Search Results (28)

Rhaponticum carthamoides (Willd.) Iljin. (Leuzea carthamoides, Maral root), a medicinally valuable species listed in the Red Book of Kazakhstan, is known for its rich phytochemical profile. However, limited data exist on its microclonal propagation. This study aimed to optimize in vitro and medium-term storage conditions using biotechnological methods. Mature seeds collected from natural populations in the Kazakhstani Altai were germinated, and tissues from the seedlings were used as explants. Sterile shoots were cultured on Murashige and Skoog (MS) medium supplemented with 3.0 mg L⁻¹ −6-benzylaminopurine and 3.0 mg L⁻¹ kinetin. For shoot induction, MS medium supplemented with 0.5 mg L⁻¹ meta-Topolin and using stem apices as explants yielded optimal results. Medium-term storage with chlorocholine chloride at 0.1–0.4 g/L effectively preserved regenerative capacity for further rooting. After 12 months of storage, plantlets were transferred to half-strength MS medium with 3.0 g/L activated carbon and at 2.0 or 5.0 mg L⁻¹ indole-3-butyric acid for rooting. Regenerated plants were successfully acclimatized ex vitro. The 20-hydroxyecdysone content in field-grown plants post-storage reached 9.24 mg/mL, 2.4-fold higher than in wild plants. Inter simple sequence repeat analysis confirmed genetic stability. Our optimized protocol ensures high-yield metabolite production and genetic fidelity, enabling in vitro conservation, nursery-scale cultivation, and the restoration of R. carthamoides natural populations. Full article

Adult trees of pedunculate oak (Quercus robur L.) are recalcitrant to vegetative propagation. In this study, we investigated the micropropagation of five oak genotypes corresponding to trees aged 60–800 years in a liquid medium. We used commercial RITA bioreactors to study the influence of the explant type, the culture medium, shoot support and number of immersions. Variables evaluated included the number of normal and hyperhydric shoots, shoot length, multiplication coefficient and number of rootable shoots per explant. All genotypes could be cultured in temporary immersion. Basal stem sections attached to callus grew better than apical sections and developed less hyperhydricity. For long-term cultivation, Gresshoff and Doy medium was the best of the three media evaluated. All genotypes produced vigorous shoots suitable for rooting and acclimation. This is the first protocol to proliferate adult oak trees in bioreactors, representing significant progress towards large-scale propagation of this and other related species. Full article

Plant-specific WUSCHEL (WUS)-related homeobox (WOX) family of transcription factors are involved in apical meristem maintenance, embryogenesis, lateral organ development, and hormone signaling. Among the members of this family, WOX1 is known to play essential roles in many species. However, the function of the peony ‘Feng Dan’ (Paeonia ostii L.) WOX1 (PoWOX1) remains unknown. The initial bioinformatic analysis revealed that PoWOX1 belongs to the modern clade of the WOX gene family and has a highly conserved homeodomain (HD), the WUS motif, the STF-box, and the MAEWEST/WOX4-box. Subsequent heterologous overexpression in Arabidopsis thaliana revealed that PoWOX1 promotes root growth, early shoot initiation, and flowering. The root vascular tissues, especially the arrangement and size of xylem cells, were different between the PoWOX1-overexpressing transgenics and the wild-type plants, and the pericycle cells adjacent to the xylem divided more easily in the transgenics than in the wild type. Furthermore, under in vitro conditions, the transgenic leaf explants exhibited more callus induction and differentiation than the wild-type leaf explants. Thus, the study’s findings provide novel insights into the role of PoWOX1 in promoting root development and callus tissue induction and differentiation, serving as a reference for developing an efficient regeneration system for the peony. Full article

Apical dominance and culture heterogeneity significantly limit the efficiency of olive micropropagation, hindering the rapid production of plantlets. This study explores how manipulating the explant origin (topophysis) and density can mitigate these challenges. Explants originating from apical and middle sections were cultivated at densities of 18, 24, and 30 explants per vessel. After 12 weeks, significant differences in the growth parameters were observed based on the explant origin and density. The middle-section explants exhibited superior shoot proliferation and node production, especially at higher densities. The callus weight also increased with the density, while the internode length remained relatively stable. Hormone analysis demonstrated the density-dependent spatial distribution pattern of aromatic and isoprenoid cytokinins. Notably, at higher densities, the aromatic free bases in the apical-section leaves showed migration toward the shoot apices, while this migration was less pronounced in the middle-section leaves. Isoprenoid cytokinins displayed complex distribution patterns, with free bases and O-glucosides often increasing toward the basal nodes. These findings demonstrate that optimizing the explant origin and density can effectively reduce apical dominance and enhance culture homogeneity in olive micropropagation. This approach offers a promising strategy for improving the micropropagation protocols for olive and potentially other woody plants, leading to more efficient and cost-effective production of high-quality plantlets for commercial use. Full article

Ballota acetabulosa (L.) Benth. (syn. Pseudodictamnus acetabulosus (L.) Salmaki and Siadati), f. Lamiaceae, the Greek horehound, is a compact evergreen small shrub native to Greece, with hairy grey-green leaves, that bears small pink-purple flowers with green conical calyxes along its erect stems in late spring. The species stands out for its high resistance in xerothermic conditions and therefore it is advisable to promote its use in xeriscaping. The aim of this study was to develop an efficient protocol for in vitro propagation of B. acetabulosa for introduction into the horticultural and pharmaceutical industries. Shoot tip and single node explants derived from in vitro seedlings were cultured on MS medium with various cytokinin types and concentrations. Explants responded at almost 100% to produce high number of shoots on a medium with 1.0 mg L⁻¹ zeatin or 6-benzyladenine. However, there was intense hyperhydricity in the cultures, which was addressed in further experiments by increasing agar concentration from 8 to 12 g L⁻¹, preserving high multiplication indices (92% response, 10.2 shoots per explant). Microcuttings with 2–3 visible nodes, either from the apical part, including the apical meristem, or from the basal part of microshoots, as well as microshoot clusters, rooted 100% on full- or half-strength MS medium, respectively, regardless of the addition of indole-3-butyric acid (ΙΒA, 0.5–4.0 mg L⁻¹) in the rooting medium. However, middle level concentrations of IBA increased the number and length of roots produced, while the higher its concentration, the more and longer axillary shoots developed in the microcuttings during the rooting period. The acclimatization of all plantlets was completely successful (100%) in ex vitro conditions on peat/perlite substrate (1:1, v/v). Thus, efficient methods of producing propagation material to promote Ballota acetabulosa as a horticultural and medicinal plant were developed. In particular, rooting of microshoot clusters or microcuttings without the shoot tip, in the presence of 1.0 mg L⁻¹ IBA, leads to a plant of suitable shape for the floricultural market, without the need for further manipulation (pruning) in the nursery. Full article

This study aims to establish efficient in vitro propagation protocols for Gymnocalycium cv. Fancy, an ornamental cactus with variegated variants, by evaluating the effects of cytokinin type and explant source on the organogenic response. Plants with different degrees of variegation (0–100%) were classified by size to obtain different explant types (apices, central discs, epicotyls, and hypocotyls). The effects of 6-Benzylaminopurine (BAP, 8 µM), Kinetin (KIN, 4 µM), and Thidiazuron (TDZ, 1 µM) were assessed on shoot production, callus formation, and rhizogenesis. Additionally, we studied the relationship between initial plant variegation and the productivity of the variegated shoots. The best shoot production results were obtained for central discs treated with 1 µM TDZ. Furthermore, a correlation was observed between the activated areole type (green, mixed, or fully colored) and shoot color percentage, enabling precise explant selection. The appearance of differently colored shoots confirms the potential for selecting new lines from this cultivar too. These findings hold significant potential not only for the breeding and propagation of ornamental cacti but also for the cultivation of other edible cacti and their relatives. Full article

Worldwide, the yellow-fleshed kiwifruit (Actinidia chinensis) is an important crop that possesses great economic significance due to its nutritional, medicinal and ornamental values. The call for the expansion of the kiwifruit industry in South Africa, due to rising local and international market demand, resulted in the introduction of new plant species in sub-mountainous areas, where soil and climate conditions are more suitable for intensive kiwifruit production than in lowland areas. Consequently, a need to develop suitable commercial protocols for mass propagation of A. chinensis emerged. This study introduces an optimized micropropagation protocol for A. chinensis, facilitating seed germination, seedling development and multiple shoot induction. For seed germination, the effect of cold stratification (CS) and gibberellic acid (GA₃) alone and in combination on in vitro germination of A. chinensis seeds was studied. Sterile seeds were stratified at 4 °C for 28 and 42 days. Batches of stratified and non-stratified (control) seeds were germinated on plant growth regulator-free Murashige and Skoog (MS) media and also on sterile filter paper bridges moistened with dH₂O and GA₃ concentrations of 500, 1000, 1500, 2000 and 2500 ppm. Seeds from the control and the CS treatments alone did not germinate on MS medium. However, on filter paper bridges, seeds cold stratified for 28 days yielded only a 20% germination percentage (GP), whereas CS for 42 days did not promote germination. A maximum GP of 64% and a mean germination time (MGT) of 27.52 days were achieved at a 2000 ppm GA₃ concentration. Cold stratification (28 days) followed by GA₃ treatments yielded an optimum GP of 80% and optimum MGT of 18.94 days at GA₃ concentrations of 500 ppm. In contrast, CS (42 days) followed by GA₃ yielded a maximum GP of 72% and MGT of 18.80 days at a GA₃ of 500 ppm. Conclusively, CS alone had little effect on germination, whereas CS (28 and 42 days) followed by GA₃ significantly (p ≤ 0.05) improved GP. Germinated seeds on moist filter paper can produce seedlings when sub-cultured on MS medium for seedling development. For multiple shoot induction, in vitro shoot culture of A. chinensis was carried out using apical and basal shoot explants from the above in vitro-produced seedlings. These explants were cultured on MS supplemented with 2.2 µM and 4.4 µM 6-Benzylaminopurine (BAP) for shoot multiplication. Axillary shoot proliferation was not observed on apical shoot explants after 4 weeks of culture on MS medium with 2.2 µM BAP. In contrast, the basal shoot explants produced 2–3 axillary shoots, tendrils and calluses at the base on the same medium. The highest number (3–4) of multiple shoots was attained from these basal shoot explants after subculture (10–12 weeks) in the same culture medium. In contrast, only elongation and rooting of apical shoot explants, without axillary shoot induction, occurred after the subculture. Regenerated plantlets derived from both apical and basal shoot explants were successfully acclimatised under a controlled environment at 24 ± 2 °C and 16 h photoperiod of 150–200 µmol m⁻² s⁻¹ light intensity. A similar response was observed for both types of explants of A. chinensis when cultured on MS with 4.4 µM BAP, although the higher concentration of BAP affected the morphological appearance of the regenerated plantlets that had shorter stems and smaller and narrower leaves compared to plantlets derived from 2.2 µM BAP. Full article

Eucalyptus cloeziana is an important, fast-growing, precious timber species in southern China, with tissue culture being the primary method for its propagation. However, the declining proliferation coefficient of adventitious shoots with multi-generation culture is a major constraint on its rapid propagation. This study aims to address this issue through the selection of suitable explants and optimizing the plant growth regulators’ formulation during the process of shoot proliferation. In this study, we cut shoots from the 21st generation of the tissue-cultured seedlings of ‘Chuanlinzhen 7523’ into apical, middle, and basal sections, and we measured their content of endogenous hormones. The proliferation coefficient (PC) and growth coefficient (GC) of explants under different concentrations of plant growth regulators were analyzed and ranked using the PCA method. The results indicated that the shoot basal sections were the best for proliferation when treated with 0.40 mg/L BAP (6-benzylaminopurine), 0.20 mg/L NAA (naphthaleneacetic acid), and 0.20 mg/L TDZ (thidiazuron), yielding a PC of 4.7 and a GC of 4.1. These basal sections ranked first with a rooting rate of up to 80%. When the rooted plantlets were acclimatized in the nursery, a survival rate of 100% was achieved. This protocol—from proliferation to acclimation—effectively improves the propagation efficiency of E. cloeziana ‘Chuanlinzhen 7523’ after multi-generation propagation. Full article

Malus ‘Baiyun’ (registration no. 20210210), a new crabapple cultivar, was registered in 2021 by the Nanjing Forestry Unversity. However, the difficult rooting has greatly limited the production of high-quality M. ‘Baiyun’ in industrialization development. There is thus a pressing need to develop an organogenesis protocol for the in vitro propagation of M. ‘Baiyun’ to alleviate a shortage of high-quality M. ‘Baiyun’ seedlings. The results showed that choosing the apical bud in mid-March was an excellent explant material. To promote proliferation, the highest proliferation (6.27) of apical shoots was cultured on Murashige and Skoog (MS) medium supplemented with 0.5 mg·L⁻¹ 6-benzylaminopurine(6-BA) + 0.05 mg·L⁻¹ indole-3-butyric acid (IBA). Subsequently, a 100% rooting rate, average number of roots per shoot of 6.2 and maximum length of roots of 4.96 cm were obtained on half-strength Murashige and Skoog (1/2 MS) medium with the application of 0.5 mg·L⁻¹ naphthaleneacetic acid (NAA) or 0.6 mg·L⁻¹ NAA + 0.7 mg·L⁻¹ IBA. Additionally, thick and lateral roots were obtained with 0.6 mg·L⁻¹ NAA + 0.7 mg·L⁻¹ IBA. Our study is the first to establish an effective organogenesis protocol for new crabapple cultivars using stem segments. Full article

In vitro conservation and regeneration of potato germplasm is important in breeding programs. The objective of this study was to assess the combined effect of paclobutrazol (PAC) and silver nanoparticles (AgNPs) during reduced-growth conservation and their in vitro ability to regenerate S. tuberosum after conservation treatments. For the conservation system, apices were used as explants in Murashige and Skoog culture media with different combinations of PAC (0, 0.5, 1 and 2 mg L⁻¹) and AgNPs (0, 50, 100 and 200 mg L⁻¹). At six months of culture, plant length, number of lateral branches, number of leaves and roots, root length, stomatal density and total chlorophyll content were assessed. For regeneration, explants were internodes cultured in medium with only 2 mg L⁻¹ benzyladenine (BA). At 60 d of culture, the response percentage, number of shoots per explant and shoot length were assessed. For in vitro conservation, the combination of 2 mg L⁻¹ PAC and 50 mg L⁻¹ AgNPs was the best treatment, whereas for in vitro regeneration, the highest number of shoots was in explants that were treated with 1 or 2 mg L⁻¹ PAC with 50 mg L⁻¹ AgNPs. In conclusion, PAC and AgNPs are alternatives for in vitro S. tuberosum conservation, and their evaluation in other species recalcitrant to the effect of ethylene is recommended. Full article

Tomato (Lycopersicon esculentum Mill.) as the most economically important vegetable crop worldwide has been investigated intensively for the development of new and improved varieties. Most of these technologies require efficient protocols for in vitro regeneration and propagation of plant material. In the present study, an efficient and reproducible in vitro regeneration system for five Romanian tomato genotypes (cvs. ‘Capriciu’, ‘Darsirius’, ‘Kristin’, ‘Pontica’ and ‘Siriana’) has been established. The tomato genotypes were selected based on their horticultural and economically valuable traits. To study the in vitro morphogenic response, various explants, such as cotyledons, cotyledonary nodes, hypocotyls, leaf explants, internodes, stem nodes and apical buds have been selected. The highest efficiency in terms of direct shoot organogenesis was obtained in cv. ‘Capriciu’ (98% for apical buds and 94% for stem nodes) on culture media with zeatin and indole-3-butyric acid. One advantage of this regeneration procedure is beside its feasibility in handling, the high percentage of regenerated shoots and their rooting. The present protocol contributes to the existing information regarding the response of tomato cultivars to in vitro culture conditions. Full article

Drug-type cannabis is often multiplied using micropropagation methods to produce genetically uniform and disease/insect-free crops. However, micropropagated plantlets often exhibit phenotypic variation, leading to culture decline over time. In cannabis, the source of these changes remains unknown, though several factors (e.g., explant’s sources and prolonged in vitro culture) can result in such phenotypical variations. The study presented herein evaluates the effects of explant sources (i.e., nodal segments derived from the basal, near-basal, middle, and apical parts of the greenhouse-grown mother plant) over multiple subcultures (4 subcultures during 235 days) on multiplication parameters and leaf morphological traits of in vitro cannabis plantlets. While initial in vitro responses were similar among explants sourced from different regions of the plant, there were significant differences in performance over the course of multiple subcultures. Specifically, explant source and/or the number of subcultures significantly impacted plantlet height, number of nodes, and canopy surface area. The explants derived from the basal and near-basal parts of the plant resulted in the tallest shoots with the greatest number of nodes, while the explants derived from the middle and apical regions led to shorter shoots with fewer nodes. Moreover, the basal-derived explants produced cannabis plantlets with shorter but wider leaves which demonstrated the potential of such explants for in vitro rejuvenation practices with minimal culture decline. This study provides new evidence into the long-term impacts of explant source in cannabis micropropagation. Full article

In this work, we studied the induction of somatic embryogenesis in Arabidopsis using IZEs as explants. We characterized the process at the light and scanning electron microscope level and studied several specific aspects such as WUS expression, callose deposition, and principally Ca²⁺ dynamics during the first stages of the process of embryogenesis induction, by confocal FRET analysis with an Arabidopsis line expressing a cameleon calcium sensor. We also performed a pharmacological study with a series of chemicals know to alter calcium homeostasis (CaCl₂, inositol 1,4,5-trisphosphate, ionophore A23187, EGTA), the calcium–calmodulin interaction (chlorpromazine, W-7), and callose deposition (2-deoxy-D-glucose). We showed that, after determination of the cotiledonary protrusions as embryogenic regions, a finger-like appendix may emerge from the shoot apical region and somatic embryos are produced from the WUS-expressing cells of the appendix tip. Ca²⁺ levels increase and callose is deposited in the cells of the regions where somatic embryos will be formed, thereby constituting early markers of the embryogenic regions. We also found that Ca²⁺ homeostasis in this system is strictly maintained and cannot be altered to modulate embryo production, as shown for other systems. Together, these results contribute to a better knowledge and understanding of the process of induction of somatic embryos in this system. Full article

Digitalis purpurea L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient propagation protocol for D. purpurea by optimizing various PGR concentrations in Murashige and Skoog (MS) medium. The genetic homogeneity of in vitro regenerants was assessed by the flow cytometric method (FCM) and Start Codon Targeted (SCoT) marker technique. Firstly, the seeds inoculated in full MS medium added with 0.5 mg/L GA₃ produced seedlings. Different parts such as hypocotyl, nodes, leaves and apical shoots were used as explants. The compact calli were obtained on BAP alone or in combinations with 2, 4-D/NAA. The hypocotyl-derived callus induced somatic embryos which proliferated and germinated best in 0.75 mg/L BAP-fortified MS medium. Scanning electron microscopic (SEM) images confirmed the presence of various developmental stages of somatic embryos. Shoot regeneration was obtained in which BAP at 1.0 mg/L and 2.0 mg/L BAP + 0.5 mg/L 2,4-D proved to be the best treatments of PGRs in inducing direct and indirect shoot buds. The regenerated shoots showed the highest rooting percentage (87.5%) with 24.7 ± 1.9 numbers of roots/shoot in 1.0 mg/L IBA augmented medium. The rooted plantlets were acclimatized in a greenhouse at a survival rate of 85–90%. The genome size and the 2C nuclear DNA content of field-grown, somatic embryo-regenerated and organogenic-derived plants were estimated and noted to be 3.1, 3.2 and 3.0 picogram (pg), respectively; there is no alteration in ploidy status and the DNA content, validating genetic uniformity. Six SCoT primers unveiled 94.3%–95.13% monomorphic bands across all the plant samples analyzed, further indicating genetic stability among in vitro clones and mother plants. This study describes for the first time successful induction of somatic embryos from hypocotyl callus; and flow cytometry and SCoT marker confirmed the genetic homogeneity of regenerated plants. Full article

This study provides alternative approaches toward ex situ conservation by means of in vitro seed germination and the multiplication of Penthorum chinense Pursh using nodal explants. An overlay of a liquid medium on top of a gelled medium significantly increased the growth of shoots and roots, while the presence of activated charcoal or growth regulators (benzyl adenine and α-naphthaleneacetic acid) decreased the growth. Shoot tips of in vitro plantlets were cryopreserved using a droplet-vitrification method. The standard procedure included preculture with 10% sucrose for 31 h and with 17.5% sucrose for 17 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 20 min, cryoprotection with alternative plant vitrification solution (PVS) A3-70% (29.2% glycerol + 11.7% DMSO + 11.7% EG + 17.4% sucrose, w/v) at 0 °C for 30 min, cooling the samples in liquid nitrogen using aluminum foil strips and rewarming by plunging into pre-heated (40 °C) unloading solution (35% sucrose) for 40 min. A three-step regrowth procedure starting with ammonium-free medium followed by ammonium-containing medium with and without growth regulators was essential for the regeneration of cryopreserved shoot tips. The species was found to be very sensitive to the chemical cytotoxicity of permeating cryoprotectants during cryoprotection and to ammonium-induced oxidant stress during initial regrowth steps. Improvement of donor plant vigor by using apical sections and liquid overlay on top of the solid medium for propagation, improved shoot tip tolerance to osmotic stress and increased post-cryopreservation regeneration up to 64% were observed following PVS B5-85% (42.5% glycerol + 42.5% sucrose) treatment for 60 min. The systematic approach used in this study enables fast optimization of the in vitro growth and cryopreservation procedure for a new stress-sensitive wild plant species. Full article