture.

During organ regeneration, differentiated cells acquire cell proliferation competence before the re-start of cell division. In Arabidopsis thaliana (Arabidopsis), CDKA;1, a cyclin-dependent kinase, RID1, a DEAH-box RNA helicase, and SRD2, a small nuclear RNA transcription factor, are implicated in the regulation of cell proliferation competence. Here, we report phytohormonal transcriptional regulation of these cell proliferation competence-associated genes during callus initiation. We can induce the callus initiation from Arabidopsis hypocotyl explants by the culture on the auxin-containing medium. By RT-quantitative PCR analysis, we observed higher mRNA accumulation of CDKA;1, RID1, and SRD2 in culture on the auxin-containing medium than in culture on the auxin-free medium. Promoter-reporter analysis showed that the CDKA;1, RID1, and SRD2 expression was induced in the stele regions containing pericycle cells, where cell division would be resumed to make callus, by the culture in the medium containing auxin and/or cytokinin. However, the expression levels of these genes in cortical and epidermal cells, which would not originate callus cells, were variable by genes and phytohormonal conditions. We also found that the rid1-1 mutation greatly decreased the expression levels of CDKA;1 and SRD2 during callus initiation specifically at 28°C (restrictive temperature), while the srd2-1 mutation did not obviously decrease the expression levels of CDKA;1 and RID1 regardless of temperature conditions but rather even increased them at 22°C (permissive temperature). Together, our results implicated the phytohormonal and differential regulation of cell proliferation competence-associated genes in the multistep regulation of cell proliferation competence.

In Vitro Culture as a Tool for Studying Plant Developmental Processes at the Physiological Level in Poland

Abstract Over the last 40 years, in vitro tissue culture has developed dynamically and has become a popular technique for scientific research in the field of biology. Initially, studies were carried out to develop procedures to obtain callus cultures, cell suspensions, and protoplasts of various plant species. Over time, these cultures have been used to analyze the course of processes and mechanisms that occur at the cellular level, including the course of embryological development, formation of cellular structures, polyploidization, signal transduction, gene expression, and responses to various stress factors. In a minireview, different nutritional, hormonal, atmospheric, and light conditions occurring in in vitro cultures, which are stressful conditions compared to those in ex vitro plant culture, were discussed. In this review, some examples of physiological studies conducted on in vitro culture by Polish scientists are presented, including studies carried out to optimize the composition of media that induce callus and plant regeneration; determine the use of in vitro culture for the preservation of endangered plant species; understand the mechanisms of resistance responses to pathogens, salinity, nutritional stress, and low temperatures; and determine the potential production of plants with different chemical compositions. The potential of sterile plant culture is large and beyond the scope of its current use. Therefore, the number and variety of applications of these cultures will be significantly greater in the future.

Improvement of cell suspension cultures of transformed and untransformed Carica papaya cell lines, towards the development of an antiparasitic product against the gastrointestinal nematode Haemonchus contortus.

Parasitic diseases have a major impact on human and animal health worldwide. Despite the availability of effective anti-parasitic drugs, their excessive and uncontrolled use has promoted the emergence of drug resistance, severely affecting ecosystems and human health. Thus, developing environmentally friendly antiparasitic treatments is urgently needed. Carica papaya has shown promising effects against infectious diseases. C. papaya embryogenic calluses were genetically modified by our research team to insert immunogenic peptides with the goal of developing an oral anti-cysticercosis vaccine. Among these callus cell lines, one labeled as CF-23, which expresses the KETc7 immunogenic peptide, induced the highest protection levels against experimental cysticercosis. In the process of designing a natural antiparasitic product based on C. papaya that simultaneously induced immunity against cysticercosis, both transformed (SF-23) and untransformed (SF-WT) suspension cultures were produced and optimized. Our results showed a better duplication time (td) for SF-23 (6.9 days) than SF-WT (13.02 days); thus, the SF-23 line was selected for scale-up in a 2-L airlift bioreactor, reaching a td of 4.4 days. This is the first time that a transgenic line of C. papaya has been grown in an airlift bioreactor, highlighting its potential for scale-up cultivation in this type of reactor. Considering the previously reported nematocidal activity of C. papaya tissues, their activity against the nematode Haemonchus contortus of aqueous extracts of SF-WT and SF-23 was explored in this study, with promising results. The information herein reported will allow us to continue the cultivation of the transgenic cell suspension line of C. papaya under reproducible conditions, to develop a new anti-parasitic product.

Multiphoton Imaging of Blood Vessels and Mesenchymal Progenitor Cells in Callus of Mice with Tibial Fracture Longitudinally

Multiphoton Imaging of Blood Vessels and Mesenchymal Progenitor Cells in Callus of Mice with Tibial Fracture Longitudinally

Comparison of Polyphenol Levels of Callus and Wild Type of Cat's Whiskers Plant ( Orthosiphon aristatus (Blume) Miq.) Purple Varieties

The cat's whiskers plant ( Orthosiphon aristatus (Blume) Miq) that grows in Indonesia has white flowers, purple flowers, and white flowers with purple patterns. The quality of the secondary metabolite content of each cat's whiskers plant is different because it depends on environmental influences such as rainfall or soil mineral content. Plant tissue culture methods such as callus culture or cell culture are used to overcome these obstacles because these methods do not depend on the environment and can produce secondary metabolites such as those produced by the original plant. Cat's whiskers contain the main secondary metabolite compounds: rosmarinic acid, eupatorin, and sinensetin. Rosmarinic acid belongs to the phenolic group, while sinensetin and eupatorin are flavonoids. This study aimed to determine the total levels of polyphenols and flavonoids in purple cat whiskers and determine the ratio of secondary metabolites of cat whiskers callus with wild-type plants—determination of polyphenol content using Folin Ciocalteau reagent. Determining flavonoid levels is based on colorimetry using AlCl 3 reagent, specific for flavone and flavonol compounds. The levels of polyphenols contained in the ethanolic extract of callus and cat whiskers of the purple variety were 16.056 ± 0.204 mgQE/g and 30.780 ± 0.425 mgQE/g, while the polyphenol content of the callus ethyl acetate extract and the purple variety of cat whiskers leaves was 15,489 ± 0.425 mgQE/g and 13,262 ± 0.044 mgQE/g. The levels of flavonoids contained in the ethanol extract of the cat whiskers leaf were 1.39±0.03 mgQE/g and callus 0.16±0.02 mgQE/g in the ethyl acetate extract of the leaves of the cat whiskers 2.16±0.012 mgQE/g and callus 2.24±0.010 mgQE/g. The polyphenols and flavonoids in the callus ethyl acetate extract were more significant than the ethyl acetate extract of the purple variety ( wild type ) cat whiskers leaf.

GhTCE1-GhTCEE1 dimers regulate transcriptional reprogramming during wound-induced callus formation in cotton.

Wounded plant cells can form callus to seal the wound site. Alternatively, wounding can cause adventitious organogenesis or somatic embryogenesis. These distinct developmental pathways require specific cell fate decisions. Here, we identify GhTCE1, a basic helix-loop-helix family transcription factor, and its interacting partners as a central regulatory module of early cell fate transition during in vitro dedifferentiation of cotton (Gossypium hirsutum). RNAi- or CRISPR/Cas9-mediated loss of GhTCE1 function resulted in excessive accumulation of reactive oxygen species (ROS), arrested callus cell elongation, and increased adventitious organogenesis. In contrast, GhTCE1-overexpressing tissues underwent callus cell growth, but organogenesis was repressed. Transcriptome analysis revealed that several pathways depend on proper regulation of GhTCE1 expression, including lipid transfer pathway components, ROS homeostasis, and cell expansion. GhTCE1 bound to the promoters of the target genes GhLTP2 and GhLTP3, activating their expression synergistically, and the heterodimer TCE1-TCEE1 enhances this activity. GhLTP2- and GhLTP3-deficient tissues accumulated ROS and had arrested callus cell elongation, which was restored by ROS scavengers. These results reveal a unique regulatory network involving ROS and lipid transfer proteins, which act as potential ROS scavengers. This network acts as a switch between unorganized callus growth and organized development during in vitro dedifferentiation of cotton cells.

Ultrasonic Extraction and Separation of Taxanes from Taxus cuspidata Optimized by Response Surface Methodology

Taxanes are natural compounds with strong antitumor activity. In this study, we first extracted taxanes from the needles of Taxus cuspidata using ultrasonic (US) extraction, and then assessed the effects of different extraction conditions on the yields of eight target compounds. Response surface methodology (RSM) was further used to optimize the extraction conditions: when the liquid-to-solid ratio was 20.88 times, ultrasonic power was 140.00 W, ultrasonic time was 47.63 min, and ethanol content in solvent was 83.50%, taxane yields reached the maximum value of 354.28 μg/g. Under these conditions, the actual extraction rate of taxanes from the needles was 342.27 μg/g. The scanning electron microscopy (SEM) results indicated that the morphology of the needles, suspension cells, and callus of Taxus cuspidata extracted by ultrasonic wave had changed, the pores of the sections of the needles extracted by ultrasonic wave had become relatively loose, and the pore diameter had obviously increased. The callus and overall structure of the suspension cells extracted by ultrasonic wave were destroyed, forming cell fragments. The components of Taxus cuspidata are complex; the high-performance liquid chromatography (HPLC) method established in this paper is suitable for the rapid and effective separation of taxanes in Taxus cuspidata. We systematically and comprehensively compared the yields of taxanes in needles, callus, and suspension cells of Taxus cuspidata, and the taxane yields were increased by the suspension cell culture.

Simulated microgravity contributed to modification of callogenesis performance and secondary metabolite production in CannabisIndica

In vitro plant culture paves the way for meeting the industrial demand of pharmaceutically valuable secondary metabolites. This study intends to monitor how callus cells of Cannabis indica respond to the simulated microgravity (clinorotation; a Man-made technology). Callus initiation resulted from the culture of the leaf explant in a medium supplemented with kinetin (0.5 mgL−1) and 2, 4-D (2 mgL−1). Calli were treated with microgravity at three exposure times (0, 3, and 5 days). The microgravity treatments increased callus biomass about 2.5-fold. The clinorotation treatments transcriptionally induced the olivetolic acid cyclase (OAC) and olivetol synthase (OLS) genes about 6.2-fold. The tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes displayed a similar upward trend in response to microgravity. The applied treatments also stimulated the expression of the ethylene-responsive element-binding proteins (ERF1B) and WRKY1 transcription factors by an average of 7.6-fold. Moreover, the simulated microgravity triggered epigenetic modification in the DNA methylation profile. The HPLC-based assessment validated the high efficacy of the clinorotation treatments to increase the concentration of cannabinoids, including Cannabigerol (CBG) and Cannabidiol (CBD). However, the clinorotated calli contained a lower concentration of Tetrahydrocannabinol (THC) than the control group. The microgravity treatments increased concentrations of proline (79%), soluble sugars (61.3%), and proteins (21.4%) in calli. The biochemical assessment revealed that the clinorotation treatments slightly increased H2O2 concentration. The upregulation in the activities of peroxidase, catalase, and phenylalanine ammonia-lyase enzymes resulted from the microgravity treatments. Both HPLC and molecular assessments validated the significant efficacy of microgravity to enhance the production of cannabinoids.

Establishment of Stem Cell-like Cells of Sida hermaphrodita (L.) Rusby from Explants Containing Cambial Meristems.

The in vitro cultures of plant stem cells and stem cell-like cells can be established from tissues containing meristematic cells. Chemical compounds—as well as their production potential—is among the emerging topics of plant biotechnology. We induced the callus cell biomass growth and characterized the parameters indicating the presence of stem cells or stem cell-like cells. Four types of explants (stem, petiole, leaf, root) from Sida hermaphrodita (L.) Rusby and various combinations of auxins and cytokinins were tested for initiation of callus, growth of sub-cultivated callus biomass, and establishment of stem cells or stem cell-like cells. Induction of callus and its growth parameters were significantly affected both by the explant type and the combination of used plant growth hormones and regulators. The responsibility for callus initiation and growth was the highest in stem-derived explants containing cambial meristematic cells. Growth parameters of callus biomass and specific characteristics of vacuoles confirmed the presence of stem cells or stem cell-like cells in sub-cultivated callus cell biomass. Establishment of in vitro stem cell or stem cell-like cell cultures in S. hermaphrodita can lead to the development of various applications of in vitro cultivation systems as well as alternative applications of this crop.