Search Results (126)

In vitro-cultured plant calli are colonized by diverse endophytes. As these endophytes are inherited from the maternal plant and appear to be highly dependent on the eco-niche of the host cells, they have been termed host-dependent endophytes (HDEs). HDEs occupy the most intimate microbial environment of plant cells. Nevertheless, our understanding of HDEs and their microenvironmental effects on host plants remains limited due to their cultivation-recalcitrant nature. In this study, grapevine (Vitis vinifera L. × V. labrusca L.) callus was subjected to long-term cultivation in media containing different antibiotics (Q: penicillin; L: streptomycin; Z: nystatin) with the intention of creating grapevine calli with different HDEs. The treated calli were then transferred to an antibiotic-free medium for continuous cultivation. After three cycles of subculture over a total period of 45 days, the endophytic microbiota of the grapevine calli were profiled and their physiological parameters were analyzed. Our results revealed that antibiotic treatments can effectively shape HDEs and create distinct bacterial and fungal HDE microbiota in grapevine calli. Compared to treatment without antibiotics (CK), the Q-treated callus contained more Gram-positive bacterial HDEs but fewer Gram-negative and stress-resistant bacterial HDEs, whereas the Z-treated callus had fewer Gram-positive bacterial HDEs and more Gram-negative, stress-resistant and potentially pathogenic bacterial HDEs. More importantly, grapevine calli with different HDE communities showed varying physiological traits such as respiratory rate, peroxidase activity and total sugar content. Correlative analyses further revealed the functional associations between HDE taxa and callus traits. This work provides an example for studying and utilizing plant HDEs. Full article

Red Fruit Ginseng (Campanumoea lancifolia), widely cultivated in Yunnan, Guizhou, Sichuan, and Guangxi, in China, is valued for its sweet-tasting fruit and medicinal potential. In this study, leaves of the Yunnan Maguan variety were used as explants to establish an efficient tissue culture system for callus induction, adventitious bud regeneration, and rooting. Different combinations of cytokinins and auxins were tested to determine the optimal balance of plant growth regulators (PGRs). Our results indicated that when the combined concentration of kinetin (KT) and 6-benzylaminopurine (6-BA) in the primary culture medium was lower than 1.0 mg/L, callus induction was the most effective, with treatments of 0.5 mg/L KT + 0.2 mg/L 6-BA and 0.2 mg/L KT + 0.8 mg/L 6-BA showing higher induction rates compared to other groups. During the subculture, 0.5 mg/L 6-BA promoted vigorous adventitious bud growth, while higher levels inhibited development. For rooting, 0.5 mg/L indole-3-acetic acid (IAA) combined with 0.2 mg/L 1-naphthaleneacetic acid (NAA) induced the highest root number and healthiest plantlets, outperforming indole-3-butyric acid (IBA). The optimized protocol resulted in over 80% explant survival and produced vigorous seedlings suitable for acclimatization. This leaf-derived regeneration method provides a reliable approach for large-scale propagation, conservation, and potential pharmacological applications of Red Fruit Ginseng, contributing to the sustainable utilization and commercial development of this valuable medicinal plant. Full article

Larix olgensis is a valuable timber species in northern China, typically propagated through somatic embryogenesis (SE). However, long-term subculture can lead to a loss of embryogenic potential. This study aimed to establish a simple and stable protocol for the cryopreservation and regeneration of L. olgensis embryogenic callus (EC) that preserves its SE potential and regenerative capacity. The slow-freezing method was employed for cryopreservation. A cryopreservation protocol for L. olgensis EC was developed by optimizing the preculture duration and conditions, cryoprotectant composition and thawing temperature. The results showed that optimal outcomes were achieved using a 24 h stepwise preculture on medium containing 0.2 and 0.4 mol∙L⁻¹ sucrose, followed by cryoprotectant treatment with 0.4 mol∙L⁻¹ sucrose, 2.5% (v/v) dimethyl sulfoxide (DMSO) and 10% polyethylene glycol 6000 (PEG₆₀₀₀), and thawing at 37 °C. EC cryopreserved using this protocol achieved a 100% recovery rate. Moreover, the cryopreserved recoverable EC successfully underwent SE, progressing through germination and rooting. Cryopreservation duration (storage duration in liquid nitrogen) did not affect cell viability and proliferation rate, confirming the protocol’s suitability for long-term cryopreservation of L. olgensis EC. This study provides a valuable reference for the cryopreservation and regeneration of L. olgensis EC, with potential applications for other coniferous species. It establishes a robust foundation for the large-scale propagation of conifers. Full article

Jujube (Ziziphus jujuba Mill.) is an important economic fruit tree in China, and its in vitro culture technology is the key to achieving large-scale seedling cultivation. PGRs (Plant growth regulators) play a central regulatory role in all stages of jujube micropropagation, including explant initiation, proliferation, and rooting. This article provides a comprehensive overview of recent advances in in vitro culture of jujube, with a focus on the recommended exogenous phytohormone ratios, their effects, and underlying regulatory mechanisms across distinct varieties during the key stages such as in vitro culture, shoot proliferation, and root formation. The primary culture of most jujube varieties usually employs the MS medium, and it is recommended that auxin and cytokinin be used in combination. During the initial cultivation stage, the use of NAA (1-naphthaleneacetic acid) or IBA (indole butyric acid) is recommended at concentrations ranging from 0.1 to 1.0 mg/L. At the same time, 6-BA (6-benzylaminopurine) is suggested, with a concentration range of 0.5 to 2.5 mg/L. In the subculture multiplication of most jujube varieties, MS medium is used, and auxin (such as NAA, IBA), and TDZ (thidiazuron) and cytokinin (e.g., 6-BA) are used in combination. The recommended concentration range for auxin remains between 0.1 and 1.0 mg/L, and for cytokinin 6-BA between 0.5 and 2.5 mg/L, while the recommended concentration of TDZ is suggested to be below 0.01 mg/L. Rooting induction for most jujube varieties has predominantly been achieved using 1/2 MS medium, with growth regulator concentrations typically ranging from 0.5 to 3.0 mg/L. Full article

Long-term subculture of embryogenic callus leads to a decline in somatic embryogenesis and germination capacity, which may be associated with increased methylation levels. 5-Azacytidine (5-AzaC), a methylation inhibitor, modulates DNA methylation and is widely involved in regulating plant growth, development, and metabolism. In order to investigate the effect of 5-AzaC on somatic embryogenesis and germination in Larix olgensis, we supplemented the proliferation medium with different concentrations of 5-AzaC. The results showed that the addition of 5-AzaC inhibited the proliferation of embryogenic callus, with the proliferation of embryogenic line N2 completely inhibited at 100 μM, while that of embryogenic line N4 ceased at 20 μM. In contrast, treatment with 10 μM and 20 μM of 5-AzaC significantly increased the somatic embryo yield in both embryogenic lines, with the peak yield observed at 20 μM for embryogenic line N2 and at 10 μM for embryogenic line N4. Furthermore, the addition of 10 μM 5-AzaC effectively reduced the deformity rate during somatic embryo germination in embryogenic line N2 and N4, by 15.91% and 13.53%, respectively. These findings demonstrate that 5-AzaC can partially restore the somatic embryogenesis potential of embryogenic callus in L. olgensis under long-term subculture. Additionally, these results suggest that its effects may be both concentration-dependent and genotype-specific. The results provide a potential approach to mitigating the decline in embryogenic competence, while also demonstrating significant potential for large-scale propagation. Full article

The family Anaplasmataceae comprises etiological agents of infectious diseases of significant importance. This study aimed to achieve the in vitro isolation and propagation of an Anaplasma sp. using tick-derived cell lines. The study was realized in Seropédica municipality, Rio de Janeiro, Brazil. Blood smears from a naturally infected bovine revealed cytoplasmic inclusions in blood cells. To isolate and propagate the organism, IDE8 and ISE6 tick cell lines derived from Ixodes scapularis were used. Two methods of inoculum preparation were employed: Histopaque^® density gradient and platelet-rich plasma separation. Following infection, cells were maintained in L-15B medium without antibiotics at 34 °C, and infection was monitored weekly by Giemsa-stained cytocentrifuge smears. After achieving ≥ 70% infection, bacteria were subcultured and successfully cryopreserved and resuscitated. PCR amplification and sequencing of 16S rDNA, 23S rDNA, rpoB, and groEL genes were performed for molecular characterization. Phylogenetic analyses revealed that the isolated strain clustered within the A. platys-like clade. This study reports the successful in vitro isolation, propagation, and cryopreservation of the ‘A. platys-like strain Natal’ bacterium in tick cell lines and provides molecular evidence supporting its phylogenetic classification. These findings contribute to the understanding of genetic variability and host–cell interactions of Anaplasma spp., laying the groundwork for future research. Full article

Background: Tissue culture might be a method supplementing traditional plant propagation in various fields, like agriculture, medicine, industry, and the active conservation of plant species. For the purpose of plant restoration, it is important that the obtained progenies are identical with the mother plants to ensure the true-to-typeness of the future population. Methods: In the present study, the stability of Aldrovanda vesiculosa regenerants obtained in vitro through phenotypic and genetic analysis was estimated. Clones of aldrovanda plants were cultivated in tissue culture in the 1/10 MS liquid medium under the same conditions for over a year, with five weeks of subculturing. Results: It was observed that two clones formed plants that displayed atypical growth structures, the shoots were shorter with many lateral shoots, and they had a lower fresh weight. They also formed fewer and smaller snap-traps, which, in the case of carnivorous plants, determines the capability of catching prey. The 35 in vitro regenerated plants and 5 specimens obtained from the natural habitat were subjected to genetic analyses with two molecular markers: start codon targeted (SCoT) polymorphism and sequence-related amplified polymorphism (SRAP). Despite the visible morphological variants, the genetic stability of all the regenerants with the individuals from natural stands was confirmed. All of them were monomorphic except three bands that were obtained for reference, where individuals were amplified with SCoT28 and me12-em13 SRAP primers. Conclusions: As shown in the presented research, it might be recommended to use different methods to evaluate the stability of in vitro cultivated plants. Full article

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

Saccharomyces cerevisiae often undergoes strain degeneration during industrial serial subculturing, though this phenomenon remains understudied. This study first conducted strain screening and biological characterization through TTC (2,3,5-triphenyltetrazolium chloride) colorimetric assays, Durham tube fermentation gas production tests, and WL medium (Wallerstein Laboratory medium) cultivation. Subsequently, the changes in intergenerational biological traits after serial subculturing were investigated. Finally, transcriptomic analysis was employed to examine differential gene expression under high-glucose stress during continuous subculturing. The experimental results demonstrated that: (1) The S. cerevisiae QDSK310-Z-07 (GenBank: PP663884), isolated from farm soil, exhibited robust growth within a temperature range of 24–36 °C, with optimal growth observed at 28 °C. It thrived in a pH range of 4–5.5 and efficiently utilized various carbon and nitrogen sources; (2) After serial subculturing, the strain’s ethanol production capacity and fermentation rate partially declined and then stabilized, while maintaining strong tolerance to high ethanol concentrations and hyperosmotic stress; (3) Transcriptomic analysis revealed significant differential expression of genes related to lipid metabolism, amino acid metabolism, and other pathways under high-glucose stress following continuous subculturing. These findings elucidate the biological trait variations in S. cerevisiae during serial subculturing and provide key metabolic regulation candidate targets for its long-term adaptive evolution under high-glucose stress. Full article

The establishment and partial characterization of three continuous cell lines from European freshwater fish species are provided. The three new cell lines, designated NPL-3, AF-1, and PF-1, were created from larvae of northern pike (Esox lucius) and fin tissues of asp (Leuciscus aspius) and European perch (Perca fluviatilis) fin tissues, respectively. All three cell lines have been subcultured more than 90 times since their establishment. Cells were optimally maintained at 25 °C in M199 medium supplemented with 10% fetal bovine serum. The NPL-3 and AF-1 cells are susceptible to spring viraemia of carp virus, pike fry rhabdovirus, ictalurid herpesvirus 2, and European catfish virus, while in the PF-1 cells, only the latter two viruses were successfully propagated. These newly established cell lines could serve as diagnostic tools for the aforementioned economically important viral diseases. They might be effective appliances for isolating novel viruses from northern pike, asp, European perch, and other closely related fish species. Full article

Pinus massoniana Lamb. (masson pine) is a critical species for afforestation in southern China but faces severe threats from pine wilt disease (PWD) caused by Bursaphelenchus xylophilus. To accelerate disease-resistant breeding, this study investigated the effects of cryopreservation on the embryonic capacity of the embryogenic callus as well as the effects of abscisic acid (ABA), polyethylene glycol 8000 (PEG 8000) and phytagel concentration on the somatic embryo’s maturation and germination. Furthermore, the impact of transplanting substrates on the survival and growth of regenerated plantlets were evaluated. The results showed that cryopreservation at −196 °C effectively maintained the embryogenic potential of the callus, with post-thaw tissues exhibiting superior somatic embryo maturation capacity compared to the long-term subcultured callus (38.4 vs. 13.2 embryos/mL). Key maturation parameters were systematically optimized: ABA concentration at 6 mg/L in the suspension culture maximized embryo yield of 24.1 somatic embryos/mL, while PEG 8000 at 130 g/L in solid medium achieved peak embryo production of 38.4 somatic embryos/mL, and the maximum of 26.6 somatic embryos/mL when the concentration of phytagel was 3.5 g/L. The highest germination rate of 29.8% was observed with 130 g/L PEG in the maturation medium. The highest survival rate (56.5%) and maximum plant height (22.3 cm) after 12 months of transplantation were achieved in substrates consisting of soil and vermiculite, which outperformed those containing varying proportions of mushroom residue. This study establishes a scalable protocol for the mass propagation of PWD-resistant P. massoniana, integrating cryopreservation and maturation media optimization, which offers dual benefits for disease-resistant breeding and sustainable germplasm conservation. Full article

Background: Nowadays, the microbial degradation of cellulose represents a new perspective for reducing cellulose waste from industry and households and at the same time obtaining energy sources. Methods: We isolated and enriched two aerobic (at 37 °C and 50 °C) and one anaerobic microbial consortium from an anaerobic bioreactor for biogas production by continuous subculturing on peptone cellulose solution (PCS) medium supplemented with 0.3% treated or untreated Whatman filter paper under static conditions. Samples were taken every 7 days until day 21 to determine the percentage of cellulose biodegradation. We determined the antimicrobial resistance of aerobic and anaerobic consortia and some single colonies by disc diffusion method, against 42 clinically applied antibiotics. PCR analyses were performed to search for the presence of eight genes for cellulolytic activity and nine genes for antibiotic resistance. By metagenomics analysis, the bacterial and fungal genus distributions in the studied populations were determined. Results: Aerobes cultured at 50 °C degraded cellulose to the greatest extent (47%), followed by anaerobes (24–38%) and aerobes (8%) cultured at 37 °C. The bacterial sequence analysis showed that the dominant phyla are Bacillota and Bacteroidetes and genera—Paraclostridium, Defluvitalea, Anaerobacillus, Acetivibrio, Lysinibacillus, Paenibacillus, Romboutsia, Terrisporobacter, Clostridium, Sporanaerobacter, Lentimicrobium, etc. in a different ratio depending on the cultivation conditions and the stage of the process. Some of these representatives are cellulolytic and hemicellulolytic microorganisms. We performed lyophilization and proved that it is suitable for long-term storage of the most active consortium, which degrades even after the 10th re-inoculation for a period of one year. We proved the presence of ssrA, ssrA BS and blaTEM genes. Conclusions: Our findings demonstrated the potential utility of the microbial consortium of anaerobes in the degradation of waste lignocellulose biomass. 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

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