Root Explants Research Articles

Spatial–Temporal Dynamics of Adventitious Roots of Typha domingensis Pers. Seedlings Grown with Auxin/Cytokinin

The spatial–temporal dynamics of an in vitro radicular system of Typha domingensis for the development of rhizofiltration technologies, with the potential for use as a phytotreatment of eutrophicated water, were studied for the first time in the roots of seedlings and in rhizotron systems. The effect of indole-3-acetic acid (AIA) in combination with kinetin (CIN) or 6-benzylaminopurine (BAP) on seedlings cultivated in the light and dark in three radicular systems and in a rhizotrophic regime for the screening of dynamic rhizogenic lines, by weekly allometric measurements of the length and number of roots, were studied. Inhibition of the elongation and branching velocities of roots by BAP and light was observed but CIN increased elongation and branching. In rhizotrons cultivated in light and dark conditions with different AIA/CIN ratios, isolated root explants remained inactive; however, roots attached to a meristematic base presented a significant increase in growth development, with values comparable to those of roots attached to seedlings cultivated in light without hormones. The results revealed that six adventitious rhizogenic root lines with basal meristems have the potential for use in a wide range of environmental and innovative applications in phytotreatment technologies involving eutrophicated water.

Optimizing In Vitro Propagation of Haworthia truncata Schönland Using Leaf, Root, and Inflorescence

Haworthia truncata, a species native to South Africa, is characterized by its limited growth and scarcity, contributing to high production costs. Countries like China and Turkey are known for exporting Haworthia globally. Tissue culture offers an efficient method for mass-producing unique and beautiful species such as H. truncata. This study tested Murashige and Skoog (MS) basal media supplemented with various concentrations of IBA (0.05–1.5 mg/L), NAA (0.05–0.25 mg/L), and BA (0.25–1.5 mg/L) to promote shoot proliferation. MS medium without plant growth regulators (PGRs) was also tested as a control. Different explant types (leaf, root, and inflorescence) were analyzed for their potential in direct and indirect regeneration. Inflorescence explants showed the highest callus induction with 1.5 mg/L IBA, while optimal shoot proliferation occurred at 1 mg/L IBA. Callus induction was optimal for leaf explants with 0.05 mg/L NAA and 0.25 mg/L BA, and shoot proliferation was highest at 0.05 mg/L NAA and 1 mg/L BA. Root explants achieved maximum callus induction with 0.25 mg/L BA and 0.25 mg/L NAA, with the best shoot proliferation using 0.05 mg/L NAA and 1 mg/L BA. The highest rooting percentage of regenerated shoots was obtained on ½ MS medium with 1.5 mg/L IBA.

Poplar transformation with variable explant sources to maximize transformation efficiency

For decades, Agrobacterium tumefaciens-mediated plant transformation has played an integral role in advancing fundamental and applied plant biology. The recent omnipresent emergence of synthetic biology, which relies on plant transformation to manipulate plant DNA and gene expression for novel product biosynthesis, has further propelled basic as well as applied interests in plant transformation technologies. The strong demand for a faster design-build-test-learn cycle, the essence of synthetic biology, is, however, still ill-matched with the long-standing issues of high tissue culture recalcitrance and low transformation efficiency of a wide range of plant species especially food, fiber and energy crops. To maximize the utility of plant material and improve the transformation productivity per unit plant form, we studied the regeneration and transformation efficiency of different types of explants, including leaf, stem, petiole, and root from Populus, a woody perennial bioenergy crop. Our results show that root explants, in addition to the above-ground tissues, have considerable regeneration capacity and amenability to A. tumefaciens and, the resulting transformants have largely comparable morphology, reporter gene expression, and transcriptome profile, independent of the explant source tissue. Transcriptome analyses mapped to regeneration stages and transformation efficiencies further revealed the expression of the auxin and cytokinin signaling and various developmental pathway genes in leaf and root explants undergoing early organogenesis. We further report high-potential candidate genes that may potentially be associated with higher regeneration and transformation efficiency. Overall, our study shows that explants from above- and belowground organs of a Populus plant are suitable for genetic transformation and tissue culture regeneration, and together with the underlying transcriptome data open new routes to maximize plant explant utilization, stable transformation productivity, and plant transformation efficiency.

Cytoplasmic G6PDs modulate callus formation in Arabidopsis root explants through regulation of very-long-chain fatty acids accumulation

Cytoplasmic G6PDs modulate callus formation in Arabidopsis root explants through regulation of very-long-chain fatty acids accumulation

The First Protocol for In Vitro Propagation of Kalanchoe beharensis Through Adventitious Shoots, a Preliminary Study

Kalanchoe beharensis, a vulnerable species according to the International Union for Conservation of Nature, is highly prized for its ornamental value and medicinal properties. Therefore, an efficient methodology to propagate this ecologically significant species would be of particular interest. The propagation of K. beharensis has traditionally been achieved by seed or cuttings, but these methods are limited in efficiency. Micropropagation provides a more efficient and controlled alternative by enabling the in vitro production of numerous plants in a small space and in a short period of time. Despite its advantages, no micropropagation protocol for K. beharensis has been reported in the literature. In this study, we report an efficient in vitro regeneration protocol for K. beharensis. In order to implement this, we evaluated the morphogenetic response of leaf and root explants in media supplemented with auxins, cytokinins, or a combination of both growth regulators. Surprisingly, the best results were observed in indole-3-acetic acid-supplemented media. Adventitious shoots were rooted in either hormone-free or auxin-supplemented media, with indole-3-acetic acid yielding the best results. Rooted plants were acclimatized in the greenhouse, achieving over 80% survival during acclimatization. This protocol improves multiplication rate, space utilization, and uniformity, providing a viable alternative to conventional propagation methods.

Dynamic changes of endogenous phytohormones and carbohydrates during spontaneous morphogenesis of Centaurium erythraea Rafn.

Common centaury (Centaurium eryhtraea Rafn) is a medicinal plant species with vigorous morphogenic potential in vitro. The process of spontaneous shoot regeneration in a solid root culture is characteristic for this plant species. In this context, the aim of this work was to investigate the dynamic changes of endogenous phytohormones and carbohydrates content in root explants at different time points (0, 2, 4, 7, 14, 21, 28, and 60 days) during spontaneous centaury morphogenesis in vitro. Detailed analysis of cytokinins (CKs) showed that trans-zeatin (tZ) was the major bioactive CK at all time points. The corresponding riboside, tZ9R, was also determined in the majority of the identified transport forms, at all time-points. Further analysis of endogenous auxin revealed a significant increase in endogenous indole-3-acetic acid (IAA) after 21 days, when a huge jump in the ratio of IAA/bioactive CKs was also observed. The maximum total soluble sugar content was measured after 14 days, while a significant decrease was determined after 21 days, when the first regenerated adventitious shoots appeared. This undoubtedly indicates an increased energy requirement prior to the actual regeneration of the shoots. The obtained results indicate that the period from day 14 to day 21 involves the most dramatic disturbances in endogenous bioactive CKs, IAA and carbohydrate balance, which are very important and valuable factors for the onset of shoot regeneration.

EXPRESIÓN EMBRIOGÉNICA DIRECTA EN Agave cupreata

<p><strong>Background:</strong> <em>Agave cupreata</em> is a specie that can only reproduce by seeds and could take up to 15 years until flowering, placing it at risk of extinction due to its agroindustrial exploitation to produce mezcal. <strong>Objective:</strong> To evaluate the type of explant, the vitamin complex and 2,4-D concentrations on the direct embryogenic response. <strong>Methodology:</strong> Explants of leaf, root and stem sections were evaluated. Also, the vitamins MS (Murashige and Skoog, 1962) and L2 (Phillips and Collins, 1979), five concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), (0.0, 0.1, 0.3, 0.6 and 0.9 mg L<sup>-1</sup>) and six concentrations of indole-3-acetic acid (IAA), (0.0, 0.1, 0.5, 1.0, 2.0 and 3.0 mg L<sup>-1</sup>) were assayed for the expression of somatic embryos directly. <strong>Results: </strong>The leaf explant, the vitamin L2 complex, and the concentration of 0.9 mg L<sup>-1</sup> of 2,4-D and 0.5 mg L<sup>-1</sup> of IAA turned out to be the most efficient for the formation of pro-embryogenic masses and the expression of direct somatic embryos in the globular state. <strong>Implications:</strong> These results lay the groundwork for the future regeneration of <em>A. cupreata</em> plants via direct somatic embryogenesis, potentially facilitating their conservation and mass propagation. This contributes to the sustainability of mezcal; boosts the local economy, preserves biodiversity, and ensures the continued production of this endangered species. <strong>Conclusions:</strong> For the first time, the successful expression of somatic embryos directly from <em>A. cupreata</em> explants was evidenced.</p>

Comparative analysis of the transcriptomes from regenerated plants and root explants of endangered Oplopanax elatus.

Oplopanax elatus is a plant of therapeutic significance in oriental medicine; however, its mass cultivation is limited owing to the difficulties in propagating it from seeds. In this study, we investigated the transcriptome profiles and transcriptional regulatory factors expressed during plantlet regeneration from root tissues of the endangered O. elatus. The RNA-seq results for the control and regenerated plants cultured in liquid medium for 8weeks showed that the clean length of the control group was 11,901,667,912 and that of the 8-week sample was 10,115,155,171, indicating a clean value of 97% for both samples. The number of mapped paired-end reads was 63,922,480 for the control group and 54,146,902 for the 8-week sample. The number of genes for which at least one clean data point was mapped was 43,177 in the control group and 42,970 in the 8-week sample. The results of the differentially expressed gene analysis indicate that the number of upregulated genes in the 8-week sample was 158, and the number of downregulated genes was 424. Gene Ontology (GO) analysis of the upregulated genes revealed that GO terms were classified into 14 categories, and genes expressed in the biological process category occurred most frequently. GO terms of the downregulated genes were evenly distributed into two categories: biological process and molecular function. From the upregulated genes, eight reference genes with significant differences in expression were selected and analyzed using real-time PCR. The Oe38836 gene (late embryogenesis abundant protein M17-like isoform X1) showed the highest expression rate that was more than tenfold that of the control. Oe40610 (auxin-responsive protein SAUR21-like) and Oe07114 (glucose-1-phosphate adenyl transferase-like protein) genes showed expression levels that were increased eightfold relative to the control. The RNA sequencing (RNA-seq) results from the plants regenerated through liquid culture of O. elatus root tissue were confirmed using real-time PCR, indicating their reliability.

Micropropagation and genetic transformation of Byblis liniflora

Byblis, a small genus of carnivorous plants predominantly found in Australia, is characterized by its passive trapping mechanism and unique floral features. The chemical composition of Byblis, including identified phenylethanoid glycosides, particularly acteoside, highlights its pharmacological potential with various biological activities. In vitro culture techniques have been established for propagation, with micropropagation protocols developed for different Byblis species. However, information on genetic transformation, vital for trait modification and enhanced pharmacological interest, remains limited. This study focuses on optimizing micropropagation, adventitious regeneration, and genetic transformation methods for Byblis liniflora. Adventitious regeneration rates were highest in medium with reduced Murashige and Skoog salts (MS/10) and sucrose (3 gL−1) concentrations. Zeatin supplementation (1 mgL−1) further improved regeneration rates and bud development with 100% of regenerated root explants and 8.8 shoots per explant. Liquid MB3 medium supplemented with indole-3-acetic acid (IAA) 5 mgL−1 facilitated efficient rooting and acclimatization. The establishment of an efficient Rhizobium-mediated genetic transformation method yielded transgenic plants expressing green fluorescent protein (GFP). Molecular analysis confirmed transgene integration, marking the first successful genetic transformation in the Byblis genus. These advancements pave the way for exploring gene function and enhancing pharmacological properties, thereby broadening our understanding and utilization of carnivorous plants like Byblis.

Antioxidant and Anticancer Activity of Tannins Isolated from Callus Cultures of Achyranthes aspera L.

In the current study, the impact of oxidative stress induced by free radicals generated during metabolic processes was investigated. It was found to be linked to a variety of diseases due to their detrimental effects on nucleic acids and proteins. The primary focus remained on investigating the free radical-scavenging properties and potential anticancer activities of tannins found in callus cultures of A. aspera. It was observed that the induction of callus formation was notably successful when using leaf and root explants, resulting in a callus index as high as 160, as compared to stem explants in the presence of auxins. The extraction yield of callus tannins was the most abundant in chloroform extracts, although the overall antioxidant activity was comparable among chloroform, methanol, and petroleum ether callus extracts. Among these extracts, the chloroform extract from stem callus cultures grown on the MSDN medium showed the highest total antioxidant activity. Notably, tannins extracted from A. aspera leaf callus extracts displayed significant anticancer effects against the Jurket cell line. These effects were evaluated through measures such as cell viability and colony formation. The anticancer activity was notably higher in callus culture extracts as compared to the control. In summary, the results showed that in vitro biomass production can be a valuable approach for augmenting the production of bioactive compounds, such as tannins, from selected medicinal plants including A. aspera. Tannins extracted from A. aspera hold promise for their potential role in the development of new medicines.

Clonal Propagation of Turmeric (Curcuma longa) and Confirmation of Genetic Fidelity of the Micropropagated Shoots by RAPD Markers

Turmeric (Curcuma longa) is used as a spice and has excellent medicinal value. However, the field multiplication rate of turmeric plants is not satisfactory. In vitro methods could be a feasible alternative to vegetative propagation in the field. This study was aimed at developing a suitable protocol for the clonal propagation of turmeric confirming the genetic fidelity of the in vitro regenerated plantlets. The explants of shoot bud (full, half, and quarter), root, rhizome, and leaf were cultivated on MS medium with BAP, NAA, and Kinetin. Cultured shoot buds showed a higher regeneration frequency and shoots per explant than those obtained from other explants. The highest shoot regeneration was 72.49% from shoot buds, followed by rhizome (68.76%) and the lowest was 53.93 % from ¼ shoot buds. There were significant effects of growth regulator combinations and concentrations in shoot regeneration from shoot bud explant. The highest shoot regeneration per shoot bud explant was 4.42 with BAP at 20.0 mg/l and NAA at 0.5 mg/l at 24 weeks. The best rooting response with 9.75 roots per explant was reported with 1.0 mg/l BAP with 1.0 mg/l NAA. The rooted plants were successfully transferred to pots for their acclimatization. Both the micropropagated shoots and their mother plant produced monomorphic bands with the six RAPD markers that confirm the genetic fidelity of micropropagated clones. These results showed that in vitro raised plantlets of turmeric had no risk of somaclonal variations and were found to be true-to-type in nature over the culture period. The protocol developed through this investigation has the potential application for the rapid multiplication of turmeric plants. Plant Tissue Cult. & Biotech. 34(1): 55-69, 2024 (June)

A single-cell transcriptome atlas reveals the trajectory of early cell fate transition during callus induction in Arabidopsis

The acquisition of pluripotent callus from somatic cells plays an important role in plant development studies and crop genetic improvement. This developmental process incorporates a series of cell fate transitions and reprogramming. However, our understanding of cell heterogeneity and mechanisms of cell fate transition during callus induction remains quite limited. Here, we report a time-series single-cell transcriptome experiment on Arabidopsis root explants that were induced in callus induction medium for 0, 1, and 4 days, and the construction of a detailed single-cell transcriptional atlas of the callus induction process. We identify the cell types responsible for initiating the early callus: lateral root primordium-initiating (LRPI)-like cells and quiescent center (QC)-like cells. LRPI-like cells are derived from xylem pole pericycle cells and are similar to lateral root primordia. We delineate the developmental trajectory of the dedifferentiation of LRPI-like cells into QC-like cells. QC-like cells are undifferentiated pluripotent acquired cells that appear in the early stages of callus formation and play a critical role in later callus development and organ regeneration. We also identify the transcription factors that regulate QC-like cells and the gene expression signatures that are related to cell fate decisions. Overall, our cell-lineage transcriptome atlas for callus induction provides a distinct perspective on cell fate transitions during callus formation, significantly improving our understanding of callus formation.

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.

Synthesis and Accumulation of Phytocompounds in Field-, Tissue-Culture Grown (Stress) Root Tissues and Simultaneous Defense Response Activity in Glycyrrhiza glabra L.

Harsh climates, i.e., drought, extreme temperatures, and toxic gases, pose issues to agriculture by altering plants’ growth and yield. Biotechnology with biochemical defense approaches is beneficial for generating new plants/varieties with extra resilience to adverse conditions. In response to stress, cultures show an enriched level of secondary metabolite synthesis. Here, an efficient in vitro propagation method using axillary shoot proliferation, along with callus formation, was established in Glycyrrhiza glabra L. The phytochemical composition of in vitro and in vivo grown tissues was analyzed using a gas chromatography–mass spectrometry (GC–MS) technique, and the biochemical attributes were measured and compared in different investigated tissues. Callus formation from root explants was achieved with a frequency of 88.89% on MS medium containing 2.0 mg/L BAP and 0.5 mg/L 2,4-D. Axillary shoot proliferation was obtained from dormant buds when cultured onto MS supplemented with BAP alone, or in combination with, IAA. The maximum shoot proliferation (94.44%) was recorded on MS with 1.0 mg/L BAP with an average shoot length of 10.5 cm. The regenerated shoots were subcultured and transferred to the root induction medium, supplemented with various concentrations of IAA/IBA, wherein 2.0 mg/L IBA resulted in the best rooting frequency (88.89%). The GC–MS-based phytocompounds analysis of the methanolic extracts of root-derived callus and in vivo- and in vitro- grown root tissues was conducted. These samples revealed the presence of more than 35 therapeutically important bioactive compounds, such as methylglabridin, sitosterol, lupeol, squalene, stearic acid, linoleic acid, etc. The biochemical parameters, like total phenolic content, flavonoid content, DPPH scavenging activity, superoxide dismutase, and peroxidase activity were also measured. All the biochemical attributes were found to be higher in in vitro derived roots than the callus and in vivo grown root (donor) samples. These findings demonstrated that callus (root derived) and in vitro roots are a stable and potent source of multiple phytocompounds, encompassing medical significance with wide applications. This study may serve as an alternative opportunity in the sustained and continuous synthesis of important compounds without harming natural vegetation and normal environment.

Echinacea purpurea L. Kallus Kültürlerinde Karbon Kaynağı, Nitrojen ve PEG'in Kafeik Asit Türevlerinin Üretimine Etkisi

This study aimed to determine the effects of polyethylene glycol (PEG) as an abiotic elicitor and nutritional factors (different ammonium/nitrate ratios, carbon source and amount) in the culture medium on the production of Caffeic Acid Derivatives (CADs) in callus cultures of Echinacea purpurea L. Petiole and root explants were cultured on MS medium modified in terms of different types (sucrose and maltose) and amounts (sucrose 15, 45, 60 g l-1, and maltose 15, 30, 45, 60 g l-1) of carbon source, different concentrations (5, 10, 15 g l-1) of PEG and ammonium nitrate ratios (0:35, 5:25, 15:15, 35:0 mM). The amounts of CADs in the callus obtained at the end of the 10-week culture period were analysed. In both explant types, the highest amount of CADs were obtained from the medium containing 15 g l-1 sucrose and 15 or 30 g l-1 maltose applications, while the highest amount of CADs was obtained in the medium containing 0:35 mM ammonium/nitrate in nitrogen applications. While the highest amount of CADs in root explant was obtained from the medium containing 10 g l-1 PEG applications, CADs content could not be obtained in petiole explant. As a result, the highest amounts of caftaric, chlorogenic, caffeic, and chicoric acids (respectively, 9.38, 0.71, 0.29, and 34.77 mg g-1) were determined at callus obtained from root explant cultured on MS medium containing 30 g l-1 sucrose and 0:35 mM ammonium/nitrate. In conclusion, optimization of culture conditions and different elicitor applications were made to increase secondary metabolite content in E. purpurea L. under in vitro conditions and the results obtained were presented comparatively.

In vitro propagation and DNA barcoding of the rare near endemic Plantago sinaica (Barnéoud) plant in Saint Katherine, Sinai

Plantago sinaica is a rare perennial shrub near-endemic to Egypt and found in Saint Katherine Protectorate in Sinai. The first successful in vitro propagation protocol was conducted to protect the plant outside its natural reserves. Shoot tip, stem node section, cotyledonary node, and root explants separated from in vitro germinated seedlings were cultured in vitro on Murashige and Skoog (MS) medium enriched with different concentrations and types of cytokinins. It was found that 6-benzyl adenine (BA) is the most efficient cytokinin. MS medium containing 3.33 µM BA and 0.54 µM α-naphthalene acetic acids (NAA) produced 10.25 and 11.30 shoots/explant using shoot tip and stem node section, respectively. Conversely, MS medium + 2.22 µM BA + 0.54 µM NAA produced 13.25 shoots from root explants. Surprisingly, the cotyledonary node explants favored MS medium free from plant growth regulators (PGRs), which produced only 4.25 shoots/explant. The multiplied shoots were rooted successfully with a 100% rooting percentage on half MS medium containing 1.23 or 2.46 µM indole-3-butyric acid (IBA). In vitro, rooted plantlets were efficiently transferred to the greenhouse with a 90% survivability. Finally, the plant was identified using three DNA barcodes; 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL), plastid photosystem II protein D1 intergenic spacer region (psbA–trnH), and Internal Transcribed Spacer (ITS) barcodes. Additionally, psbA–trnH and ITS were novel and submitted to the GenBank databases for the first time for Plantago sinaica. Our study supports the United Nations Sustainable Development Goal number 15, which is to preserve, restore and reinstate sustainable usage of terrestrial ecosystems and to stop biodiversity loss.

In Vitro Propagation of Commercially Used Krymsk 5® (Prunus fruticosa × Prunus lannesiana) Cherry Rootstock: Impact of Sugar Types and pH Levels

In the present study, the effects of different types of sugars and cultivation medium pH levels on the micropropagation of Krymsk 5® cherry rootstock were investigated. During the proliferation stage, the effects of four sugars (sucrose, fructose, glucose, and sorbitol) both separately and in two combinations were studied, along with the effects of pre-adjusted pH (4.5, 5.0, 5.2, 5.5, 5.8, 6.0, 6.2, or 6.5) on shoot proliferation parameters, growth medium’s post-autoclaving and post-cultivation pH, and their relations. Similarly, during the rooting stage, the effects of four sugars (sucrose, glucose, fructose, or sorbitol) at three concentrations (1% w/v, 2% w/v, or 3% w/v) without any auxin inclusion were studied as well as the effects of two sugars (sucrose or fructose) at six pre-adjusted pH levels (4.8, 5.2, 5.8, 6.2, or 6.5), also in the absence of auxin, on rooting parameters. Explants cultivated in fructose-supplemented growth mediums exhibited superior proliferation performance, characterized by the highest values of shoots per explant, shoot length, and nodes per explant. Generally, the medium’s pH decreased after autoclaving, and proliferation performance was favored by low pH values (either pre-adjusted or post-autoclaving). As far as rooting is concerned, fructose inclusion induced a higher rooting percentage (88%) compared to sucrose. The highest rooting was obtained in fructose-supplemented rooting mediums at concentrations of 2% or 3% w/v (95% rooting in both cases), in the absence of auxins. Post-autoclaving pH in fructose-supplemented rooting mediums was lower and buffered in low pH levels than in sucrose-supplemented ones, and the rooting of explants in all pH combinations with fructose exceeded 75%. In addition, rooting was negatively correlated with the post-autoclaving pH. These findings underscore the significance of both the sugar type and the post-autoclaving pH of the medium in both proliferation and rooting stages, highlighting their possible physiological, biochemical, or hormonal effects. Additionally, rooting without the use of auxin, but with the correct choice of sugar, emerges with both financial and environmental benefits, whereas fructose could be potentially used as a buffering agent.

Suspension culture of stem cells established of Calendula officinalis L.

Plant stem cell cultures have so far been established in only a few plant species using cambial meristematic cells. The presence of stem cells or stem cell-like cells in other organs and tissues of the plant body, as well as the possibility of de novo generation of meristematic cells from differentiated cells, allow to consider the establishment of stem cell cultures in a broader range of species. This study aimed to establish a stem cell culture of the medicinal plant Calendula officinalis L. Callus tissues were induced from leaf and root explants, and already at this stage, stem and dedifferentiated cells could be identified. The cell suspension cultures established both from the root- and leaf-derived calli contained a high proportion of stem cells (92–93% and 72–73%, respectively). The most effective combination of growth regulators for the development of stem cells in calli as well as cell cultures was 1.0 mg/L 2,4-D and 0.5 mg/L BAP. The highest amount of stem cells (5.60–5.72 × 105) was in cell suspension derived from the roots. An effective protocol for the establishment of marigold stem cell suspension culture was developed. The ratio of root-derived stem cells against dedifferentiated cells exceeded 90%.

Direct Shoot From Root and True-to-Type Micropropagation of Limonium "Misty Blue" in Partially Immersed Culture on an Aluminum Mesh Raft.

Commercial plant tissue culture now primarily serves the ornamental horticulture industry. The main pillars of the commercial tissue culture business are scalability of production, cost reduction, limited labor involvement, high quality, and genetic homogeneity of propagated plants. Based on these requirements, the current protocol employs a partially immersed liquid culture medium supported by a flexible aluminum mesh raft with a wire stand to facilitate shoot organogenesis from the horizontally placed root explants and hold the plants upright for shoot multiplication and rooting of Limonium Misty Blue. It is a florist crop that is in high demand as both dried and fresh flower fillers in various floral decorations. The majority of cultivated Limonium or statice cultivars are heterozygous in nature and propagate commercially through in vitro propagation to cater to the huge demand for planting materials needed for flower production. This is the first protocol to describe direct shoot organogenesis from the roots in a liquid half-component of Murashige and Skoog's (1962) (MS) basal medium supplemented with 1.6μM NAA and 1.1μM BA. The regenerated shoots are multiplied and rooted at the same time on the raft in a MS-based liquid culture medium that included 0.44μM BA and 1.07μM NAA. In comparison to agar-gelled medium, plants cultured in liquid medium grow more quickly without any signs of hyperhydricity. In liquid medium, a clump of 4-5 shoots is formed from a single shoot explant within 4weeks and are rooted simultaneously within 6weeks. On average, seven explants may fit on each raft, so on average, 25 healthy plants are produced from a single bottle. The regenerated plants are easily hardened in the greenhouse, and using ISSR-based molecular markers, the genetic homogeneity of the randomly selected hardened plants can be determined.

Multi-drops and Cross-sections, Efficient Methods for Establishing Cell Suspension Culture of Cuminum cyminum L. and Plant Regeneration

The current study aims of the current study is to establish cell suspension cultures of the medicinal plant, cumin Cuminum cyminum L. by the application of multiple drops and cross-section techniques. These methods were used in cultivating cell suspensions and in vitro plant regeneration. Leaf, stem, hypocotyl and root explants were cultured in Murashige and Skoog (MS) medium containing different concentrations (0.05, 0.5 and 1.0 mg.l-1) of naphthalene acetic acid (NAA) and (0.05, 0.1, 1.0 and 2.0 mg.l-1) benzyl adenine BA for callus production. The results indicated the high response of cumin, as the percentage of callus initiation was 100%.The plant regeneration percentage reached 91.6%. Moreover, a friable callus of hypocotyls was appropriate for initiation of cell suspension culture in MS medium with 0.5mg.l-1 NAA and 1.0 mg.l-1benzyl adenine (BA). The best density for primordial callus formation was 51.0 ×105 cells ml-1 in both multiple-drops and cross-sections embedding methods. Callus that had been produced from multi drops and sectors had the ability for shoot regeneration. This study clarified the efficiency of these techniques in establishing cell suspension culture and shoot regeneration, which could be promising sources for high production of active compounds in cumin plant.