Search Results (119)

Agave species are plants with great economic value and multiple possibilities of use as ornamentals, medicinal plants, and fibers, as well as being significant sources of bioethanol. However, their long life cycles hinder their conventional breeding. Therefore, biotechnology tools are the most effective means for clonal propagation and genetic improvement. In vitro micropropagation of A. sisalana via axillary shoot proliferation from bulbil explants was attained using Murashige and Skoog medium (MS) supplemented with cytokinins (CKs), such as 6-benzyladenine (BA), kinetin (KIN), or thidiazuron (TDZ). The optimum significant shoot proliferation (14.67 shoots/explant) was achieved on 1.0 mg L⁻¹ TDZ. The carry-over effect of CKs on subsequent rooting could be detected. Control and KIN treatments could enhance the rooting of shoots on shoot proliferation media. The regenerated plantlets were acclimatized directly with 100% survival. To mitigate this carry-over effect, that causes hindering further root growth and development, and promote healthy growth of roots, subculturing shoots onto a CK-free medium is a recommended practice. The shoots induced on all BA treatments, and TDZ at 0.5 and 1.0 mg L⁻¹ could be rooted after two subcultures on CK-free medium, then they were acclimatized with 100% survival. However, the higher concentrations of TDZ inhibited in vitro rooting even after two subcultures on CK-free medium, and the acclimatization percentage was reduced by increasing the TDZ concentration recorded from 10 to 0%. Full article

Essential oils (EOs) are widely recognized for their antifungal properties, but their efficacy against specific phytopathogenic fungi associated with banana (Musa paradisiaca) rot remains underexplored. This study aimed to evaluate the antifungal potential of EOs from Origanum vulgare, Salvia rosmarinus, Syzygium aromaticum, Thymus vulgaris, Cinnamomum verum, and Ocimum basilicum against five fungal species isolated from infected banana peels. Fungal isolates were obtained using PDA medium supplemented with chloramphenicol and were purified by weekly subculturing. Morphological and microscopic characterization was complemented by molecular identification based on ITS sequencing and phylogenetic reconstruction using Neighbor-Joining and UPGMA methods in MEGA v11. In vitro and ex vivo antifungal assays were performed at EO concentrations ranging from 200 to 1000 ppm. Thyme oil exhibited the strongest inhibitory effect, with complete growth suppression at 1000 ppm. Cinnamon and oregano also demonstrated effective inhibition at 600 ppm, while clove, rosemary, and basil were markedly less effective. Statistical analysis confirmed significant effects of EO type and concentration on fungal growth (p < 0.001). Molecular results showed strong phylogenetic support for isolate identification, with bootstrap values above 93% in most clades. These findings support the selective use of specific EOs as sustainable alternatives to synthetic fungicides in the postharvest management of banana diseases and provide a molecularly supported basis for their targeted application in integrated control strategies. 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

Sophora koreensis Nakai, listed as endangered on the IUCN Red List, is a species native to Korea, specifically found in parts of Gangwon-do. Recent research highlights its potential in hangover relief and as an antioxidant source, sparking interest in enhancing its components through mutation for commercial purposes. Given its limited distribution, micropropagation of S. koreensis is essential for its economic exploitation. This study focuses on in vitro culture to develop an elongation system for micropropagation. The hormonal combination of 6-benzylaminopurine (2 μM), thidiazuron (2 μM), and indole-3-butyric acid (0.5 μM) produced the highest number of shoots (14) with an average length of 0.7 cm compared to the control. Additionally, adjusting photoperiod conditions under specific culture media further increased shoot length to 0.6 cm, which was also higher than that of the corresponding control group under standard light conditions. However, survival rates were generally low across all treatments during subculture. Isolating and individually culturing induced explants resulted in shorter shoots and lower survival rates. Improvements were noted when explants with 10 shoots were subcultured, achieving an 83% survival rate, with an average of 4.93 shoots at 0.95 cm in length. Rooting was most successful with 10 μM IBA, also showing the highest root number, indicating a potential pathway for enhancing micropropagation efficiency. Full article

Hemerocallis middendorffii is widely used in the landscaping of Northern China for its exceptional ornamental and ecological attributes. It is also the focus of a substantial body of germplasm development and stress tolerance research. However, the absence of an efficient regeneration and genetic transformation system has been a critical barrier to conducting gene function studies on this species. In this research, the aerial parts of seed-derived H. middendorffii plantlets were used as explants, and the callus induction, proliferation, subculture, differentiation, and rooting conditions in the in vitro regeneration process were optimized. A callus induction rate of 95.6% was achieved, with a regeneration rate of 84.4%. Based on this procedure, a simple and effective Agrobacterium-mediated genetic transformation system was preliminarily developed using a hygromycin-based selection system. The system comprised an Agrobacterium tumefaciens culture solution optical density at 600 nm (OD₆₀₀) of 0.6, an acetosyringone concentration of 100 μmol·L⁻¹ in both the A. tumefaciens infection solution and the co-cultivation medium, a sterilization culture with Timentin at 300 mg·L⁻¹, and a selection culture with hygromycin at 9 mg·L⁻¹. Transgenic H. middendorffii T0 rooted plants were produced within a 5-month period, with a transformation rate of 11.9% and positive rate of 32.8%. The regeneration and genetic transformation system established in this study should help advance functional gene research and genetic improvement in H. middendorffii. However, the genetic transformation was only validated in the T0 plants. To confirm stable integration and long-term transgene stability, future research on the phenotypic and molecular characterization of T1 progeny, including segregation analysis and Southern blot verification, will be conducted. 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

Biochar, a by-product of agri-food waste, has shown benefits in plant growth and soil health. However, its use in vitro remains underexplored. This study investigates the impact of biochar supplementation in the culture medium, alone or in combination, with 6-benzylaminopurine (BAP), on kiwifruit (Actinidia chinensis var. deliciosa), cv. Tomuri proliferation. Kiwifruit explants were cultured on media enriched with 0, 4, or 6 g/L biochar, without or with BAP (0.2 mg/L), over two subcultures (SUB1 and SUB2). Parameters such as shoot and root number and length, fresh and dry weight, as well as plantlets’ total phenolic content and antioxidant activity, were measured and analyzed. Biochar enhanced plantlets proliferation, particularly with BAP. In SUB1, at 4 g/L, biochar promoted shoot production (2.00 vs. 1.63) and their length (1.50 cm vs. 0.98), independently of the presence of BAP. The presence of biochar in the BAP-free media, favored rhizogenesis; particularly in SUB2, where on average, 5.58 roots per plantlets were recorded. Biochar increased the plantlets’ total phenolic content and antioxidant activity, especially in BAP-free media. The addition of biochar as an additive to the culture medium during the kiwifruit in vitro proliferation phase could be a breakthrough outcome for the nursery sector. Full article

Rumen bacteria have the ability to efficiently degrade and acidify lignocellulosic biomass, among which rumen solid-phase bacteria are more dominant. However, the effectiveness of in vitro cultured ruminal solid-phase bacteria in producing volatile fatty acids (VFA) during lignocellulosic biomass degradation remains unclear. This study presents a feasibility analysis of the long-term subculture of rumen solid-phase bacteria in vitro for VFA production. The results indicated that VFA production could reach 0.20–0.30 g/g dry matter. After 40 generations (200 days) of subculturing, the bacterial community underwent alterations. The relative abundance of certain fiber-degrading, acid-producing bacteria, which were less abundant in rumen solids, such as Oribacterium and Victivallis, was significantly upregulated following subculturing in vitro. The success of this study in subculturing rumen solid-phase bacteria in vitro over an extended period and achieving efficient VFA production is of considerable importance for the practical application of rumen microorganisms in production settings. 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

Prunus cerasus var. Marasca (Rosaceae) is an important Croatian cultivar, known wordwide for the production of Luxardo maraschino liqueur, which occurs in the eastern Po Valley of Italy. Besides liqueur, Marasca is attractive for its beneficial effects on human health and well-being. The undifferentiated in vitro cell cultures of Marasca were investigated as a source of healthy products. The in vitro conditions for obtaining callus and suspension cultures under photoperiod were defined. The cell lines were evaluated for growth rate, total phenol and proanthocyanidin contents, and antioxidant activities via colorimetric assays. The best cell lines were also subcultured in darkness, studying the importance of the light parameter in the possible industrial scaling-up. The juices extracted from the obtained biomasses were analyzed by LC-DAD-MS and six flavanone derivatives, among which naringenin and its glucoside were identified. The quantitative analysis, pursued during the cell growth cycle, revealed that the flavanone content was higher at the end of the growth cycle (28th day) and that the total content of identified flavanone compounds varied from 17.22 to 79.22 μg/mL of juice. The results of the antioxidant and anti-inflammatory activities on Caco-2 cells support the potential applications of this material in human health. Full article

Suspension growth can greatly increase the cell density and yield of cell metabolites. To meet the requirements of aquatic industries, a culture model derived from Anguilla anguilla skin was developed using the explant outgrowth and enzyme-digesting passaging methods. These cells were kept in vitro continuously for over 12 months and subcultured 68 times. This heteroploid cell line, designated as ES, can naturally adapt to adherent and suspension growth reversibly under certain temperatures, serum percentages, and inoculum densities, without the need for any microcarriers or special medium additives. The ES cells can continue being highly productive under a temperature range of 15–37 °C and a serum percentage ranging from 3 to 15%. An inoculum density higher than 5 × 10⁵ cells·mL⁻¹ is necessary for the ES cells to turn into suspension efficiently. The green fluorescent protein (GFP) reporter gene was successfully expressed in the ES cells. The ES cells demonstrated susceptibility to Anguillid herpesvirus (AngHV) and red-spotted grouper nervous necrosis virus (RGNNV). ES is the first natural suspension growth model of aquatic origin; it does not require the processes of suspension domestication and carrier dissolution, making it a promising and cost-effective model for vaccine production, bio-pharmaceutical manufacturing, and cellular agriculture. 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

Aging is a major risk factor for osteoarthritis (OA), but the specific mechanisms connecting aging and OA remain unclear. Although chondrocytes rarely divide in adult articular cartilage, they undergo replicative senescence in vitro, offering a model to study aging-related changes under controlled conditions. OA cartilage was obtained from an 80-year-old male and a 72-year-old female, while normal cartilage was sourced from a 26-year-old male. Chondrocyte cultures were established and sub-cultured to their Hayflick limit. Bulk RNA sequencing on early- and late-passage human articular chondrocytes identified transcriptomic changes associated with cellular aging. Early-passage OA chondrocytes already showed senescent phenotypes, unlike normal chondrocytes. All three cultures underwent 30 population doublings before replicative exhaustion, at which point all cells displayed senescence. During this process, cells lost their ability to form cartilaginous pellets. Differential gene expression analysis revealed distinct transcriptomic profiles between early- and late-passage chondrocytes and between normal and OA-derived cells. Genes related to matrix synthesis, degradation, inflammation, and the senescence-associated secretory phenotype (SASP) showed significant expression changes. Despite being a small pilot study, these findings suggest that further research into the molecular and metabolic changes during chondrocyte senescence could provide valuable insights into OA pathobiology. Full article

The major factors affecting the in vitro immature embryo rescue efficiencies from Prunus persica or P. armeniaca accessions have been identified, along with improving the feasibility. Variations in the woody plant medium (WPM) were used depending on the embryo size. Embryos less than 5 mm long were cultured in WPM supplemented with 1 μM BAP and 1 μM GA₃, while embryos bigger than 5 mm long were cultured in hormone-free medium, with or without vermiculite. The environmental in vitro culture conditions consisted of three phases: a (I) stratification at 4 °C during a 3- to 5-month-long period in the dark, followed by (II) growth of germinated embryos at 14 °C for a 4-week-long period, with 12 h light a day, which favors plantlet development, and finally, (III) growth at 24 °C, with 16 h light a day, until the plantlets were acclimatized in the greenhouse. The germination of smaller embryos, at the end of phase I, ranged from 82.2% to 22.1% for apricots and flat peaches, respectively, whereas for bigger embryos, the germination varied from 97.3% to 53.2% for the same species. The embryo germination for peaches and nectarines ranged from 40.1% to 30.3% for smaller embryos, and from 91.9% to 63.0% for bigger embryos. Endo- and epiphytic contamination, affecting from 7.4% to 52.9% of cultured embryos, depending on the fruit type and conservation conditions, and the capacity to acclimate to soil conditions, ranging from 50.4% to 93.2%, were the two most important factors influencing the protocol’s efficiency and feasibility. Considering the overall efficiencies, expressed as hardened plants transferred to field plots over clean uncontaminated embryo, the values ranged from 55.8% for nectarines, 54.0% for peaches, 45.6% for apricots, and 23.3% for flat fruits. The addition of vermiculite to the culture medium significantly improved the plantlet development, avoiding subculture to fresh medium when an extension of phase III was required before acclimatization. Compared to laboratory glassware, the use of food glass containers with air-permeable sealing film, along with vermiculite-containing medium, significantly reduced the costs when handling the large number of embryos required for breeding programs. Full article

The growth of high-quality in vitro potato plants (Solanum stenotomum subsp. stenotomum, Solanum stenotomum subsp. goniocalyx, and Solanum tuberosum subsp. andigena) is affected by multiple biological, operational, and environmental factors. Research on in vitro culture is frequently focused on the species, explant, composition of the culture medium, and incubation conditions, but only limited information is available on the effect of the gas exchange rate and volume of in vitro culture vessels under variable planting densities. In the present study, these factors were evaluated with a set of seven diverse potato landraces. The results were compared to the plants’ responses in routinely used in vitro culture vessels, i.e., 13 × 100 mm and 25 × 150 mm test tubes, and GA7^® magenta vessels. In vitro potato plants grown in plastic vessels equipped with a HEPA filter delivering a high gas exchange rate developed thicker stems (0.95 mm), a higher total average leaf area (2.51 cm²), increased chlorophyll content in leaves (32.2 ppm), and lower moisture content in their tissues (90.1%) compared to filter systems with lower gas exchange rates. A high planting density of 10 × 10 plants per vessel (360 and 870 mL) negatively affected the average stem width and root length but increased the plant height (3.4 cm). High fluctuations of ion-uptake of NO₃⁻, Ca⁺⁺, K⁺, and Na⁺ were observed between genotypes, with some accessions having a 4.6-times higher Ca⁺⁺-ion concentration in their tissues (190–234 ppm). The in vitro plants developed more robust stems, longer roots, and larger leaves within in vitro culture vessels equipped with a HEPA filter (high gas exchange rate) compared to the control vessels, in contrast to the chlorophyll content in leaves, which was higher in plants grown in narrow test tubes. Depending on the purpose of the subculture of in vitro plants, their growth and development can be molded using different gas exchange rates, planting densities, and vessel volumes. Full article