Tissue Culture Research Articles

Blood culture bottles meet the operating room: enhancing the diagnostic accuracy of infectious spondylitis through open microsurgical biopsy and intraoperative inoculation.

Infectious spondylitis is caused by hematogenous seeding or adjacent soft tissue infection. No study has provided evidence that incubating biopsy specimens in blood culture bottles could enhance detection rates, nor has any study compared this method with conventional culture techniques. We aimed to assess the diagnostic yield of open microsurgical biopsies for infectious spondylitis and the efficacy of various culture media in the presence and absence of pre-biopsy antibiotic therapy. This retrospective study, which was conducted at a university-affiliated teaching hospital in Korea, enrolled 165 adult patients with suspected infectious spondylitis between February 2014 and September 2020. The diagnostic yield of open biopsy was compared among three culture media, namely, blood culture bottles, swab culture using transport media, and tissue culture using plain tubes, while considering preoperative antibiotic exposure. Causative bacteria were identified in 84.2% of all cases. Blood culture bottles had the highest positivity rate (83.5%), followed by swab cultures (64.4%) and tissue cultures (44.9%). The differences in positivity rates were significant (P < 0.001). Preoperative antibiotic therapy reduced detection rates across all media, particularly in tissue cultures. We established the high diagnostic yield of open microsurgical biopsy using blood culture bottles, suggesting that pre-biopsy antibiotic therapy significantly affects bacterial detection, thereby underscoring the importance of culture medium selection in the diagnosis of infectious spondylitis.

The gendered value chain of matooke banana and its implications for tissue culture adoption in Uganda

ABSTRACT Tissue culture banana is promoted as a form of micro-propagation that can aid farmers in managing pests and disease in Uganda, the country with the largest per capita consumption of banana in the world. But uptake amongst smallholder farmers remains low. This study recruited 71 farmers from five banana-growing districts in Uganda to assess how gender dynamics of matooke production impact the adoption and sustained use of tissue culture. We collaborated with farmers and co-produced a three-stage, participatory methodological protocol for mapping value chains. Our analysis reveals a gendered division of labour and limited access to resources that shape farmers’ ability to benefit from this technology. Women farmers are interested in tissue culture banana but face challenges related to demands for land preparation, accessing plantlets, and relevant knowledge, while men are more reluctant due to their fragility, high cost, and the additional labour demands needed to ensure the crop's survival. This study highlights the necessity of understanding the gendered division along a crop’s value chain to ensure the success of new breeding technologies.

A RapidAgrobacterium-Mediated Transformation Method Using Maize B104 Immature Embryos

Maize genetic transformation is a critical tool for functional genomics and crop improvement. Many laboratories, however, continue to face multiple challenges in attempting to achieve routine genetic transformation of maize inbred genotypes. Here, we describe a rapid and robust maize B104 transformation method using immature embryos as explants. This method uses anAgrobacteriumternary vector system, which includes a conventional T-DNA binary vector (pCBL101-RUBY) and a compatible ternary helper plasmid (pKL2299) that carries extra copies of essential virulence genes. The T-DNA binary vector carries theneomycin phosphotransferase II(NptII) gene for selection and a betalain biosynthesis marker,RUBY,for visual screening. We provide step-by-step instructions for immature embryo explant preparation,Agrobacteriuminfection, tissue culture procedures, and greenhouse care for acclimatization of regenerated plantlets.

Recent Advances inAgrobacterium-Mediated Maize Genetic Transformation Using Immature Embryos and Seedling Leaf Whorl Explants

The introduction of maize genetic transformation in the 1990s brought forth a powerful tool for crop improvement and a deeper understanding of plant genetics. Despite decades of genetics research, however, and the promise of CRISPR-mediated gene editing, maize transformation currently faces several challenges, such as genotype dependence and limitations in explant availability. Indeed, although the most commonly used method, immature embryo transformation, has been improved through optimization of tissue culture media composition and selection methods, the approach is only applicable to a limited number of public genotypes, including B104 and Hi II. Recently, genotype-flexible methods have been developed using coexpression cassettes of morphogenic transcription factors (MTFs)Baby boom(Bbm) andWushel2(Wus2), which have enabled the successful transformation of many previously recalcitrant maize lines. This MTF-based transformation method has also allowed for the use of alternate explants, such as seedling leaf whorl, whose production is cost-effective and requires only minimum controlled growth space. In this review, we summarize recent advances inAgrobacterium-mediated maize transformation methods that use immature embryos or seedling leaf whorls as starting material.

Investigation of Persistent Photoconductivity of Gallium Nitride Semiconductor and Differentiation of Primary Neural Stem Cells

A gallium nitride (GaN) semiconductor is one of the most promising materials integrated into biomedical devices to play the roles of connecting, monitoring, and manipulating the activity of biological components, due to its excellent photoelectric properties, chemical stability, and biocompatibility. In this work, it was found that the photogenerated free charge carriers of the GaN substrate, as an exogenous stimulus, served to promote neural stem cells (NSCs) to differentiate into neurons. This was observed through the systematic investigation of the effect of the persistent photoconductivity (PPC) of GaN on the differentiation of primary NSCs from the embryonic rat cerebral cortex. NSCs were directly cultured on the GaN surface with and without ultraviolet (UV) irradiation, with a control sample consisting of tissue culture polystyrene (TCPS) in the presence of fetal bovine serum (FBS) medium. Through optical microscopy, the morphology showed a greater number of neurons with the branching structures of axons and dendrites on GaN with UV irradiation. The immunocytochemical results demonstrated that GaN with UV irradiation could promote the NSCs to differentiate into neurons. Western blot analysis showed that GaN with UV irradiation significantly upregulated the expression of two neuron-related markers, βIII-tubulin (Tuj-1) and microtubule-associated protein 2 (MAP-2), suggesting that neurite formation and the proliferation of NSCs during differentiation were enhanced by GaN with UV irradiation. Finally, the results of the Kelvin probe force microscope (KPFM) experiments showed that the NSCs cultured on GaN with UV irradiation displayed about 50 mV higher potential than those cultured on GaN without irradiation. The increase in cell membrane potential may have been due to the larger number of photogenerated free charges on the GaN surface with UV irradiation. These results could benefit topical research and the application of GaN as a biomedical material integrated into neural interface systems or other bioelectronic devices.

Agrobacterium-mediated Transformation of Tropical Maize Using Seedling Leaf Whorl Explants

Conventional maize transformation has largely relied on immature embryos as explants, and is thus often hampered by the limited access to high-quality immature embryos year-round. Here, we present a detailed protocol using seedling leaf whorls as alternative explants for tropical maize inbred transformation. This approach involves the use of a cassette that drives the expression of the morphogenic transcription factors (MTFs)Baby boom(Bbm) andWuschel2(Wus2), which have been shown to greatly enhance transformation efficiency. We outline here the steps required for the preparation of seedling leaf whorl explants and subsequentAgrobacteriuminfection, and describe the tissue culture regimen that results in transgenic plant regeneration. Because constitutive expression ofBbmandWus2prevents normal plant regeneration and the production of fertile plants, the cassette containing these genes must be excised. As such, we include the steps for theCre/loxP-mediated excision of the MTF gene cassette. The protocol outlines a year-round, more affordable, and efficient approach for carrying out maize transformation for crop improvement.

Insight into the emerging insect to human pathogen Photorhabdus revealing geographic differences in immune cell tropism

BackgroundPhotorhabdus asymbiotica is a species of the insect pathogenic Photorhabdus genus that has been isolated as an etiological agent in human infections. Since then, multiple isolates have been identified worldwide; however, actual clinical infections have so far only been identified in North America, Australia, and Nepal. Previous research on the clinical isolates had shown that the strains differed in their behaviour when infecting cultured human cells.MethodsIn this study, we investigate the differences between the pathogenic activities of P. asymbiotica isolates from different geographic locations. Pathogenicity was analysed using infection assays with both cultured cell lines (THP-1, CHO, and HEK cells) and primary immune cells, and peripheral blood mononuclear cells (PBMCs) isolated from human blood.ResultsHere, we present the findings from the Australian (Kingscliff) and North American (ATCC43949) clinical isolates, and non-clinical soilborne nematode isolates from Thailand (PB68) and Northern Europe (HIT and JUN) of P. asymbiotica. We also show the first findings from a new clinical isolate of P. luminescens (Texas), the first non-asymbiotica species to cause a human infection, confirming its ability to infect and survive inside human immune cells.ConclusionHere for the first time, we show how P. asymbiotica selectively infects certain immune cells while avoiding others and that infectivity varies depending on growth temperature. We also show that the tropism varies depending on the geographic location a strain is isolated from, with only the European HIT and JUN strains lack the ability to survive within mammalian cells in tissue culture.

Multipurpose Impacts of Silver Nitrate on Direct Organogenesis of Begonia rex cv. DS-EYWA via Transverse Thin Cell Layering (tTCL) Technique

Begonia rex cv. DS-EYWA is an important plant for indoor and outdoor cultivation, and cv. DS-EYWA is a rare unique cultivar with curly, colorful leaves. Due to their importance, applying plant tissue culture techniques for mass and healthy production in a short period of time without seasonal limitation is of immense economic value. Applying several concentrations of silver nitrate (AgNO3) in combination with varied concentrations of cytokinins including 6-benzylaminopurine (BAP), thidiazuron (TDZ) (0, 0.5, 1, 1.5 mgL−1), and 1-naphthaleneacetic acid (NAA) auxin (0, 0.5, 1 mgL−1) via focusing on transverse thin cell layer (tTCL) petiole explants for high-scale production was used to establish an efficient in vitro propagation protocol. Our results showed that even low concentrations (25 mgL−1) can control internal bacterial infection and increase shoot direct regeneration efficiency. A combination of 1.5 mgL−1 BAP, 0.5 mgL−1 NAA, and 25 mgL−1 AgNO3 was the best treatment to increase the number of direct regenerated shoots, and a lower concentration of BAP (0.5 mgL−1) can be suggested for shoot elongation. Elongated shoots were successfully rooted in MS basal medium and acclimatized in a 1:1 peat moss/perlite sterilized pot mixture.

3D printed gelatin methacryloyl hydrogels for perfusion culture of human trabecular meshwork cells and glaucoma studies.

Glaucoma, a progressive eye disease leading to irreversible blindness, currently affects over 70 million people globally. Elevated intraocular pressure (IOP) is implicated in its development. IOP is carefully regulated by the trabecular meshwork (TM). However, studying TM behavior has been limited to traditional tissue culture studies or costly ex vivo cultures of animal and donor eyes. Developing novel functional TM models could enhance cell/tissue behavior understanding and aid therapeutic development for glaucoma. In this study, we 3D printed a simplified and reproducible model of the human TM (hTM) and studied hTM cell behavior under static and dynamic cultures. Gelatin Methacryloyl bioinks proved suitable for printing with viable and proliferative hTM cells expressing crucial marker genes in response to glucocorticoid induction. This, to our knowledge, is the first functional 3D printed hTM model and aims to facilitate TM research. Moreover, this easily reproducible model could also be applicable in the study of numerous other cell types throughout the body.

Endothelial extracellular vesicles enhance vascular self-assembly in engineered human cardiac tissues.

The fabrication of complex and stable vasculature in engineered cardiac tissues represents a significant hurdle towards building physiologically relevant models of the heart. Here, we implemented a 3D model of cardiac vasculogenesis, incorporating endothelial cells (EC), stromal cells, and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) in a fibrin hydrogel. The presence of CMs disrupted vessel formation in 3D tissues, resulting in the upregulation of endothelial activation markers and altered extracellular vesicle (EV) signaling in engineered tissues as determined by the proteomic analysis of culture supernatant. miRNA sequencing of CM- and EC-secreted EVs highlighted key EV-miRNAs that were postulated to play differing roles in cardiac vasculogenesis, including the let-7 family and miR-126-3p in EC-EVs. In the absence of CMs, the supplementation of CM-EVs to EC monolayers attenuated EC migration and proliferation and resulted in shorter and more discontinuous self-assembling vessels when applied to 3D vascular tissues. In contrast, supplementation of EC-EVs to the tissue culture media of 3D vascularized cardiac tissues mitigated some of the deleterious effects of CMs on vascular self-assembly, enhancing the average length and continuity of vessel tubes that formed in the presence of CMs. Direct transfection validated the effects of the key EC-EV miRNAs let-7b-5p and miR-126-3p in improving the maintenance of continuous vascular networks. EC-EV supplementation to biofabricated cardiac tissues and microfluidic devices resulted in tissue vascularization, illustrating the use of this approach in the engineering of enhanced, perfusable, microfluidic models of the myocardium.

Effect of plant growth regulators in inducing Vitisvinifera Callus and increased production of flavonoids in vitro

This study was aimed to use of plant tissue culture technology to induce callus from grapevine Vitisvinifera and stimulate it to increase the production of flavonoids. The study was carried out in two stages after the sterilization process was carried out: The first included establishing callus farms by cultivating cuttings containing a single node after completing their sterilization and cultivating them on MS medium containing 2,4-D and BA at different concentrations (0, 1, 2, 3,4) mg L -1and BA in concentrations (0, 0.1, 0.2, 0.4) in independent experiments. The second phase included studying the effect of growth regulators on the concentrations of carbohydrates and flavonoids in the callus induced from the first experiment. The results of the study showed the superiority of auxien 2,4-D at a concentration of 2mg L -1as it achieved the highest average fresh and dry weight of callus, reaching (3.32 and 1.03) mg respectively, while BA at a concentration of 0.2 mg L -1 achieved the highest average fresh and dry weight of callus was (1.91 and 0.51) mg respectively. The results also showed that the MS medium prepared with a concentration of 4 mg L -1of 2,4-D was significantly superior in the average concentration of the compounds Hesperdine, Nargnine, Proanthocyanin, and Rutin, reaching (64.94, 62.56, 55.63, and 71.22)% respectively.While the medium prepared with a concentration of 3 mg L -1outperformed 2,4-D achieved the highest concentration of Quercetin compounds reaching 75.36% compared to the neutral treatment, which achieved the lowest concentrations.

Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus

IntroductionThe critical early stages of infection and innate immune responses to porcine epidemic diarrhea virus (PEDV) at the intestinal epithelium remain underexplored due to the limitations of traditional cell culture and animal models. This study aims to establish a porcine enteroid culture model to investigate potential differences in susceptibility to infection across segments of the porcine small intestine (duodenum, jejunum, and ileum).MethodsIntestinal crypt cells from nursery pigs were cultured in Matrigel to differentiate into porcine enteroid monolayer cultures (PEMCs). Following characterization, PEMCs were enzymatically dissociated and subcultured on transwell inserts (PETCs) for apical surface exposure and infection studies. Characterization of region-specific PEMCs and PETCs included assessment of morphology, proliferation, viability, and cellular phenotyping via immunohistochemistry/immunocytochemistry and gene expression analysis. Subsequently, PETCs were inoculated with 105 TCID50 (50% tissue culture infectious dose)/mL of a high pathogenic PEDV non-S INDEL strain and incubated for 24 h. Infection outcomes were assessed by cytopathic effect, PEDV N protein expression (immunofluorescence assay, IFA), and PEDV N-gene detection (quantitative reverse transcription polymerase chain reaction, RT-qPCR).ResultsNo significant morphological and phenotypical differences were observed among PEMCs and PETCs across intestinal regions, resembling the porcine intestinal epithelium. Although PETCs established from different segments of the small intestine were susceptible to PEDV infection, jejunum-derived PETCs exhibited higher PEDV replication, confirmed by IFA and RT-qPCR.DiscussionThis segment-specific enteroid culture model provides a reliable platform for virological studies, offering a controlled environment that overcomes the limitations of in vivo and traditional cell culture methods. Standardizing culture conditions and characterizing the model are essential for advancing enteroid-based infection models.

Heritable gene editing in tomato through viral delivery of isopentenyl transferase and single-guide RNAs to latent axillary meristematic cells

Realizing the full potential of genome editing for crop improvement has been slow due to inefficient methods for reagent delivery and the reliance on tissue culture for creating gene-edited plants. RNA viral vectors offer an alternative approach for delivering gene engineering reagents and bypassing the tissue culture requirement. Viruses, however, are often excluded from the shoot apical meristem, making virus-mediated gene editing inefficient in some species. Here, we developed effective approaches for generating gene-edited shoots in Cas9-expressing transgenic tomato plants using RNA virus-mediated delivery of single-guide RNAs (sgRNAs). RNA viral vectors expressing sgRNAs were either delivered to leaves or sites near axillary meristems. Trimming of the apical and axillary meristems induced new shoots to form from edited somatic cells. To further encourage the induction of shoots, we used RNA viral vectors to deliver sgRNAs along with the cytokinin biosynthesis gene, isopentenyl transferase. Abundant, phenotypically normal, gene-edited shoots were induced per infected plant with single and multiplexed gene edits fixed in the germline. The use of viruses to deliver both gene editing reagents and developmental regulators overcomes the bottleneck in applying virus-induced gene editing to dicotyledonous crops such as tomato and reduces the dependency on tissue culture.

An Unusual Case of Blastomycosis Presenting as "Punched Out" Ulcerations

Background: Cutaneous blastomycosis commonly presents as verrucous, hyperkeratotic nodules or plaques. Less commonly, it presents as ulcerations with accompanying scale crusting or verrucous features. Case Report: A healthy 34-year-old incarcerated male with a history of drug abuse presented with several, well-defined (punched out), deep ulcerating cutaneous lesions with undermined borders. There was no crusting, or verrucous features. The clinical differential diagnoses included pyoderma, vasculitic ulcers, and pyoderma gangrenosum. The histopathology from a gluteal ulceration demonstrated pseudoepitheliomatous hyperplasia with acute, chronic, and granulomatous inflammation. PAS stain of the specimen exhibited thick-walled yeast demonstrating broad-based budding typical of blastomycosis and tissue cultures grew Blastomyces dermatitidis after one month. Conclusion: Ulceration without verrucous features can portend cutaneous blastomycosis. Diagnosis using fungal culture of the wound or tissue takes weeks to result. A prompt biopsy and histopathologic examination or broad-range PCR testing of a wound swab, tissue or urine specimen can lead to a prompt diagnosis.

PSXIII-14 Effect of melatonin supplementation during in vitro embryo culture on cleavage rates following in vitro maturation and fertilization of bovine oocytes

Abstract In vitro embryo production (IVEP) is an important advanced reproductive technology and has great potential in enhancing livestock production. However, in vitro culture (IVC) of embryos exposes them to stress resulting in compromising quality and efficiency of embryo production. Melatonin has been reported in several studies to reduce oxidative stress and improve the efficiency of IVEP. Our objective was to evaluate the effect of melatonin supplementation in IVC media on cleavage rates of bovine embryos. Bovine ovaries (n = 22) were obtained from local abattoir with ovarian weight of 7.29 ± 3.31 g, and size of 3.65 ± 0.69 cm. Cumulus-oocyte complexes (COCs) were recovered from 2 to 8 mm size follicles by aspiration using an 18-gauge needle attached with a 10 mL syringe. COCs were matured, fertilized, and cultured in vitro using IVF Bioscience bovine media and their protocols. Oocytes were in vitro matured for 23 h at 38.8°C, 5.5% CO2 in a humidified atmosphere and assessed for maturation by assessing COC expansion. Fully expanded and partially expanded COCs were fertilized for 20 h in a 4-well dish with frozen/thawed bull semen. Following IVF, the cumulus cells were removed by vertexing, and the presumptive zygotes were cultured in vitro with and without 1 µM melatonin in 4-well dishes in triplicates under the oil for embryo development at 38.8°C and 5.5% CO2. Cleavage rates were determined on d 3 of culture. The percentage of cleaved embryos was calculated as (number of zygotes cleaved/total number of fertilized oocytes) x100. Data were analyzed by student’s unpaired t- test with Welch’s correction using GraphPad Prism software. The cleavage rate was greater in melatonin supplemented group (54.17 ± 11.24%) than the control (49.83 ± 7.36%) but the difference was not significant (P &amp;gt; 0.05). In conclusion, under our experimental conditions, melatonin supplementation during embryo culture, following IVM/IVF of the bovine oocytes, did not significantly improve cleavage rates.

The value of current diagnostic techniques in the diagnosis of fracture-related infections: Serum markers, histology, and cultures

Although fracture-related infection (FRI) is a serious complication following bone fractures, a comprehensive definition and diagnostic criteria have only emerged in recent years. According to this consensus definition, the diagnosis of FRI is based on preoperative and intraoperative suggestive or confirmatory criteria. Serum markers, histology, and microbiological cultures are considered to play a crucial role in the FRI diagnostic pathway. However, at the time of publication of the FRI consensus definition in 2018 and its update in 2020, limited data was available on the accuracy of these diagnostic methods. This review aims to provide an overview of recent publications and discuss whether new evidence has been obtained regarding the value of these current diagnostic techniques.Meanwhile, several studies have confirmed the limited prognostic value of C-reactive protein, erythrocyte sedimentation ratio, and white blood cell count. Other serologic markers for preoperative diagnosis of FRI with promising diagnostic performance are d-dimer, plasma fibrinogen, platelet count to mean platelet volume ratio, and a risk prediction model that includes soft tissue injury type and fracture complexity in addition to blood markers. However, their true diagnostic value in daily clinical practice needs to be investigated in further studies. Data on histology in FRI diagnosis is still limited, but its potential as a confirmatory criterion seems to lie in its high specificity. Recent studies indicate that tissue culture exhibits moderate sensitivity and high specificity, with sensitivity improvements achieved by sampling of five specimens and long-term culture. Implant sonication also appears to enhance the sensitivity of culture and the detection rate of polymicrobial infections.In conclusion, the true value of diagnostic techniques is difficult to assess, in part because it is measured against a gold standard that is itself imperfect and still evolving, but also because of methodological differences in sample processing or the use of different thresholds. Nevertheless, this review has identified that the value of current diagnostic techniques is high when used in combination. To draw more accurate conclusions about the value of serum markers, histology, and culture including sonication, future studies should be prospective and utilize a greater standardization in sampling and methodological protocols.

β-glucans, SAM, and GSH fluctuations in barley anther tissue culture conditions affect regenerants’ DNA methylation and GPRE

BackgroundMicrospore embryogenesis is a process that produces doubled haploids in tissue culture environments and is widely used in cereal plants. The efficient production of green regenerants requires stresses that could be sensed at the level of glycolysis, followed by the Krebs cycle and electron transfer chain. The latter can be affected by Cu(II) ion concentration in the induction media acting as cofactors of biochemical reactions, indirectly influencing the production of glutathione (GSH) and S-adenosyl-L-methionine (SAM) and thereby affecting epigenetic mechanisms involving DNA methylation (demethylation—DM, de novo methylation—DNM). The conclusions mentioned were acquired from research on triticale regenerants, but there is no similar research on barley. In this way, the study looks at how DNM, DM, Cu(II), SAM, GSH, and β-glucan affect the ability of green plant regeneration efficiency (GPRE).ResultsThe experiment involved spring barley regenerants obtained through anther culture. Nine variants (trials) of induction media were created by adding copper (CuSO4: 0.1; 5; 10 µM) and silver salts (AgNO3: 0; 10; 60 µM), with varying incubation times for the anthers (21, 28, and 35 days). Changes in DNA methylation were estimated using the DArTseqMet molecular marker method, which also detects cytosine methylation.Phenotype variability in β-glucans, SAM and GSH induced by the nutrient treatments was assessed using tentative assignments based on the Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. The effectiveness of green plant regeneration ranged from 0.1 to 2.91 plants per 100 plated anthers. The level of demethylation ranged from 7.61 to 32.29, while de novo methylation reached values ranging from 6.83 to 32.27. The paper demonstrates that the samples from specific in vitro conditions (trials) formed tight groups linked to the factors contributing to the two main components responsible for 55.05% of the variance (to the first component DNM, DM, to the second component GSH, β-glucans, Cu(II), GPRE).ConclusionsWe can conclude that in vitro tissue culture conditions affect biochemical levels, DNA methylation changes, and GPRE. Increasing Cu(II) concentration in the IM impacts the metabolism and DNA methylation, elevating GPRE. Thus, changing Cu(II) concentration in the IM is fair to expect to boost GPRE.

Optimizing cannabis cultivation: an efficient in vitro system for flowering induction

BackgroundCannabis sativa L. is a versatile medicinal plant known for its therapeutic properties, derived from its diverse array of secondary metabolites synthesized primarily in female flower organs. Breeding cannabis is challenging due to its dioecious nature, strict regulatory requirements, and the need for photoperiod control to trigger flowering, coupled with highly dispersible pollen that can easily contaminate nearby female flowers. This study aimed to develop a protocol for in vitro flowering in cannabis, investigate factors affecting in vitro flower production, and generate viable in vitro seeds, potentially offering a method for producing sterile cannabinoids or advancing breeding techniques.ResultsWe show that the life cycle of cannabis can be fully completed in tissue culture; plantlets readily produce inflorescences and viable seeds in vitro. Our findings highlight the superior performance of DKW medium with 2% sucrose in a filtered vessel and emphasize the need for low light intensity during flower induction to optimize production. The improved performance in filtered vessels suggests that plants conduct photosynthesis in vitro, highlighting the need for future investigations into the effects of forced ventilation to refine this system. All tested lines readily developed inflorescences upon induction, with a 100% occurrence rate, including male flowering. We revealed the non-dehiscent trait of in vitro anthers, which is advantageous as it allows for multiple crosses to be conducted in vitro without concerns about cross-contamination.ConclusionThe current work developed and optimized an effective protocol for in vitro flowering and seed production in cannabis, potentially providing a platform for sterile cannabinoid production and an efficient tool for breeding programs. This system allows for the full and consistent control of plant growth conditions year-round, potentially offering the reliable production of sterile molecules suitable for pharmacological use. As a breeding strategy, this method overcomes the complex challenges of breeding cannabis, such as the need for large facilities, by enabling the production of hundreds of lines in a small facility. By offering precise control over factors such as plant growth regulators, light intensity, photoperiod, and temperature, this system also serves as a valuable tool for studying flowering aspects in cannabis.

Programmable soft DNA hydrogels stimulate cellular endocytic pathways and proliferation

Hydrogels are pivotal in tissue engineering, regenerative medicine, and drug delivery applications. Existing hydrogel platforms are not easily customizable and often lack precise programmability, making them less suited for 3D tissue culture and programming of cells. DNA molecules stand out among other promising biomaterials due to their unparalleled precision, programmability, and customization. In this study, we introduced a palette of novel cellular scaffolding platforms made of pure DNA-based hydrogel systems while improving the shortcomings of the existing platforms. We showed a quick and easy one step synthesis of DNA hydrogels using thermal annealing based on sequence specific hybridization strategy. We also demonstrated the formation of multi-armed branched supramolecular scaffolds with custom mechanical stiffness, porosity, and network density by increasing or decreasing the number of branching arms. These mechanically tuneable DNA hydrogels proved to be a suitable suitable platform for modulating the physiological processes of retinal pigment epithelial cells (RPE1). In-vitro studies showed dynamic changes at multiple levels, ranging from a change in morphology to protein expression patterns, enhanced membrane traffic, and proliferation. The soft DNA hydrogels explored here are mechanically compliant and pliable, thus excellently suited for applications in cellular programming and reprogramming. This research lays the groundwork for developing a DNA hydrogel system with a higher dynamic range of stiffness, which will open exciting avenues for tissue engineering and beyond.

Novel mechanism of MicroRNA408 in callus formation from rice mature embryo.

Mature embryos are the main explants of tissue culture used in rice transgenic technology. However, the mechanism of mature embryo callus formation remains unclear. In this study, a microRNA-mediated gene regulatory network of rice calli was established using degradome sequencing. We identified a microRNA, OsmiR408, that regulates the formation of the callus derived from the mature rice embryo. OsUCLACYANIN 30 (OsUCL 30), a target gene of OsmiR408, was the most abundant cleavage mRNA in rice callus. OsUCL17 was verified as a target gene of OsmiR408 using RNA ligase-mediated 5'-RACE. In analysis of the OsmiR408 promoter reporter line and pri-miR408 transcript level, the promoter activity and transcript level of MIR408 were increased dramatically during callus formation. In phenotypic observations, OsmiR408 knockout caused severe defects in mature embryo callus formation, whereas OsmiR408 overexpression promoted callus formation. Transcriptome analysis demonstrated that OsUCLs and certain genes related to the plant hormone signal transduction and phenylpropanoid-flavonoid biosynthesis pathway had different differential expression patterns between OsmiR408 knockout and overexpression calli. Thus, OsmiR408 may regulate callus formation mainly by affecting plant hormone signal transduction and phenylpropanoid-flavonoid biosynthesis pathway. Our findings provide insight into OsmiR408/UCLs module function in callus formation.