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Medicinal plants have been used historically for various treatments and in human nutrition. Due to their natural occurrence and biodegradability, medicinal plants can be an eco-friendly alternative to toxic chemicals such as pesticides, fungicides, and herbicides. An in vitro experiment was conducted to test the impact of six medicinal plant extracts on tomato growth and callus induction. Two concentrations of each extract (50 and 100 mg L-1) were added to the Murashige and Skoog (MS) medium culture. Results showed that Juniperus sabina extract (50 mg L-1) accelerated tomato seed germination by 100% in vitro, compared to the control group in MS basal medium. Callus growth index (CGI) and callus weight (CW) increased by 50% and by more than 200% when using Taraxacum officinale (100 mg L-1) and Conocarpus erectus (50 mg L-1) extracts, respectively. Biochemical analysis revealed that the extracts were rich in phenolic compounds (348 mg g-1 of total phenol), flavonoids (162 mg g-1), antioxidants (61%), and auxin. A rise in antioxidant activity, plant growth regulators (PGR), and plant defense elicitation probably contributed to these outcomes. Plant extracts also affected the biochemical content of calluses, except for their total phenol. We recommend using these plant extracts to increase growth, accelerate seed germination, and promote callus induction. Optimizing concentrations and combinations of medicinal plant extracts require further research to maximize their benefits to different plant species and their growth-related values. [ABSTRACT FROM AUTHOR]

An essential aspect of ensuring availability and stability of mesenchymal stem/stromal cells (MSCs) products for clinical use is that these cells are cryopreserved before individual infusion into patients. Currently, cryopreservation of MSCs involves use of a cryoprotectant solution containing dimethyl sulfoxide (DMSO). However, it is recognized that DMSO may be toxic for both the patient and the MSC product. In this Production Assistance for Cellular Therapies (PACT) and Biomedical Excellence for Safer Transfusion (BEST) Collaborative study, we compared a novel DMSO-free solution with DMSO containing cryoprotectant solutions for freezing MSCs. A DMSO-free cryoprotectant solution containing sucrose, glycerol, and isoleucine (SGI) in a base of Plasmalyte A was prepared at the University of Minnesota. Cryoprotectant solutions containing 5–10% DMSO (in-house) were prepared at seven participating centers (five from USA, one each from Australia and Germany). The MSCs were isolated from bone marrow or adipose tissue and cultured ex vivo per local protocols at each center. The cells in suspension were frozen by aliquoting into vials/bags. For six out of the seven centers, the vials/bags were placed in a controlled rate freezer (one center placed them at -80°C freezer overnight) before transferring to liquid nitrogen. The cells were kept frozen for at least one week before thawing and testing. Pre- and post-thaw assessment included cell viability and recovery, immunophenotype as well as transcriptional and gene expression profiles. Linear regression, mixed effects models and two-sided t-tests were applied for statistical analysis. MSCs had an average viability of 94.3% (95% CI: 87.2–100%) before cryopreservation, decreasing by 4.5% (95% CI: 0.03–9.0%; P : 0.049) and 11.4% (95% CI: 6.9–15.8%; P < 0.001), for MSCs cryopreserved in the in-house and SGI solutions, respectively. The average recovery of viable MSCs cryopreserved in the SGI was 92.9% (95% CI: 85.7–100.0%), and it was lower by 5.6% (95% CI: 1.3–9.8%, P < 0.013) for the in-house solution. Additionally, MSCs cryopreserved in the two solutions had expected level of expressions for CD45, CD73, CD90, and CD105 with no significant difference in global gene expression profiles. MSCs cryopreserved in a DMSO-free solution containing sucrose, glycerol, and isoleucine in a base of Plasmalyte A had slightly lower cell viability, better recovery, and comparable immunophenotype and global gene expression profiles compared to MSCs cryopreserved in DMSO containing solutions. The average viability of MSCs in the novel solution was above 80% and, thus, likely clinically acceptable. Future studies are suggested to test the post-thaw functions of MSCs cryopreserved in the novel DMSO-free solution. [ABSTRACT FROM AUTHOR]

Eucheuma denticulatum and Kappaphycus alvarezii are two of the most commercially important, cultivated carrageenophytes in Southeast Asia. Their large-scale farming through repeated vegetative propagation and virtual monocropping has resulted in a variety of phyconomic issues. The efficacy of two European, commercial liquid extracts from the brown seaweeds, i.e., Ascophyllum nodosum (ANE), a fucoid, and Laminaria digitata (LDE), a kelp, on the direct formation of axes and survival of these two eucheumatoids was examined using tissue culture techniques. Seaweed apical sections (2–3 mm long) were cultured for 45 days, with different concentrations of ANE and LDE (i.e., 0.005, 0.05, 0.5, 1.5, and 2.5 mL L−1) and compared with a control (UV-filtered seawater). Lower concentrations (< 1.5 mL L−1) of both liquid extracts promoted morphogenesis and growth of E. denticulatum and K. alvarezii in tissue culture, with the dilution rate of 0.05 mL LDE L−1 resulting in a high percentage survival (i.e., 95.3 ± 0.9%), formation of direct axes (i.e., 95.3 ± 0.9%), and the longest length (i.e., 10.0 ± 0.3 mm) for E. denticulatum after a typical 45-day culture period. A 0.5 mL ANE L−1 concentration was found to be beneficial for the tissue culture of K. alvarezii, given the relatively high final survival and direct axes formation (i.e., 96.2 ± 2.2%) and axes length (i.e., 8.2 ± 0.1 mm). This study supports the application of these temperate, brown seaweed-derived extracts as a phycobiostimulant enrichment in eucheumatoid micropropagation for mass production of plantlets for out-planting purposes. [ABSTRACT FROM AUTHOR]

Several studies have been conducted in the last 3 years to evaluate tilapia's parasitic and bacterial infection in cultures in Mexico. The present work evaluates the severity, prevalence, and health condition through the histopathological index based on the damage in the organs and tissues of tilapia cultured in ponds and cages during 2018 and 2019. Seven hundred and eight fish were collected from ponds and cages of tilapia farms in Chiapas, Oaxaca, and Guerrero and preserved and processed for histological observations. The prevalence of histopathological alterations found in each biological system was calculated and expressed in percentage. The histopathological index of each fish (HIfish) was calculated based on two different semi-quantitative methods. Histologically, the tilapia showed alterations as inflammation, melanomacrophage centers, hyperplasia, telangiectasias, and epitheliocystis unrelated to specific infectious pathogenic agents in both years but suggest effects associated with diverse factors in different biological systems. The prevalence was higher in all the parameters evaluated in 2018. The pathological manifestations in both years show that the health condition of the fish was affected regardless of the type of culture (cages and ponds), with the respiratory and digestive systems the most affected. These results show a baseline of the health condition of the tilapias cultured in southeast Mexico. The observed damages were not lethal but reflected a constant exposition to stressful conditions to trigger chronic inflammatory responses in the fish and make them susceptible to pathogens present in the environment. [ABSTRACT FROM AUTHOR]

The potential of nanotechnology revolutionized the different fields of modern sciences, enabling researchers to develop nanomaterial with extraordinary functionalities making life easier. Nanoparticles such as ZnO NPs and Ag NPs along with sterilizing agents found great potential for controlling contamination in banana tissue culture. Banana provides food for millions of people worldwide even though its origin was South East Asia and the Indian subcontinent. The emergence of tissue culture technology replaces the conventional banana propagation method, i.e., using suckers. Contaminations persist in tissue culture due to nutrient-rich media, thus decreasing its efficiency and profitability. The motive of this study is to make banana tissue culture more profitable by eradicating the contamination problem. Surface-sterilized explant may not be germ-free from inside due to covert bacteria (endogenous bacteria), which reside inside the plant in a passive state but can contaminate the experimental setup at later stages. In the present study, an experiment is designed by using various sterilizing agents (ethanol, NaOCl, H2O2, and HgCl2) for different periods to optimize the best combination of sterilizing agents for banana tissue culture. After treatment with these chemicals, the explant was inoculated to Murashige and Skoog (MS) media with plant growth hormones such as kinetin and indole acetic acid (IAA) and placed into a growth chamber for 1 week and observed regularly for appearance of contamination. The result shows that when the explant was treated with 70% ethanol for 1 min, 20% NaOCl for 10 min, and 0.2% HgCl2 for 2 min, we found it to be the most effective with the least contamination, i.e., 20%. In a separate experiment, zinc oxide (ZnO) and silver (Ag) nanoparticles were used to control endophytic contamination with different concentrations (60 mg/liter, 90 mg/liter, 120 mg/liter). Both nanoparticles show promising results in controlling endophytic contamination, but using silver nanoparticles as a part of media controls up to 100% contamination. Dual treatment of ZnO and Ag nanoparticles as sterilizing agents as an additional component of media successfully removes the contamination but becomes the major cause of tissue death in banana tissue culture. Chemical sterilization was good but not efficient as compared to the combination with nanoparticle application. Blends of chemicals and nanoparticles are effective against microbes not only from the surface but also from the inside of the plant, so using available nanoparticles with optimized concentration may open a new way in tissue culture technology. The result of this research explains the success of nanoparticles (Ag NPs and ZnO NPs) in controlling exo and endophytic contaminations in banana tissue culture, thus creating a unique way of banana explant sterilization. [ABSTRACT FROM AUTHOR]

【Objective】The study was carried out to establish a technical system suitable for tissue culture and rapid propagation of Pholidota chinensis Lindl.【Method】With the whole plant of P. chinensis Lindl as test material, the growth of explants was studied, the best explant and culture medium were selected, and the culture conditions such as seed germination and seedling proliferation were optimized.【Result】The best explant for tissue culture of P. chinensis Lindl. was the seed, and its roots, rhizomes and pseudobulbs did not germinate, but the seeds could germinate. The optimum culture formula of P. chinensis Lindl. was 1/2MS + 6-BA 1.5 mg/L + NAA 0.8 mg/L+6-KT 0.1 mg/L and the protocorm of P. chinensis Lindl. turned green under this condition. The organic matter selection of strong seedlings was conducted by adding banana puree (50 g/L), mashed potato (50 g/L) and coconut water (50 mL/L) to the basal medium, and it was found that the overall growth of P. chinensis Lindl. in the medium with banana puree was good and uniform with many pseudobulbs, and the plant height and leaf length were better than those of the P. chinensis Lindl. with mashed potato or coconut water added to the medium. Finally, 50 g/L banana puree was selected as the best organic matter added to the medium in the subsequent experiment. The best culture formula for the optimization of strong seedlings was 1/2MS + 50 g/L banana puree + 6-BA 0.5 mg/L + NAA 0.5 mg/L + 6-KT 0.3 mg/ L. Under this condition, the P. chinensis Lindl. grew well and the plant height was better. The best medium formula for rooting of strong seedlings was MS + 50 g/L banana puree + IBA 0.5 mg/L + NAA 0.5 mg/L. Under this condition, there were more pseudobulbs and more aerial roots on the seedlings.【Conclusion】To establish a technical system suitable for tissue culture and rapid propagation of P.chinensis Lindl. seeds, the best formula for the germination culture of P. chinensis Lindl. seeds is 1/2MS + 6-BA 1.5 mg/L + NAA 0.8 mg/L+6-KT 0.1 mg/L; the optimum culture formula for the optimization of strong seedlings is 1/2MS + 50 g/L banana puree + 6-BA 0.5 mg/L + NAA 0.5 mg/L + 6-KT 0.3 mg/L; the best medium formula for rooting is MS + 50 g/L banana puree + IBA 0.5 mg/L + NAA 0.5 mg/L. The study results lay a foundation for the resource conservation and utilization of P. chinensis Lindl. and its large-scale production.

Abstract The development and production of secondary metabolites from priceless medicinal plants are restricted by drought stress. Mentha pulegium L. belongs to the Lamiaceae family and is a significant plant grown in the Mediterranean region for its medicinal and aesthetic properties. This study investigated the effects of three polyethylene glycol (PEG) (0, 5, and 10%) as a drought stress inducer and four silicon nanoparticle (SiNP) (0, 25, 50, and 100 ppm) concentrations as an elicitor to overcome the adverse effect of drought stress, on the growth parameters and bioactive chemical composition of M. pulegium L. plants grown in vitro. The experiment was performed as a factorial experiment using a completely randomized design (CRD) consisting of 12 treatments with two factors (3 PEG × 4 SiNPs concentrations), 6 replicates were used for each treatment for a total of 72 experimental units. The percentage of shoot formation was inversely proportional to the PEG concentration; for the highest PEG concentration, the lowest percentage of shoot formation (70.26%) was achieved at 10% PEG. SiNPs at 50 ppm enhanced shoot formation, the number of shoots, shoot height, fresh and dry weight, rosmarinic acid, total phenols, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. The methanol extract from M. pulegium revealed the presence of significant secondary metabolites using gas chromatography‒mass spectrometry (GC–MS). The principal constituents of the extract were limonene (2.51, 2.99%), linalool (3.84, 4.64%), geraniol (6.49, 8.77%), menthol (59.73, 65.43%), pulegone (3.76, 2.76%) and hexadecanoic acid methyl ester or methyl palmitate (3.2, 4.71%) for the 0 ppm SiNPs, PEG 0% and 50 ppm SiNPs, and PEG 10%, respectively. Most of the chemical components identified by GC‒MS in the methanol extract were greater in the 50 ppm SiNP and 10% PEG treatment groups than in the control group. SiNP improves drought tolerance by regulating biosynthesis and accumulating some osmolytes and lessens the negative effects of polyethylene glycol-induced drought stress. Based on the results, the best treatment for most of the parameters was 50 ppm SiNPs combined with 10% PEG, the morphological and chemical characteristics were inversely proportional to the PEG concentration, as the highest PEG concentration (10%) had the lowest results. Most parameters decreased at the highest SiNP concentration (100 ppm), except for the DPPH scavenging percentage, as there was no significant difference between the 50 and 100 ppm SiNPs.

Nanomaterials (NMs) have found extensive applications in the realm of ornamental plants due to their unique properties. This article comprehensively discusses four main aspects of NM utilization in ornamental plants: 1) providing new insights into challenging problems in tissue culture, 2) exploring their regulatory effects on the growth of ornamental plants, 3) enhancing the resistance to biotic and abiotic stressors, and 4) discussing their widespread application and mechanisms in cut flower harvesting. Furthermore, potential issues and future directions are explored, providing a deeper theoretical basis for the application of novel NMs in the realm of ornamental plants.

Removing contaminants from plant material with minimal damage to plant cells using different chemical solutions represents very important and one of the most critical steps in establishing plant tissue culture in vitro. Our study deals with the optimisation of the protocol for successful surface sterilization of initial explants in two raspberry cultivars, ‘Meeker’ and ‘Willamette’ in two experimental years. The main objective during the first experimental year was to examine the protocols for surface sterilization which included different combinations of disinfectants (mercuric chloride or commercial bleach solution each in combination with 70% ethanol). In the following year, the impact of the two most effective sterilization treatments on the establishment of aseptic cultures over the four-month period (May, June, July, and August) was investigated in both raspberry cultivars. Aseptic cultures were established on Murashige and Skoog medium (MS) containing 2.0 mg/l N6-Benzyladenine (BA), 0.5 mg/l indole-3-butyric acid (IBA) and 0.1 mg/l gibberellic acid (GA3). The highest percentage of explants initiation in ‘Meekerʼ (79.3%) was achieved with surface sterilization in 70% ethanol (1 min) combined with 10% (v/v) commercial bleach solution (20 min), whereas sterilization in 70% ethanol (1 min) followed by HgCl2 (3 min) gave the best results (38.3%) in ‘Willametteʼ. The most optimal month for the establishment of aseptic culture was May for ‘Meeker’, while June was the most suitable month for the ‘Willametteʼ cultivar.

Abstract Stevia rebaudiana (stevia) is a plant in the Asteraceae that contains several biologically active compounds including the antidiabetic diterpene glycosides (e.g. stevioside, rebaudioside and dulcoside) that can serve as zero-calorie sugar alternatives. In this study, an elicitation strategy was applied using 5% polyethylene glycol (PEG), sodium chloride (NaCl; 50 and 100 mM) and gibberellic acid (2.0 and 4.0 mg/L GA3) to investigate their effect on shoot morphogenesis, and the production of phenolics, flavonoids, total soluble sugars, proline and stevioside, as well as antioxidant activity, in shoot cultures of S. rebaudiana. Herewith, the media supplemented with 2 mg/L and 4 mg/L GA3 exhibited the highest shooting response (87% and 80%). The augmentation of lower concentrations of GA3 (2 mg/L) in combination with 6-benzylaminopurine (BAP) resulted in the maximum mean shoot length (11.1 cm). The addition of 100 mM NaCl salts to the media led to the highest observed total phenolics content (TPC; 4.11 mg/g-DW compared to the control 0.52 mg/g-DW), total flavonoids content (TFC; 1.26 mg/g-DW) and polyphenolics concentration (5.39 mg/g-DW) in shoots cultured. However, the maximum antioxidant activity (81.8%) was observed in shoots raised in media treated with 50 mM NaCl. The application of 2 mg/L of GA3 resulted in the highest accumulation of proline (0.99 μg/mL) as compared to controls (0.37 μg/mL). Maximum stevioside content (71 µL/mL) was observed in cultures supplemented with 100 mM NaCl and 5% PEG, followed by the 4 mg/L GA3 treatment (70 µL/mL) as compared to control (60 µL/mL). Positive correlation was observed between GA3 and stevioside content. Notably, these two compounds are derived from a shared biochemical pathway. These results suggest that elicitation is an effective option to enhance the accumulation of steviosides and other metabolites and provides the groundwork for future industrial scale production using bioreactors.

Purpose: The objective of the present study was to examine the impact of explant type and varying concentrations of 2,4-Dichlorophenoxyacetic acid and 6-Benzyladenine growth regulators on the San Pedro cactus callus morphological and biochemical characteristics. Research method: Four types of explants were used i.e. explants with areola, without areola, with truncated areola, and with central tissue. Additionally, five combinations of BA and 2,4-D, were tested (0 mg/L BA + 2 mg/L 2,4-D, 2 mg/L BA + 2 mg/L 2,4-D, 3 mg/L BA + 3 mg/L 2,4-D, 4 mg/L BA + 4 mg/L 2,4-D, 0 mg/L BA + 0 mg/L 2,4-D). Findings: The results indicated that callus formation induced in all treatments 6 days after inoculation. There were significant differences in growth parameters, including fresh weight, volume, moisture, tissue firmness, total phenols, total flavonoids and antioxidant activity of the callus (P < 0.01) and dry weight of callus (P < 0.05). Explants holding a segment of central tissue, yielded the least favorable results in most of experimental treatments, and the application of 2,4-D in the absence of BA had an inhibitory and toxic effect on the San Pedro cactus explants. Research limitations: No limitations were found. Originality/Value: Specifically, use of 2 mg/L BA + 2 mg/L 2,4-D and explants with areola resulted in callus with higher fresh weight, volume and total flavonoids, as well as good tissue integrity and firmness. The reported results are a valuable resource for future research related to cell tissue culture and the elicitation of secondary metabolites in Echinopsis spp.

Background: Xerostomia is a pathological condition characterized by decreased salivation due to salivary gland dysfunction and is frequently attributed to irreversible damage as a side effect of radiation therapy. Stem cell–derived organoid therapy has garnered attention as a promising avenue for resolving this issue. However, Matrigel, a hydrogel commonly used in organoid culture, is considered inappropriate for clinical use due to its undefined composition and immunogenicity. In this study, we aimed to develop a method for culturing collagen-based human salivary gland organoids (hSGOs) suitable for clinical applications and evaluated their therapeutic effectiveness. Methods: Human salivary gland stem cells were isolated from the salivary gland tissues and cultured in both Matrigel and collagen. We compared the gene and protein expression patterns of salivary gland–specific markers and measured amylase activity in the two types of hSGOs. To evaluate the therapeutic effects, we performed xenogeneic and allogeneic transplantation using human and mouse salivary gland organoids (hSGOs and mSGOs), respectively, in a mouse model of radiation-induced xerostomia. Results: hSGOs cultured in Matrigel exhibited self-renewal capacity and differentiated into acinar and ductal cell lineages. In collagen, they maintained a comparable self-renewal ability and more closely replicated the characteristics of salivary gland tissue following differentiation. Upon xenotransplantation of collagen-based hSGOs, we observed engraftment, which was verified by detecting human-specific nucleoli and E-cadherin expression. The expression of mucins, especially MUC5B, within the transplanted hSGOs suggested a potential improvement in the salivary composition. Moreover, the allograft procedure using mSGOs led to increased salivation, validating the efficacy of our approach. Conclusions: This study showed that collagen-based hSGOs can be used appropriately in clinical settings and demonstrated the effectiveness of an allograft procedure. Our research has laid the groundwork for the future application of collagen-based hSGOs in allogeneic clinical trials. [ABSTRACT FROM AUTHOR]

Prenatal exposure to toxins can adversely affect long-term health outcomes of the offspring. Though chemotherapeutics are now standard of care for treating cancer patients during pregnancy, certain compounds are known to cross the placenta and harm placental tissue. The consequences for the fetus are largely unexplored. Here we examined the responses of newborn cord blood mononuclear cells in tissue culture to two chemotherapeutic drugs, cyclophosphamide and epirubicin, when either directly exposed to these drugs, or indirectly after crossing a placenta trophoblast bilayer barrier. Cord blood mononuclear cells exposed to the conditioned media obtained from cyclophosphamide-exposed trophoblast barriers showed a significant 2.4-fold increase of nuclear ROS levels compared to direct exposure to cyclophosphamide. Indirect exposure to epirubicine-exposed trophoblast barriers not only enhanced nuclear ROS levels but also significantly increased the fraction of cord blood cells with double strand breaks, relative to directly exposed cells. Neither apoptosis nor proliferation markers were affected in cord mononuclear blood cells upon direct or indirect exposure to cyclophosphamide or epirubicin. Our data suggests that trophoblast cells exposed to cyclophosphamide or epirubicine may induce an indirect 'bystander' effect and can aggravate genotoxicity in the fetal compartment. [ABSTRACT FROM AUTHOR]

Background: Although sonication is a valuable diagnostic tool for periprosthetic joint infections (PJI), it is not commonly utilized. We analyzed sonicate and intraoperative tissue culture results obtained from three hospitals to define the microbial etiology of PJIs in Korea. Furthermore, we investigated necessity of conducting regular fungal and mycobacterial cultures. Methods: We retrospectively analyzed data for patients with suspected orthopedic-related infections between 2017 and 2022, who had undergone prostheses removal surgery. We included 193 patients with suspected PJIs, and bacterial (n = 193), fungal (n = 193), and mycobacterial (n = 186) cultures were conducted on both sonicate and intraoperative tissue samples. The diagnosis of PJI was based on the European Bone and Joint Infection Society (EBJIS) criteria. Results: Out of 193 patients, 121 (62.7%) had positive sonicate cultures, while 112 (58.0%) had positive periprosthetic tissue cultures. According to EBJIS criteria, a total of 181 patients were diagnosed with PJI, and 141 patients received microbiological confirmation through sonicate fluid culture or tissue culture. Of the 181 patients, 28 were classified with acute PJI (within 3 months of implantation) and 153 with chronic PJI. Among 141 patients, staphylococci were the most common organisms, accounting for 51.8% of cases, followed by Gram-negative organisms (15.6%), fungus (8.5%), and mycobacteria (3.5%). Nearly 91.7% of fungal isolates were Candida species, which also grew in bacterial cultures. In total, 11 cases cultured positive only in tissue culture, whereas 20 cases cultured positive only in sonicate culture. The antibiotic treatment plans were adjusted according to culture results. Conclusions: Utilizing sonicate culture has greatly assisted in identifying pathogens responsible for chronic indolent PJIs, allowing suitable antimicrobial treatment. Based on few cases involving non-Candida and mycobacterial infections, it appears that routine fungal and mycobacterial cultures may not be necessary. [ABSTRACT FROM AUTHOR]

Myrteae is the most species-rich tribe in the Myrtaceae family, represented by a range of socioeconomically and ecologically significant species. Many of these species, including commercially relevant ones, have become increasingly threatened in the wild, and now require conservation actions. Tissue culture presents an appropriate in vitro tool to facilitate medium-term and long-term wild germplasm conservation, as well as for commercial propagation to maintain desirable traits of commercial cultivars. So far, tissue culture has not been extensively achieved for Myrteae. Here, tissue culture for Eugenia, one of the most species-rich genera in Myrteae, is reviewed, giving directions for other related Myrteae. This review also focuses on ex situ conservation of Australian Myrteae, including using seed banking and field banking. Despite some progress, challenges to conserve these species remain, mostly due to the increasing threats in the wild and limited research. Research into in vitro methods (tissue culture and cryopreservation) is paramount given that at least some of the species are 'non-orthodox'. There is an urgent need to develop long-term in vitro conservation for capturing the remaining germplasm of threatened Myrteae. [ABSTRACT FROM AUTHOR]

Johne's disease (JD), a chronic, infectious enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), affects wild and domestic ruminants. There is no cure or effective prevention, and current vaccines have substantial limitations, leaving this disease widespread in all substantial dairy industries causing economic, and animal welfare implications. Mycobacteriophages (MPs) have been gaining interest in recent years and are proposed as a promising solution to curtailing MAP infection. Using a well-validated infection model, we have demonstrated the preventative potential of MPs to protect dairy calves against MAP infection. Calves were supplemented daily with a phage cocktail from birth till weaning at 2 m of age and inoculated with MAP at 2 wk of age. Infection status was measured for 4.5 mo through blood, fecal, and postmortem tissue samples. Our findings highlight the remarkable efficacy of orally administered MPs. Notably, fecal shedding of MAP was entirely eliminated within 10 wk, in contrast to the infected control group where shedding continued for the entirety of the trial period. Postmortem tissue culture analysis further supported the effectiveness of MPs, with only 1 out of 6 animals in the phage-treated group testing positive for MAP colonized tissues compared to 6 out of 6 animals in the infected control group. Additionally, plaque assay results demonstrated the ability of phages to persist within the intestinal tract. Collectively, these results underscore the potential of orally administered MP cocktails as a highly effective intervention strategy to combat JD in dairy calves and by extension in the dairy industry. [ABSTRACT FROM AUTHOR]

Volume electron microscopy encompasses a set of electron microscopy techniques that can be used to examine the ultrastructure of biological tissues and cells in three dimensions. Two block face techniques, focused ion beam scanning electron microscopy (FIB-SEM) and serial block face scanning electron microscopy (SBF-SEM) have often been used to study biological tissue samples. More recently, these techniques have been adapted to in vitro tissue culture samples. Here we describe step-by-step protocols for two sample embedding methods for in vitro tissue culture cells intended to be studied using SBF-SEM. The first focuses on cell pellet embedding and the second on en face embedding. En face embedding can be combined with light microscopy, and this CLEM workflow can be used to identify specific biological events by light microscopy, which can then be imaged using SBF-SEM. We systematically outline the steps necessary to fix, stain, embed and image adherent tissue culture cell monolayers by SBF-SEM. In addition to sample preparation, we discuss optimization of parameters for data collection. We highlight the challenges and key steps of sample preparation, and the consideration of imaging variables. [ABSTRACT FROM AUTHOR]

Centrosomes are the main microtubule-organizing centers in animal cells. Due to the semiconservative nature of centrosome duplication, the two centrosomes differ in age. In asymmetric stem cell divisions, centrosome age can induce an asymmetry in half-spindle lengths. However, whether centrosome age affects the symmetry of the two half-spindles in tissue culture cells thought to divide symmetrically is unknown. Here, we show that in human epithelial and fibroblastic cell lines centrosome age imposes a mild spindle asymmetry that leads to asymmetric cell daughter sizes. At the mechanistic level, we show that this asymmetry depends on a cenexin-bound pool of the mitotic kinase Plk1, which favors the preferential accumulation on old centrosomes of the microtubule nucleation--organizing proteins pericentrin, γ-tubulin, and Cdk5Rap2, and microtubule regulators TPX2 and ch-TOG. Consistently, we find that old centrosomes have a higher microtubule nucleation capacity. We postulate that centrosome age breaks spindle size symmetry via microtubule nucleation even in cells thought to divide symmetrically. [ABSTRACT FROM AUTHOR]

【Objective】The study was carried out to establish a technical system suitable for tissue culture and rapid propagation of Pholidota chinensis Lindl.【Method】With the whole plant of P. chinensis Lindl as test material, the growth of explants was studied, the best explant and culture medium were selected, and the culture conditions such as seed germination and seedling proliferation were optimized.【Result】The best explant for tissue culture of P. chinensis Lindl. was the seed, and its roots, rhizomes and pseudobulbs did not germinate, but the seeds could germinate. The optimum culture formula of P. chinensis Lindl. was 1/2MS + 6-BA 1.5 mg/L + NAA 0.8 mg/L+6-KT 0.1 mg/L and the protocorm of P. chinensis Lindl. turned green under this condition. The organic matter selection of strong se edlings was conducted by adding banana puree (50 g/L), mashed potato (50 g/L) and coconut water (50 mL/L) to the basal medium, and it was found that the overall growth of P. chinensis Lindl. in the medium with banana puree was good and uniform with many pseudobulbs, and the plant height and leaf length were better than those of the P. chinensis Lindl. with mashed potato or coconut water added to the medium. Finally, 50 g/L banana puree was selected as the best organic matter added to the medium in the subsequent experiment. The best culture formula for the optimization of strong seedlings was 1/2MS + 50 g/L banana puree + 6-BA 0.5 mg/L + NAA 0.5 mg/L + 6-KT 0.3 mg/ L. Under this condition, the P. chinensis Lindl. grew well and the plant height was better. The best medium formula for rooting of strong seedlings was MS + 50 g/L banana puree + IBA 0.5 mg/L + NAA 0.5 mg/L. Under this condition, there were more pseudobulbs and more aerial roots on the seedlings.【Conclusion】To establish a technical system suitable for tissue culture and rapid propagation of P.chinensis Lindl. seeds, the best formula for the germination culture of P. chinensis Lindl. seeds is 1/2MS + 6-BA 1.5 mg/L + NAA 0.8 mg/L+6-KT 0.1 mg/L; the optimum culture formula for the optimization of strong seedlings is 1/2MS + 50 g/L banana puree + 6-BA 0.5 mg/L + NAA 0.5 mg/L + 6-KT 0.3 mg/L; the best medium formula for rooting is MS + 50 g/L banana puree + IBA 0.5 mg/L + NAA 0.5 mg/L. The study results lay a foundation for the resource conservation and utilization of P. chinensis Lindl. and its large-scale production. [ABSTRACT FROM AUTHOR]

Background: Lilium (Lilium longiflorum Thunb.) belongs to the family Liliaceae and is a native of Northern Hemisphere (up to South Canada and Siberia). Conventionally, lilium can easily be propagated by sexual and asexual methods of propagation but these prevalent methods are not capable of meeting the increasing demand in domestic and global market. Generally, lilium is propagated through bulbs which has bottlenecks like high cost of planting material, infected from seed borne and other diseases like mosaic, grey mould, etc. So, in vitro multiplication of lilium is utmost important. Lily can be easily regenerated from explants The objective of present study was to identify the best media and the explant among scales and nodal segments belonging to the three different cultivars viz., Elite, Brunello, Cordelia where maximum number of shoots could be regenerated. Methods: The present investigation was carried out in the Tissue Culture Laboratory of the Centre for Research and Application in Plant Tissue Culture. Twenty-five media combinations were used to study their effect on in vitro shoot regeneration from various explants of Lilium longiflorum cultivars. Two type of explants viz. scales and nodal segments were aseptically excised from Elite, Brunello, Cordelia cultivars of Lilum and cultured on Murashige and Skoog (MS) medium containing different concentrations and combinations of 6-benzyl amino purine (BAP), kinetin and Naphthalene Acetic Acid (NAA). Result: In cultivars Elite and Brunello, the maximum number of shoots were observed when nodal segments were used on media combination i.e., LM-8 (MS+BAP 3.0 mg/l+NAA0.1 mg/l). In cultivar Cordelia, scales showed the maximum response on media LM-24 (MS+Kinetin 2.0 mg/l+NAA 0.1 mg/l). [ABSTRACT FROM AUTHOR]

Ajuga L., a member of the Lamiaceae family, is one of the most popular species due to its medicinal, decorative, and pharmacological properties. One of them is Ajuga vestita BOISS. which is endangered and placed in the "Endangered (EN)" category. In the present study, using in vitro tissue culture techniques, Ajuga vestita BOISS. seeds were germinated in hormone-free 1/4 MS medium, and callus were regenerated from leaf explants of germinated seeds in 1/1 MS medium containing BAP (benzylaminopurine) and Kin (kinetin). It was aimed at comparatively examining the fatty acid content of Ajuga vestita BOISS. callus. When the fatty acid content of the callus obtained from the control (hormone-free), BAP, and Kin media were examined, major saturated fatty acid amounts were palmitic acid in all three extracts (59.137%, 38.836%, and 24.295%) for control, BAP, and Kin respectively. The major unsaturated fatty acid amounts were octadecanoic acid (21.097%, 32.283%) for control and BAP, respectively, and linoleic acid (21.209%) for Kin considering that high amounts of unsaturated fatty acids make the plants more reliable in terms of health, the higher amount of unsaturated fatty acid content determined in the Kin extract in our study compared to the control will shed light on future studies in this sense that can be done with tissue culture techniques. [ABSTRACT FROM AUTHOR]

Pogostemon cablin, an oil-producing plant, proceeded vegetative propagation due to limited natural pollination, leading to lower genetic variations. The presented study investigated the morphological diversity and productivity, specifically the ninth mutant vegetative (MV9) generation resulting from polyploid mutation induced by colchicine in the P. cablin var. Sidikalang. The study transpired between August 2022 and April 2023 at the Tissue Culture Laboratory, the Cikabayan Experimental Station, IPB University, Bogor, Indonesia. The experiment had a randomized complete block design (RCBD) arrangement with eight genotypes, including control as the single factor and three replications. Various characteristic measurements included essential oil yield growth and potential. The findings revealed that aneuploid mutants exhibited more leaves, primary, and secondary branches than the control plants and polyploid mutants (P < 0.05). Polyploid mutants (tetraploid and mixoploid) displayed longer and broader leaves, larger leaf surfaces, thicker leaves, and greater stem diameter (P < 0.05). Accumulation of leaves, primary, and secondary branches caused an increase in the patchouli plant's fresh weight. Patchouli oil production per hectare indicated a correlation to leaf number (r = 0.48), primary branches (r = 0.41), secondary branches (r = 0.48), and essential oil yield (r = 0.87). The study also revealed the considerable genetic diversity among the patchouli mutants, paving the way to develop promising new plant lines, specifically to obtain superior patchouli with the highest biomass and oil. [ABSTRACT FROM AUTHOR]

Background: Astragalus membranaceus Mongholicus (Astragalus) is one of the most widely used medicinal plants in the world. It is a traditional Chinese medicine plant that has been used to treat various human diseases and conditions, such as inflammation, gastrointestinal bacterial infections and malignant tumors. With the expansion of artificial planting areas, the demand for high-quality Astragalus seedlings is also increasing. The traditional seedling-raising methods of direct seeding and induced callus have problems such as a long growth period, unstable genetic traits, strict sampling and rooting difficulties. Therefore, it is very important to multiply the excellent germplasm by optimizing the tissue culture regeneration system. Methods: In this study, Astragalus field cultivated plants, which are easily available, were used as materials and the method of tissue culture cuttings was used to select the direct inoculation method of stem segments as explant materials to establish a plant regeneration system of Astragalus and the best sampling period, sampling site and optimal medium for regeneration of field cultivated plants were screened. Result: The best sampling period for plant regeneration of Astragalus in the field was about 40 d after the emergence of the 3-yearold Astragalus and the rooting rate of the regenerated plants was 100.00%. The best sampling site is the middle section of the stem and the regeneration system using the middle section of the stem as the explant is better than using the top and bottom of the stem as the explant and the rooting rate of the regenerated plants is 100.00%; the best medium for the regenerated plants is N22: 1/2MS+1 mg/L NAA and T28: 1/2MS+1 mg/L ABT, the rooting rates were 76.67% and 96.67%, respectively. The regenerated plants established with the stems of the field cultivated plants of Astragalus as explants were all able to induce seedlings after 1 week and the regenerated seedlings start to take root after 3 weeks of growth and can be transplanted and propagated after 6 weeks of culture. The regeneration system established in this study by direct cuttings from cultivated Astragalus stems is more rapid and efficient, breaking through the difficulties of limited access to material and long growth period of regenerated seedlings in the traditional regeneration system and providing technical support for the conservation and utilization of wild resources of Astragalus and rapid expansion of new varieties and germplasm conservation. [ABSTRACT FROM AUTHOR]

Juglans mandshurica is an important component of Korean pine mixed and secondary forests in Northeast China and is also a precious timber and economic species. In this study, plant regeneration of J. mandshurica has been established using stems with bud and mature zygotic embryos as explants. The results showed that placing the stems with buds in low-temperature conditions (0 to 4°C) and in darkness for 5 d inhibited the browning of the explants and promoted bud sprouting; Na2S2O3 also inhibited the browning of mature zygotic embryos significantly. The suitable basal media and hormones for bud sprouting and zygotic embryo germination were (1) WPM medium (woody plant medium) containing 2.0 mg·L−1 6-benzylaminopurine (6-BA) and 0.01 mg·L−1 3-indolebutyric acid (IBA) and (2) DKW medium (Driver and Kuniyuki medium) containing 2.5 mg·L−1 6-BA and 0.1 mg·L−1 IBA. The suitable basal medium and hormone for rooting of shoots from buds was 1/2 Murashige and Skoog (MS) medium (the macro-elements were half-strength of MS medium) supplemented with 1.0 mg·L−1 IBA, and the suitable basal medium and hormone for rooting of seedlings derived from zygotic embryo was 1/2 MS medium containing 1.0 mg·L−1 IBA, and DKW medium with 2.5 mg·L−1 6-BA, 0.1 mg·L−1 IBA, 0.2 mg·L−1 gibberellin A3 (GA3), and 0.2 g·L−1 activated carbon (AC). About 95% of the rooted plantlets survived after acclimation and transplantation. These results provided a foundation for the scale propagation to meet the demands of forestry production of the species. [ABSTRACT FROM AUTHOR]

Axonopus compressus (Swartz) Beauv., a perennial herb in the Poaceae that has been introduced to a number of tropical and subtropical countries and regions, can serve as a lawn ground cover while its leaves can be developed as a bioenergy resource. It also displays some resistance to heavy metals, allowing it to be planted in areas and urban green spaces polluted by heavy metals. A. compressus is also used as a traditional Chinese medicine. This study is the first report on tissue culture of A. compressus. Stem explants induced shoot clusters on 6-benzyladenine (BA)-containing Murashige and Skoog (MS) medium, with 2.0 mg L–1 BA inducing a shoot proliferation coefficient (SPC) of 10.89 within 30 d, while MS medium containing 2.0 mg L–1 BA and 0.1 mg L–1 α-naphthaleneacetic acid (NAA) amplified SPC to 12.88 within 30 d. SPC on MS medium supplemented with kinetin (KIN) or thidiazuron (TDZ) was not as high as on medium with BA and never exceeded 3.57 within 30 d. A. compressus formed adventitious roots easily, within 15 d, most efficiently on ½MS medium supplemented with 0.1 to 0.5 mg L–1 IAA, NAA, or IBA, or even on auxin-free medium. Resulting plantlets displayed a high survival rate (> 98%) when transplanted to a substrate containing humus, or humus mixed with vermiculite or sand. Callus was also successfully induced from leaf sheaths on MS medium containing 1.0 mg L–1 2,4-dichlorophenoxyacetic acid, even more so when 1.15 g L–1l-proline was added. Induced callus was able to differentiate into adventitious shoots on MS medium with 0.1 to 0.5 mg L–1 KIN or BA. An efficient system of shoot proliferation, callus induction, and regeneration in A. compressus has been established. [ABSTRACT FROM AUTHOR]

Mass propagation of cassava on several hectares of arable land due to increasing demand for its starch is not feasible due to land availability, pests and disease invasion, and long cultivation period. Plant cell culture technology is a promising solution despite the scarcity of cassava callus culture for starch production applications. Therefore, a systematic mapping study (SMS) was performed to identify the applications of cassava tissue culture and its prospects in starch production and investigate the important parameters for cassava callus culture initiation. The SMS began with formulating research questions (RQs), conducting searches on various databases, collecting and screening related articles, and extracting and mapping the selected articles. A total of 56 of 589 articles in the initial searching phase were chosen to be used as references to answer each RQ. The extracted data indicates that cassava tissue culture was mostly used for micropropagation, while starch production from its tissue culture is still limited. Basal medium and plant growth regulators influence cassava callus culture initiation most. The findings of the SMS offer a better understanding of cassava tissue culture and the prospects of producing cassava starch. [ABSTRACT FROM AUTHOR]

Botryosphaeriaceae fungi cause infections that generate disease symptoms in plants in extreme environments. The present study identified the causal agent of dieback disease on lebbeck trees in Saudi Arabia. Albizia lebbeck trees showed widespread dieback, decline and cracking symptoms of samples taken during a Qassim University, Saudi Arabia survey. The survey showed that over 80% of lebbeck trees showed wilted roots, stem cankers and death of wilted trees. Fungal colonies were obtained from symptomatic tissues cultured on water agar for 3 weeks at 25°C. Elongation factor alpha (EF1‐728 F, AL33R), ITS (ITS4) and LSU (128) regions of the rDNA operon and the partial beta‐tubulin gene (tub2; Bt2aF, Bt2bR) were sequenced for molecular identification. Based on morphological and molecular characterization, the pathogen was identified as Neoscytalidium dimidiatum. During the pathogenicity investigation, the fungus re‐isolated from the infected seedlings expressed the same morphological characteristics on the culture media as the N. dimidiatum isolate. A host range study involving six tree species inoculation with N. dimidiatum caused wilting and death in three plants. To our knowledge, this study is the first to report on N. dimidiatum in Saudi Arabia. [ABSTRACT FROM AUTHOR]

Background: Infectious endocarditis (IE) remains a critical condition despite all the medical advances in recent decades. Reliable pathogen identification is indispensable for precise therapy. The aim of this study was to evaluate the diagnostic and therapeutic benefit of additional polymerase chain reaction (PCR) in comparison with microbiological culture alone based on intraoperative tissue sampling for patients operated on due to IE. Methods: A total of 224 patients diagnosed with acute or subacute IE were analyzed. Intraoperatively resected infectious tissue was analyzed using both PCR and microbiological culture. Subsequently, the results of the detection of bacteria obtained based on intraoperative measurements from tissue via culture and PCR were compared with preoperative blood culture results. Furthermore, we evaluated the therapeutic impact of the culture and/or PCR results obtained from cardiac tissue. Results: The 224 patients were 63 ± 17 years old, and 64 (29%) were female. In total, 149 (67%) suffered from aortic valve endocarditis, 45 (45%) had mitral valve endocarditis, and 39 (18%) were afflicted with double-valve endocarditis. Prosthetic valve endocarditis was present in 70 (31%) patients. Pathogens were detected in 70% of the cases analyzed via PCR using cardiac valve tissue and in 25% of those analyzed via a culture of cardiac valve tissue; this figure was only 64% for preoperative blood culture. Overall, a pathogen was identified in 197 patients (88%), leading to antibiotic therapy. Targeted antibiotic therapy, based on the PCR results, was carried out in 37 cases and was conducted based on a culture from cardiac valve tissue in three cases. Finally, in 12% of patients, the causative pathogen remained unclear. Conclusions: For patients suffering endocarditis, PCR analysis is indispensable and superior to preoperative blood culture and intraoperative culture in detecting bacteria. Based on PCR testing, antibiotic therapy can be individually adjusted. The high precision of pathogen identification may lead to a significant reduction in IE-associated morbidity and mortality. [ABSTRACT FROM AUTHOR]

The current toolkit for genetic manipulation in the model animal Drosophila melanogaster is extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogaster using the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111-bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into 4 independent long noncoding RNA genes as well as the male-specific splice variant of doublesex using scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3′ fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila , with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes. [ABSTRACT FROM AUTHOR]

Pacific oysters (Crassostrea or Magallana gigas) are one of the most economically important aquaculture species globally. Over the past two decades, ostreid herpesvirus (OsHV-1) has become a major pathogen of cultured Pacific oysters, resulting in widespread mortality with a global distribution. Experimental use of OsHV-1 is challenging for many reasons, including both complexity of host–pathogen dynamics and a lack of functioning model systems. The goal of this study was to improve the tools available for working with OsHV-1 in both whole animals and in tissue explants established from oysters maintained in controlled laboratory conditions. Tissue explants were taken from oysters originating from two different sources that have different levels of mortality in experimental OsHV-1 infections and were exposed to OsHV-1. A whole-animal infection experiment was run concurrently as a comparison. Quantitative PCR and electron microscopy were used to confirm that the explants were capable of replicating OsHV-1. Furthermore, the quantitative PCR results suggest that the source of the oysters was significant in determining the outcome of infection in the explants, supporting the validity of the explant model for OsHV-1 infection. This tissue explant approach for studying OsHV-1 allows for the control of confounding factors in the disease outcome that is not possible in whole-animal experiments, providing a new tool for the study of OsHV-1 in Pacific oysters. [ABSTRACT FROM AUTHOR]

Dendrobium species, esteemed as a group of orchid plants with important ornamental and medicinal value, are currently experiencing significant developmental advancements. This research encompasses a comprehensive review of Dendrobium tissue culture literature spanning 2008 to 2023, sourced from the Web of Science Core Collection database. Employing the bibliometric analysis tool CiteSpace, we conducted a visualization and presentation of data encompassing research institutions, authorship patterns, key terminology, and emergent themes, elucidating the current status, hotspots, and frontiers of Dendrobium tissue culture research. After thorough screening, a total of 261 papers were included in this study. The publication count exhibited a consistent annual increase. Chinese Acad Sci, Univ Sains Malaysia, North Eastern Hill Univ, Univ Debrecen, and Univ Putra Malaysia emerged as primary research institutions, while a core team led by J.A. Teixeira da Silva, S. Sreeramanan, S. Kumaria, and N. Chen played a dominant role. Central research themes revolve around optimizing the tissue culture system of medicinal Dendrobium, and exploring the genetic stability and gene function. Furthermore, the field is witnessing a trajectory towards future multidisciplinary cross-collaboration. [ABSTRACT FROM AUTHOR]

• Successfully established the callus induction system of Saxifraga stolonifera Meerb. • The best sterilization method of explants and the ratio of plant growth regulators for callus induction were selected. • SA and SNP could increase the content of secondary metabolites in callus. • This study has certain reference significance for the commercial production of Saxifraga stolonifera Meerb. Elicitation is a well-known strategy for increasing the accumulation of secondary metabolites in callus cultures. In this study, a callus induction system for Saxifraga stolonifera Meerb. was developed, and elicitors were used to enhance secondary metabolite production. Tender leaves of S. stolonifera were used as explants for in vitro culture. We examined callus induction characteristics of S. stolonifera and its response to various elicitors by assessing the sterilization conditions of the explants, varying hormone ratios, and determining the optimal concentrations of salicylic acid (SA), Sodium nitroprusside (SNP) and methyl jasmonate (MeJA). The results revealed that sterilizing the explants with 70% ethanol for 40 s, followed by 0.1% mercury chloride for 10 min, resulted in a survival rate of 82.22% during tissue culture. Following these sterilization methods, callus formation was successfully induced on Murashige and Skoog medium supplemented with 2 mg/L 6-benzylaminopurine and 0.15 mg/L naphthaleneacetic acid, achieving an induction rate of 88.88%. Additionally, the study identified SA as the most effective elicitor for increasing the levels of gallic acid, protocatechuic acid, and quercitrin in the callus, with optimal concentrations of salicylic acid being 60, 40, and 60 μM, respectively. SNP at 40 μM, 80 μM, and 20 μM concentrations was identified as the best elicitor for promoting the accumulation of bergenin, chlorogenic acid, and quercetin, respectively. This study established appropriate conditions for cultivating S. stolonifera callus and identified the optimal elicitor concentrations for maximizing secondary metabolite production. [ABSTRACT FROM AUTHOR]

Potatoes (Solanum tuberosum L.) constitute one of the most economically important annual crops. In terms of tissue culture, potato microtubers (MTs) have a number of advantages over conventional plants. These advantages include their small size, which greatly facilitates storage, transport, and germplasm exchange compared to in vitro plants. One effective solution for the production and mass propagation of healthy MTs is the use of temporary immersion systems (TISs). In this study, in a SETISTM system containing kinetin/gibberellic acid (GA)/indole-3-butyric acid (IBA) hormones, we investigated the effects of different nutrient media on the morphological characteristics of potato explants and MTs. We determined the optimal cycling duration (3 h) with an immersion frequency of 2 min. The results revealed that the optimal nutrient medium for culturing single-node potato explants in a SETISTM bioreactor was the M7 medium containing kinetin (2 mg/L), GA (0.5 mg/L), and IBA (0.5 mg/L). The optimal nutrient medium for obtaining potato MTs was the M1 medium (hormone-free) with a high concentration of sucrose (9%) at 18 °C under dark growing conditions. Thus, a universal nutrient medium, employed in a bioreactor, was selected for the mass propagation of potato MTs for both domestic and foreign potato varieties. [ABSTRACT FROM AUTHOR]

We developed a technique that can measure corneal transepithelial electrical resistance (TER) in mice, which was used for evaluating corneal toxicity induced by ophthalmic drugs. We used a tissue culture well and its insert to mount the mouse globe and separated the cornea from the rest of the globe to enable corneal TER measurements to be taken. The explanted mouse eyes were divided into groups, and the corneal epithelia were exposed to different concentrations of BAC. Half of these eyes were fixed for transmission electron microscopy (TEM) examination and the other for ZO-1 immunohistochemical (IHC) evaluation. After exposure to control, 0.1%, 0.2%, and 0.5% BAC, the TER was 100 ± 0%, 91 ± 14%, 83 ± 13%, and 34 ± 12% of the pre-exposure TER at 1 min, respectively, with a statistically significant decrease in the 0.5% group. After 3 min, the TER showed a statistically significant decrease in the 0.2% and 0.5% groups. The TEM examinations showed a loss of epithelial tight junctions between superficial cells in the 0.2% and 0.5% groups. The IHC examination showed decreased ZO-1 staining of the corneal epithelium of the same groups as compared to the control. To the best of our knowledge, we succeeded in developing an innovative technique for corneal TER measurement in mice. [ABSTRACT FROM AUTHOR]

Candida albicans is a major fungal pathogen of humans that can cause serious systemic infections in vulnerable immunocompromised populations. One of its virulence attributes is its capacity to transition between yeast and filamentous morphologies, but our understanding of this process remains incomplete. Here, we analyzed data from a functional genomic screen performed with the C. albicans Gene Replacement And Conditional Expression collection to identify genes crucial for morphogenesis in host-relevant conditions. Through manual scoring of microscopy images coupled with analysis of each image using a deep learning-based method termed Candescence, we identified 307 genes important for filamentation in tissue culture medium at 37°C with 5% CO2. One such factor was orf19.5963 , which is predicted to encode the prenyltransferase Nus1 based on sequence homology to Saccharomyces cerevisiae. We further showed that Nus1 and its predicted interacting partner Rer2 are important for filamentation in multiple liquid filament-inducing conditions as well as for wrinkly colony formation on solid agar. Finally, we highlight that Nus1 and Rer2 likely govern C. albicans morphogenesis due to their importance in intracellular trafficking, as well as maintaining lipid homeostasis. Overall, this work identifies Nus1 and Rer2 as important regulators of C. albicans filamentation and highlights the power of functional genomic screens in advancing our understanding of gene function in human fungal pathogens. [ABSTRACT FROM AUTHOR]

Immortalized keratinocytes can offer a low‐cost experimental platform for human skin research, with increased cell yield compared to primary cultures. However, the usefulness of these surrogate cell models is highly dependent on their ability to retain the phenotypic attributes of the parent cells. Keratins K14 and K5 are the hallmarks of undifferentiated, mitotically active basal keratinocytes. We observed occasional progressive loss of K14 expression in growing keratinocyte cell lines, with persistent retention of K5 and an epithelial phenotype, and investigated possible reasons for this. We show that K14 repression occurs by DNA promoter methylation of KRT14 gene and is compounded by histone deacetylation and by the presence of EGF. In vivo, keratinocytes shut down K14 synthesis as they commit to terminal differentiation and move from the basal to spinous layer, but by laser‐capture microdissection of human epidermis we could detect no evidence of increased selective KRT14 methylation in this normal process. Loss of K14 expression suggests that epidermal identity of cultured keratinocytes can be compromised in certain tissue culture situations, possibly due to the immortalization method and persistent EGF supplementation. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What is the pathological mechanism involved in a thin endometrium, particularly under ischaemic conditions? SUMMARY ANSWER Endometrial dysfunction in patients with thin endometrium primarily results from remodelling in cytoskeletons and cellular junctions of endometrial epithelial cells under ischemic conditions. WHAT IS KNOWN ALREADY A healthy endometrium is essential for successful embryo implantation and subsequent pregnancy; ischemic conditions in a thin endometrium compromise fertility outcomes. STUDY DESIGN, SIZE, DURATION We recruited 10 patients with thin endometrium and 15 patients with healthy endometrium. Doppler ultrasound and immunohistochemical results confirmed the presence of insufficient endometrial blood perfusion in patients with thin endometrium. Organoids were constructed using healthy endometrial tissue and cultured under oxygen–glucose deprivation (OGD) conditions for 24 h. The morphological, transcriptomic, protein expression, and signaling pathway changes in the OGD organoids were observed. These findings were validated in both thin endometrial tissue and healthy endometrial tissue samples. PARTICIPANTS/MATERIALS, SETTING, METHODS Endometrial thickness and blood flow were measured during the late follicular phase using transvaginal Doppler ultrasound. Endometrial tissue was obtained via hysteroscopy. Fresh endometrial tissues were used for the generation and culture of human endometrial organoids. Organoids were cultured in an appropriate medium and subjected to OGD to simulate ischemic conditions. Apoptosis and cell death were assessed using Annexin-V/propidium iodide staining. Immunofluorescence analysis, RNA sequencing, western blotting, simple westerns, immunohistochemistry, and electron microscopy were conducted to evaluate cellular and molecular changes. MAIN RESULTS AND THE ROLE OF CHANCE Patients with thin endometrium showed significantly reduced endometrial thickness and altered blood flow patterns compared to those with healthy endometrium. Immunohistochemical staining revealed fewer CD34-positive blood vessels and glands in the thin endometrium group. Organoids cultured under OGD conditions exhibited significant morphological changes, increased apoptosis, and cell death. RNA-seq identified differentially expressed genes related to cytoskeletal remodeling and stress responses. OGD induced a strong cytoskeletal reorganization, mediated by the RhoA/ROCK signaling pathway. Additionally, electron microscopy indicated compromised epithelial integrity and abnormal cell junctions in thin endometrial tissues. Upregulation of hypoxia markers (HIF-1α and HIF-2α) and activation of the RhoA/ROCK pathway were also observed in thin endometrial tissues, suggesting ischemia and hypoxia as underlying mechanisms. LARGE SCALE DATA none. LIMITATIONS AND REASONS FOR CAUTION The study was conducted in an in vitro model, which may not fully replicate the complexity of in vivo conditions. WIDER IMPLICATIONS OF THE FINDINGS This research provides a new three-dimensional in vitro model of thin endometrium, as well as novel insights into the pathophysiological mechanisms of endometrial ischaemia in thin endometrium, offering potential avenues for identifying therapeutic targets for treating fertility issues related to thin endometrium. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the National Natural Science Foundation of China (81925013); National Key Research and Development Project of China (2022YFC2702500, 2021YFC2700303, 2021YFC2700601); the Capital Health Research and Development Project (SF2022-1-4092); the National Natural Science Foundation of China (82288102, 81925013, 82225019, 82192873); Special Project on Capital Clinical Diagnosis and Treatment Technology Research and Transformation Application (Z211100002921054); the Frontiers Medical Center, Tianfu Jincheng Laboratory Foundation(TFJC2023010001). The authors declare that no competing interests exist. [ABSTRACT FROM AUTHOR]

Introduction: Although antifungal supplementation reduces the fungal load in the corneal storage medium, consensus is lacking on the influence of dosing and temperature. The study aims to evaluate the impact of eye bank warming protocol and timing of antifungal supplements on efficacy in Optisol-GS and tissue. Methods: Corneoscleral rims contaminated with Candida albicans (C. albicans) were incubated in Optisol-GS, either without antifungal agents or with the addition of amphotericin B or voriconazole at various concentrations (2 ×, 5 ×, 10 ×, and 20 × MIC), at different time points, and under various preservation temperatures (2–8 °C versus 2 h-room temperature exposure). Antifungal efficacy was evaluated by counting viable yeast colonies cultured from Optisol-GS samples. Tissue sterility was determined through direct tissue culture and histological examination of the contaminated rims after a 14-day incubation period. Results: Room temperature exposure did not increase colony growth at the same multiple MIC of antifungal agents. Although antifungal addition reduced C. albicans growth in a concentration-dependent manner, yeast growth was still observed in all Optisol-GS samples with a single supplementation after a 14-day incubation. Only groups with additional antifungal supplementation on either day 2 or day 6 showed a 99% or greater reduction of C. albicans growth in Optisol-GS samples and yielded negative results in direct tissue culture. Conclusions: The eye bank warming protocol did not compromise antifungal efficacy. To sustain the required concentration and effectively reduce C. albicans growth in Optisol-GS and contaminated tissue, additional antifungal supplementation on either day 2 or day 6 was necessary during a 2-week preservation period. [ABSTRACT FROM AUTHOR]

The present study investigated in vitro multiplication and bulbification in one imported garlic accession i.e. VFG158 and eleven local garlic accessions of Mauritius viz. Beeharry, Boodnah, Bondah, Gooniah, Haulkhory, Ramdhuny, Ramjee, Rampall, Sujeebun, Sujeebun 2 and Unuth through meristem culture. The explants were subjected to fourteen shoot multiplication and three bulbification treatments. Positive results for shoot proliferation and suppression of hyperhydricity were noted on six shoot multiplication media: MS basal media with various growth regulators {G0 (0.25mg/L NAA+0.5mg/L 2iP), G1 (1.5mg/L BAP+0.5mg/L NAA), G2 (0.3mg/L NAA+3mg/L 2iP), G6 (0.5mg/L NAA+2mg/L 2iP), G14(2mg/L BAP+2mg/L NAA) and G15(1mg/L BAP+0.5mg/L NAA)}. The highest number of shoot formation was observed in G2 (0.3mg/L NAA+3mg/L 2iP) and the lowest number of shoot formation was observed in G14 (2mg/L BAP+2mg/L NAA). Genotypic difference in shoot multiplication and hyperhydricity on different media formulation was observed. The highest shoot proliferation was observed in the garlic accession Ramdhuny, while the lowest shoot proliferation was recorded in groups of similar accessions namely, Ramjee, Sujeebun and VFG 158. Bulblet formation was earlier on bulbification medium B2 (MS enriched with 12% sucrose). Largest and heavier bulblets were obtained on medium B5 (MS supplemented with 2mg/L BAP+1mg/L GA3 and enriched with 90% sucrose). A reliable protocol for rapid shoot regeneration and multiplication from meristem-tip culture and bulblet formation from multiple shoot clumps was optimised. [ABSTRACT FROM AUTHOR]

Gotu kola (Centella asiatica L.) is one of the valuable medicinal plants, whose biodiversity is facing the risk of extinction in the world due to the limitation of distribution areas and the reduction of surface waters. In order to investigate somatic embryogenesis in gotu kola, an experiment was carried out in 2018 in the tissue culture laboratory of the Faculty of Agriculture of Gilan University. Leaves were used as explants and MS medium as basal medium. The effect of different concentrations of plant growth regulators (2,4-dichlorophenoxyacetic acid (2,4-D) in five concentrations of 0, 0.25, 0.5, 1, 1.5 and 2 mg/L in combination with benzyl adenine (BAP) in four concentrations of 0, 0.25, 0.5 and 1 mg/L) were tested to determine the appropriate culture medium for inducing gotu kola embryogenic callus. For the induction of somatic embryos the good status obtained calli were transferred to different concentration of 2,4-D including 0, 0.1, 0.2 and 0.3 mg/L. In the next step the derived somatic embryos were subjected to 0, 0.25, 0.5 and 1 mg/L of BAP for germination. The results showed that the treatment with 2 mg/L of 2,4-D had the highest percentage of callus formation (96.61%), fresh weight (3.96 g/cm2 of leaf) and average callus diameter (3.56 cm) in dark conditions. Also, the treatment of 0.1 mg/L of 2,4-D had the highest average number of embryos formed in different stages of spherical, heart, and torpedo shaped, and the treatment of 0.1 mg/L of 2,4-D had the highest percentage of embryogenesis (55.27%), which was not significantly different from the treatment of 0.2 mg/L of 2,4-D. The results showed that the treatment of 1 mg/liter of BAP had the highest percentage (56.8%) of embryo germination and also the highest average number (12.4) of produced seedlings. In general, the use of 2,4-D to create embryogenic calli and BAP to germinate the formed embryos was satisfactory. [ABSTRACT FROM AUTHOR]

Cibotium barometz (L.) J. Sm., a fern, holds significant value in traditional Chinese medicine. This research aimed to establish a rapid tissue breeding technology for Cibotium barometz (L.) and provide a theoretical basis for its artificial breeding and industrial planting. The optimum conditions for spores germination were selected, including both the best sterilization time and the ideal spore germination medium. Subsequently, the optimal medium for proliferation and differentiation were chosen, and the optimal concentration of plant hormones was screened to determine the optimal medium. The rooting experiment was performed in two single-factor experiments by using differentiated sporophytes as materials. These experiments showed that the optimal sterilization time was 11 min and the best medium for germination was 1/8MS + 0.02 mg/L IAA. MS medium was suitable for proliferation, while WPM medium was suitable for sporophyte differentiation. The best medium for growth was MS + 1.0 mg/L IAA + 0.5 mg/L 6-BA. In the absence of sucrose, the seedlings barely grew and failed to differentiate into sporophytes. They showed optimal growth and differentiation at a sucrose concentration of 20 g/L. The plant growth regulators plant growth regulators used, including IAA, NAA, 6-BA, and GA3, showed more effective differentiation, wherease KT had a slightly weaker effect. The rooting rate reached the maximum when the concentrations of ABT and IBA were at 1.5 mg•L− 1 and IAA was at 1.0 mg•L− 1. The IBA had a significant effect on the average and the longest root length, while the three PGRs had no obvious effect on the average or maximum diameter. Plant tissue culture seedlings still have good genetic stability after subsequent generation. In this study, the optimal cultivation conditions for rapid breeding technology in tissue culture, covering stages from germination and growth to differentiation and rooting, were obtained. Moreover, sporophytes were successfully transplanted to the external environment. This study provided a theoretical reference for the industrial planting of C. barometz.Key message: In this study, a more systematic, complete and efficient tissue culture rapid propagation system was formed from spore germination, proliferation, differentiation, and took roots, along with the sporophytes for transplanting and culture. [ABSTRACT FROM AUTHOR]

Neolamarckia cadamba (N. cadamba) is an evergreen tree species known for its rapid growth, remarkable wood properties, and significant value in medicine, feeding, and landscape. In order to clone a N. cadamba individual with excellent genotype, a plant regeneration protocol was successfully established with leaves wrapped by bud scales as explants. The optimal sterilization method for the leaves was 0.1% Mercury Chloride (HgCl2) treatment for 1 min before culturing on Murashige and Skoog’s medium (MS) supplemented with 3.0 mg/L Thidiazuron (TDZ), 0.1 mg/L 2–4 Dichlorophenoxyacetic acid (2-4D), 0.05 mg/L α-Naphthaleneacetic acid (NAA) and 1 mL/L Plant Preservative Mixture (PPM) to induce calluses. The medium containing 1 mL/L PPM could effectively inhibit explant contamination without an unfavorable impact on the final induction rate of callus from the leaves. Three types of calluses were induced from the leaves cultured on the above medium. Among them, only the Type II callus, which was green and nodular, had few particle masses, could differentiate into adventitious shoots on the MS medium supplemented with 1.5 mg/L 6–Benzylaminopurine (6-BA) and 0.05 mg/L NAA, with the induction rate of 78.89% and adventitious shoot number per callus of 11.67. The adventitious shoots were proliferated on the MS medium supplemented with 1.0 mg/L 6-BA and 0.05 mg/L Indole-3- butyric acid (IBA) with the proliferation coefficient of 3.37. And the micro-shoots developed roots in the MS medium supplemented with 0.05 mg/L NAA and 0.05 mg/L IBA. The regeneration protocol can be used in the propagation and large scale production of seedlings with the same genotype as an excellent individual of N. cadamba in the field.Key message: A plant regeneration protocol was successfully established with leaves wrapped by bud scales in the field, furthermore it can be used in the propagation and large scale production of seedlings with the same genotype as an excellent individual of N. cadamba. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Solanum nigrum (Solanaceae family) is widely consumed as a fruit or local leafy vegetable after boiling; it also serves as a medicinal plant. Although Agrobacterium-mediated genetic transformation has been established in S. nigrum, the transformation period is long. Specifically, induction of roots takes approximately five weeks for tetraploid and hexaploid S. nigrum, and 7 weeks for diploid Solanum americanum. In this study, we developed an improved rooting-induced method that requires only about 1 week and avoids the use of tissue culture. After generating the transgenic shoots, they were directly transplanted into the soil to facilitate root formation. Remarkably, 100% of the transgenic shoots developed roots within 6 days. Our improved method is time-saving (saving more than 1 month) and simpler to operate. The improved rooting-induced step can be applied to induce roots in various plants using tissue culture, exemplified by the carnation (Dianthus caryophyllus L.). Furthermore, we applied the improved method to generate S. americanum plants expressing AcMYB110 from kiwifruit (Actinidia chinensis spp.). This method will contribute to speeding up gene functional analysis and trait improvement in S. nigrum and might have potential in fast plant molecular breeding processes in crops and rapid rooting induction in tissue culture. [ABSTRACT FROM AUTHOR]

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy, with relapse being a major obstacle to successful treatment. Our understanding of the mechanisms driving chemotherapy resistance and ultimately relapse in leukemia remains incomplete. Herein, we investigate the impact of the tumor microenvironment on leukemia cell drug responses using human plasma-like media (HPLM), designed to mimic physiological conditions more accurately ex vivo. We demonstrate that while most chemotherapeutics maintain an efficacy in HPLM comparable to standard tissue culture media, the thiopurines 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) exhibit significantly reduced potency and efficacy against both B- and T-leukemia cells in HPLM. By merging our understanding of thiopurines' mechanism of action with the metabolites supplemented in HPLM compared to standard media, we proposed and subsequently validated the hypothesis that hypoxanthine, a purine derivative, is responsible for conferring resistance to the thiopurines. Importantly, the concentration of hypoxanthine required for resistance is comparable to physiological levels found in vivo, supporting clinical relevance. Our findings demonstrate the utility of a more physiologic media in identifying and characterizing mechanisms by which the microenvironment can enable resistance. Understanding such interactions may inform strategies to overcome drug resistance and improve therapeutic outcomes in pediatric leukemia. [ABSTRACT FROM AUTHOR]

Cymbidium faberi Rolfe is one of the traditional Chinese orchids with important ornamental value, and the cultivation of Cymbidium faberi Rolfe mutant strains with different appearances is essential to increase its economic value. However, at present, their acquisition largely relies on natural mutation. The objectives of this research were to mutagenize Cymbidium faberi Rolfe protocorm-like bodies (PLBs) and shoots in vitro using sodium azide (NaN3) and to screen and evaluate mutants in the mutagenized seedlings using morphological characteristics. Cymbidium faberi Rolfe PLBs and shoots were used as mutagenic materials. Mutations were induced by the addition of 0.0 (control), 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg·L−1 and 0.0 (control), 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 mg·L−1 NaN3 to a shoot-growth-inducing medium. The mortality rates of the PLBs and shoots increased with an increase in the NaN3 concentration. At 14 d of co-cultivation, the PLBs and shoots were most efficiently mutagenized with 1.5 mg·L−1 and 4.0 mg·L−1 NaN3, respectively. After the explants were cultured for 3 months, changes in leaf and flower morphology were observed in some mutants: shorter and thicker leaves, shorter node length, reduced height, and mid-translucent leaves compared with controls. Some Cymbidium faberi Rolfe bloomed prematurely, with single flowers with large, thick petal sepals and small inflorescences. Colors included light green throughout, with some exhibiting purple stamens. This suggests that NaN3 can effectively mutagenize Cymbidium faberi Rolfe PLBs and shoots to satisfy people's demand for this plant's ornamental properties while increasing its economic value. [ABSTRACT FROM AUTHOR]