Search Results (44)

In this study, the effect of growth regulators on shoot proliferation and rooting were evaluated to develop an efficient micropropagation protocol for the Cannabis sativa L. cultivars ‘Cherry Soda’ and ‘Purple’. Apical meristems were isolated from actively growing shoots of stock plants and transferred to Driver and Kuniyuki Walnut (DKW) culture medium containing either 0.0, 0.5, 1.0, 2.0, or 5.0 μM meta-Topolin to study their shoot proliferation response. Resulting shoot cultures were transferred to medium containing varying levels of Indole Acetic Acid (IAA), Indole Butyric Acid (IBA), or Naphthalene Acetic Acid (NAA), solely or in combination, and were subjected to a 10-day dark incubation followed by a 16 h/8 h light/dark period to identify the best treatment for root production. Among the different shoot proliferation treatments studied, the maximum number of shoots was produced on the control medium that was devoid of any meta-Topolin. Cultures grown on medium containing 5.0 μM meta-Topolin exhibited hyperhydricity, where shoots appeared translucent and pale green in color; were characterized by water-soaked lesions; and leaves appeared curled and brittle in contrast to healthy looking cultures. Among the various rooting treatments studied, shoots grown in the dark for 10 days exhibited the highest frequency of rooting on medium containing 4.0 μM NAA or 6.0 μM IBA + 1.0 μM NAA. Full developed plants with a robust shoot and root system were transferred to soil, acclimatized under conditions for high humidity, and then transferred to ambient conditions in 4 weeks. The micropropagation protocol developed here allows for rapid multiplication of disease-free plants in C. sativa cultivars. Full article

Halophytes are salt-tolerant plants with ethnomedicinal value and growing potential in food and cosmetics; their adaptability to extreme conditions makes them promising candidates for sustainable agriculture and crop development in salt-affected areas. In vitro plant tissue culture further supports this by enabling resilient plant production in the face of climate and food security challenges. In this study, in vitro cultures of two medicinal halophytes from the genus Plantago (P. coronopus and P. crassifolia) were established to optimize their micropropagation protocol. Seed germination percentages, growth parameters, micropropagation rates, rooting efficiency, and physiological condition were evaluated. Growth media (modified MS medium) differed in the type of cytokinin. The seed germination efficiency was monitored at weekly intervals for 8 weeks, and other growth parameters were evaluated in 6- and 12-week cultures. Differences in both the rate and efficiency of in vitro germination between the two species were observed, with approximately 73% germination reached by P. coronopus and 47% by P. crassifolia after 4 weeks, and 80% and 53% after 8 weeks, respectively. The addition of 0.5 mg dm⁻³ kinetin plus 0.5 mg dm⁻³ IAA (indole acetic acid) proved to be effective in promoting growth in P. coronopus, resulting in longer plantlets and higher multiplication rates, while the addition of meta-topolin (mT) was a better stimulator of shoot and root growth in P. crassifolia. The highest multiplication coefficient, 6.22 for P. coronopus and 4.90 for P. crassifolia, was obtained on the P1 medium for both species. Importantly, medium with mT also had a stimulating effect on rooting in both species over the long term (12-week culture). The developed PTC enables efficient propagation and trait selection in halophytes, supporting sustainable large-scale production of the studied Plantago species, and facilitating future research on salt stress tolerance. Full article

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

This study evaluated biomass accumulation and phenolic compound production in seven Hypericum species (H. androsaemum, H. calycinum, H. hirsutum, H. kalmianum, H. olympicum, H. perforatum, and H. triquetrifolium) cultivated in vitro under varying growth regulator treatments and culture periods. Shoots were grown on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) or meta-topoline (mT) and analyzed after 40 and 60 days. MS medium supplemented with 0.2 mg/L BA was the most effective condition for promoting biomass across all species, with shoot fresh weight increasing significantly at 60 days, particularly in H. olympicum, H. perforatum, and H. triquetrifolium. High-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) identified 13 phenolic compounds, including flavonols, hydroxycinnamic acids, anthocyanins, phloroglucinols, and naphthodianthrones. Phenolic profiles were species-specific and influenced by culture period. H. kalmianum accumulated the highest total phenolic content (37.6 mg/g DW), while H. olympicum was the top producer of hypericin and pseudohypericin. These results highlight the crucial role of culture conditions in regulating both biomass and phytochemical production and provide a promising approach for producing bioactive metabolites in Hypericum species through in vitro systems. Full article

In vitro propagation of avocado faces several limitations. To optimize the establishment phase, we evaluated three plant material types: etiolated shoots, 30-day covered field shoots, and uncovered field shoots, collected at two time points. Biochemical and anatomical analyses were conducted to understand material performance during establishment. Across both collection times, etiolated shoots exhibited minimal oxidation, enhanced bud sprouting, reduced malondialdehyde (MDA) and reactive oxygen species (ROS) levels, increased peroxidase (POD) activity, and improved xylem development, consistently outperforming field-derived materials. Using etiolated shoots, we optimized disinfection and in vitro multiplication protocols. Pre-disinfection with 3 mL L⁻¹ Phyton 27^® and 2% sodium hypochlorite yielded the highest survival rates. In multiplication experiments, varying concentrations of 6-benzylaminopurine (BAP) and meta-topolin (MT), supplemented with gibberellic acid (GA₃), did not significantly affect growth variation. However, 8.88 µM BAP with 0.29 µM GA₃ resulted in the greatest number of sprouted buds. Full article

The production of healthy propagating material of the potato (Solanum tuberosum L.) is based on in vitro micropropagation. In vitro conditions, however, can cause stress leading to reduced quality, growth and development of in vitro plantlets. The effects of aeration and chemical additives on the in vitro growth and development and quality of potato plantlets were investigated. Four different jar closure types were tested, i.e., an intact metal cap (control), two layers of semi-permeable plastic foil, a metal cap with a single hole, or a metal cap with three holes. Under tightly sealed conditions (intact metal cap) the effects of silver nitrate (2.0 mg L⁻¹) and 1-naphtylacetic acid (0.1 mg L⁻¹) alone or in combination with each other, meta-topoline (0.1 mg L⁻¹), ascorbic acid (10.0 mg L⁻¹), salicylic acid (0.1 mg L⁻¹), jasmonic acid (0.1 mg L⁻¹) and glutamic acid (0.3 mg L⁻¹) were studied. Morpho-physiological parameters were measured at the end of the subculture. Leaf development was a good indicator of the presumed ethylene effect. The development and quality of the plantlets were best in cultures sealed with three-holed caps. Of the chemicals applied, only the presence of silver nitrate resulted in high-quality plantlets. The combined application of silver nitrate and 1-naphthaleneacetic acid promoted root growth and development. Full article

Turmeric (Curcuma longa L. cv. Trang 1), a high-value cultivar known for its elevated curcuminoid and volatile oil content, holds significant potential in pharmaceutical and food applications. However, its commercial propagation is constrained by low rhizome productivity and the limitations of conventional vegetative propagation. This study aimed to improve the propagation efficiency of turmeric cv. Trang 1 by developing optimized protocols for explant sterilization, shoot proliferation, root induction, and acclimatization. Sprouted rhizome buds were sterilized and cultured on a Murashige and Skoog (MS) medium supplemented with various plant growth regulators, including cytokinins (benzyladenine [BA], thidiazuron [TDZ], and meta-topolin [mT]) and auxins (indole-3-butyric acid [IBA] and 1-naphthaleneacetic acid [NAA]). The shoot induction (4.60 ± 1.47 shoots per explant) and shoot height (2.34 ± 0.61 cm) were observed on the MS medium with 3.0 mg/L BA, while the TDZ, at 0.5 mg/L, also induced a high number of shoots (5.22 ± 0.64). When using single shoots derived from bud explants, mT at 1.5 mg/L significantly enhanced the shoot formation. For the root induction, 2.0 mg/L IBA yielded the highest number of roots (7.33 ± 1.49), while NAA was less effective. The plantlets acclimatized in a 1:1 soil and peat moss mixture showed the highest survival rate (86.67%). This improved protocol enables the efficient production of turmeric plantlets, supporting commercial deployment. Full article

Synthetic seed technology is an innovative in vitro technique that provides improved storage capabilities for vegetative propagules. Its success mostly depends on the encapsulation matrix’s composition and the encapsulation procedure. The present study focuses on optimizing an encapsulation protocol for short-term storage and germplasm exchange using micro-cuttings of Salix tetrasperma. Among the different synthetic seed types evaluated, double-layered synthetic seeds (DLSs) exhibited the highest re-growth (93.6%) on MS medium supplemented with meta-Topolin (mT) (5.0 µM) and α-naphthalene acetic acid (NAA) (0.5 µM) after 8 weeks of culture. Viability assessment of non-embryogenic synthetic seeds during low-temperature storage (4 °C) demonstrated the enhanced resilience of double-layered synthetic seeds (DLSs) compared to their single-layered (SLS) counterparts. Following acclimatization in Soilrite^®-filled cups, 82% of the plantlets were successfully established in a greenhouse after four weeks. The increased activity and concentration of antioxidants in DLS-derived plantlets suggest the potential role of the extra layer of alginate in mitigating the effects of low-temperature stress during storage. SCoT molecular analysis confirmed the genetic integrity of the synthetic seed-derived plants. Full article

This study investigates the impact of light conditions on the growth, rooting, and photosynthetic performance of in vitro cultivated Cistus creticus L. explants. Initially, different plant growth regulators were tested for multiple shoot growth [5 and 10 μM 6-Benzylaminopurine (BA), 5 and 10 μM meta-Topolin (mT), and 0.5 and 1 μM melatonin (Mel)] and rooting [5 and 10 μM mT, 0.5 and 1 μM Mel, and 0.5 and 1 μM Indole-3-butyric acid (IBA)]. The media with the best results were Murashige and Skoog (MS) with 5 μM BA or 5 μM mT for shoot formation and 0.5 μM Mel or 1 μM IBA for rooting. Also, the explants were cultured under red (R), blue (B), or white (W) LED lights or fluorescent (FL) lamps. After four weeks, the photosynthetic rate, chlorophyll concentration, fluorescence (Fv/Fm), and shoot/root measurements were recorded. The optimal plant regulator for shoot generation was 5 μM mT under a W LED. For rooting, 1 μM IBA combined with a R LED resulted in 100% rooting, 3.53 roots/explant, and a 1.25 cm root length. The B LED led to the highest photosynthetic rate, while the chlorophyll concentration was highest with 5 μM BA under a FL lamp (CCI = 3.03). During acclimatization, a R LED and 1 μM IBA yielded the highest survival rate (70%). The current findings could reinforce the in vitro cultivation of the species for use in the floriculture industry, as well as for medicinal and other industrial purposes. Growth rooms equipped with automated LED lighting systems could optimize the micro-environment and create suitable climatic conditions to enhance in vitro plant growth. Full article

(1) Background: Humulus lupulus L. plants constitute a rich source of bioactive compounds. The synthesis of bioactive compounds in plants is often triggered by the activation of secondary metabolism, which can be induced by biotic or abiotic elicitors. In vitro, the effect of the elicitors can be studied in a controlled environment and in a small space, independently of seasonal variations. Cytokinins are frequently used in plant tissue culture for bud regeneration, branching and shoot elongation due to their role in cell division enhancement. This study aimed to investigate the effects of different cytokinins on the growth parameters, total (poly)phenolic content and antioxidant capacity of in vitro-grown hop plants to evaluate hop vitro-derived biomass as a potential source of bioactive compounds. (2) Methods: unimodal hop (cvs. Cascade and Columbus) explants were cultured on media enriched with four cytokinins (kinetin, 6-benzylaminopurine, meta-topolin and 6-(γ,γ-dimethylallylamino)-purine) at four concentrations. (3) Results: A genotype-dependent response to different cytokinins was encountered. (4) Conclusions Columbus explants could root in culture media auxin-free, providing valuable opportunities for commercial nurseries. Moreover, cytokinins were confirmed to be valuable elicitors to stimulate the bioactive compound biosynthesis in micropropagated hop plants, making them a precious source for various industries. Full article

Viola ucriana Erben & Raimondo is a rare and endangered taxon, endemic to a limited area on Mount Pizzuta in northwestern Sicily, Italy. Its population is significantly threatened by anthropogenic activities, including fires, overgrazing, and habitat alterations. Temporary immersion systems (TISs) have proven effective for large-scale propagation in various protected species, offering potential for ex situ conservation and population reinforcement of V. ucriana. This study aimed to establish a bioreactor-based micropropagation protocol for shoot multiplication and compare the efficacy of a TIS with that of conventional solid culture medium (SCM). Three different plant growth regulators (PGRs) were also compared: 6-benzylaminopurine (BA), zeatin, and meta-topolin-9-riboside (mTR). The starting material originated from seeds collected from mother plants in their natural environment. The best growth outcomes (in terms of shoot multiplication, shoot length, and relative growth rate) were achieved using THE RITA® TIS, with BA (0.2 mg/L) and mTR (0.5 or 0.8 mg/L) outperforming SCM. Anomalous or hyperhydric shoots were observed with all zeatin treatments (especially with 0.8 mg/L) in both the TIS and SCM, suggesting that this cytokinin is unsuitable for V. ucriana biomass production. The rooting phase was significantly improved by transferring propagules onto rockwool cubes fertilized with Hoagland solution. This approach yielded more robust roots in terms of number and length compared to the conventional agar-based medium supplemented with indole-3-butyric acid (IBA). Flow cytometry analysis confirmed the genetic fidelity of the regenerants from the optimal PGR treatments, showing that all plantlets maintained the diploid ploidy level of their maternal plants. Over 90% of the in vitro derived plantlets were successfully acclimatized to greenhouse conditions. This paper represents the first report of V. ucriana biomass multiplication using a RITA^® bioreactor. The stability of the regenerants, confirmed by nuclei quantification via cytofluorimetry, provides guidance in establishing a true-to-type ex situ population, supporting conservation and future reinforcement efforts. Full article

Actinidia kolomikta (Maxim) Maxim. is a winter-hardy species of the genus Actinidia Lindl., whose fruits are valued for their high content of vitamin C and other bioactive compounds. The use of biotechnological propagation methods significantly accelerates the production of quality planting materials for this crop. This study revealed the regeneration features of promising A. kolomikta cultivars. The main morphometric parameters of explants were determined in regard to the effect of different iron chelates (FeEDTA and FeEDDHA) and cytokinins (6-benzylaminopurine, meta-Topolin, and 2-izopentyladenine) in the Quoirin and Lepoivre medium. FeEDTA-supplemented media were optimal for explant culture. Meta-Topolin was found to promote the formation of adventitious microshoots at the base of explants and bud activation, which increased the multiplication rate by 1.5 and 1.7 times compared to the media with 6-benzylaminopurine and 2-izopentyladenine. The morpho-anatomical studies revealed the structural organization of assimilation tissues and the stomatal apparatus of A. kolomikta under different culture conditions (field, in vitro, and ex vitro). The stomata in vitro were round and had a larger area, lower thickness, and a lower layer number of mesophyll compared to field conditions. The transfer from in vitro to ex vitro caused gradual normalization of the leaf structure: a decrease in the stomatal number and area, changes in shape (from round to elliptical), and an increase in the mesophyll thickness. Full article

Robinia pseudoacacia L., commonly known as black locust, is a nitrogen-fixing species characterized by multiple uses. Among these uses, black locust is of special interest to beekeepers due to its abundant flowering and delicious honey. Given the great importance of honey production in Italy, beekeepers are looking for genotypes that have a delayed flowering time. As a consequence, the aim of the present study was to develop a complete protocol of micropropagation for genotypes, which have been selected in the Veneto region due to their delayed flowering, i.e., about 3 months, in comparison with the normal flowering time (from late April to early June). The subsequent steps of the micropropagation protocol (explant decontamination, shoot induction, proliferation, and rooting) were investigated and optimized. The most effective decontamination treatment of explants (axillary buds from shoots developed in a greenhouse) was obtained using 50 mg/L AgNO₃ for 20 min. This method resulted in the highest survival and regeneration rates for the explants (90%), although contamination was slightly higher than when using HgCl₂ and NaOCl. The best medium for shoot establishment was MS with 1 mg/L of mT, which achieved 100% regeneration of the explants. In comparison with BA, mT at 1 mg/L was shown to be the best stimulator of shoot proliferation, especially in combination with 0.7 mg/L GA₃ (Proliferation Rate, 4.7). An intermediate 2 h treatment with AgNO₃, in combination with mT, was shown to be beneficial in improving the shoot proliferation and quality in the subsequent subculture in a gelled medium. As for shoot rooting, the shoots that were pre-treated in NH₄NO₃-free and mT-free MS medium gave the highest ex vitro rooting percentage in a cell tray (80%) and the highest number of roots per shoot (3.6). This optimized protocol opens the door to the massive micropropagation of valuable genotypes of black locust selected for delayed flowering. This is an outcome of extraordinary importance for beekeepers. Full article

In recent years, interest in Lonicera caerulea production has grown significantly because of its nutritional and pharmaceutical benefits, leading to rapid expansion in its cultivation. L caerulea var. emphyllocalyx is a lesser-known botanical variety. Due to differences between plants of the Lonicera genus and the lack of scientific reports on micropropagation, it is necessary to determine the possibilities of in vitro propagation. The aim of this study was to elaborate a micropropagation protocol of two new breeding clones of Lonicera caerulea var. emphyllocalyx: ‘21–17’ and ‘139–24’. The experiments were carried out on in vitro cultures grown on MS medium supplemented with 1 mg·dm⁻³ BA or 1 mg·dm⁻³ mT. Two types of explants were used during the experiment: nodal fragments (NFs) and shoot-tips (STs). Before acclimatisation, some rooted microshoots were subjected to cooling at 4 °C for 4 weeks. Significantly more ST explants than NF explants started to grow at the proliferation stage. The application of BA resulted in much better proliferation and health of cultures. Cold storage of micropropagated ‘139–24’ plantlets significantly increased their survival in acclimatisation in contrast to ‘21–17’ plantlets but weakened further growth of the plants. In future in vitro studies on L. caerulea var. emphyllocalyx, BA can be used as the primary growth regulator due to its effectiveness and low cost. Nodal fragments should be considered as the main propagation material since they promote better growth rates. Additionally, further research is required to explore the effects of low-temperature storage on the growth and physiology of these plants. The results obtained in this research may contribute to the development of micropropagation technology in the future for L. caerulea var. emphyllocalyx. Full article

In vitro mass propagation of apple plants plays an important role in the rapid multiplication of genetically uniform, disease-free scions and rootstocks with desired traits. Successful micropropagation of apple using axillary shoot cultures is influenced by several factors, the most critical of which is the cytokinin included in the culture medium. The impact of medium composition from single added cytokinins on shoot proliferation of apple scion Húsvéti rozmaring cultured on agar-agar gelled Murashige and Skoog medium fortified with indole butyric acid and gibberellic acid was investigated. The optimum concentration for efficient shoot multiplication differs according to the type of cytokinin. The highest significant multiplication rate (5.40 shoots/explant) was achieved using 2.0 μM thidiazuron while the longest shoots (1.80 cm) were observed on the medium containing benzyladenine at a concentration of 2.0 μM. However, application of either thidiazuron or benzyladenine as cytokinin source in the medium resulted in shoots of low quality, such as stunted and thickened shoots with small leaves. In the case of benzyladenine riboside, the 8 μM concentration was the most effective in increasing the multiplication rate (4.76 shoots/explant) but caused thickened stem development with tiny leaves. In the present study, meta-topolin was shown to be the most effective cytokinin that could be applied to induce sufficient multiplication (3.28 shoots/explant) and high-quality shoots along with shoot lengths of 1.46 cm when it was applied at concentrations of 4 μM. However, kinetin was the least active cytokinin; it practically did not induce the development of new shoots. The superior cytokinin for in vitro axillary shoot development of apple scion Húsvéti rozmaring with high-quality shoots was the meta-topolin, but it may be different depending on the variety/genotype under study. Full article