Basal Medium Research Articles

In Vitro Regeneration from Leaf Explants of Helianthus verticillatus, a Critically Endangered Sunflower.

Helianthus verticillatus (Asteraceae), a whorled sunflower, is a perennial species restricted to a few locations in the southeastern United States and is now considered endangered. Therefore, restoring and protecting H. verticillatus as a species is a priority. This study introduces a highly efficient in vitro adventitious plant regeneration system from leaf explants, utilizing five diverse specimens of H. verticillatus, each representing distinct genotypes with phenotypic variations in leaf and stem morphology. Key factors influencing in vitro morphogenesis, including genetic constitution, explant source, and plant growth regulators (PGRs), were identified. The study revealed a remarkably strong genotype-dependent impact on the regeneration efficiency of the investigated H. verticillatus genotypes, ranging from a lack of regeneration to highly effective regeneration. The selection of two genotypes with varying regeneration abilities provides valuable models for genetic analyses, offering insights into factors influencing the regeneration potential of this endangered species. Optimum adventitious shoot regeneration results were achieved using Murashige and Skoog basal media (MS) supplemented with 8.8 µM N6-benzyladenine (BA) and 1.08 µM α-naphthalene acetic acid (NAA). This combination yielded the highest adventitious shoot production. Subsequent successful rooting on ½ MS medium without PGRs further solidified the efficiency of the developed protocol. Regenerated plantlets, demonstrating robust shoots and roots, were successfully acclimatized to greenhouse conditions with a 95% survival rate. The protocol developed in this study is the first such report for this endangered species and is expected to contribute to future genetic manipulation and modification studies.

Angiogenesis is promoted by hypoxic cervical carcinoma-derived extracellular vesicles depending on the endothelial cell environment

IntroductionCancer needs perfusion for its growth and metastasis. Cancer cell-derived extracellular vesicles (CA-EVs) alter the tumor microenvironment (TME), potentially promoting angiogenesis. We hypothesize that conditions in the tumor, e.g., hypoxia, and in the target cells of the TME, e.g., nutrient deprivation or extracellular matrix, can affect the angiogenic potential of CA-EVs, which would contribute to explaining the regulation of tumor vascularization and its influence on cancer growth and metastasis. MethodsCA-EVs were isolated and characterized from cervical carcinoma cell lines HeLa and SiHa cultured under normoxia and hypoxia, and their angiogenic potential was evaluated in vitro in three endothelial cells (ECs) lines and aortic rings, cultured in basal (growth factor-reduced) or complete medium. ResultsHypoxia increased EV production 10–100 times and protein content 2–4 times compared to normoxic CA-EVs. HeLa-EVs contained six times more RNA than SiHa-EVs, and this concentration was not affected by hypoxia. Treatment with CA-EVs increased tube formation and sprouting in ECs and aortic rings cultured in basal medium and long-term stabilized the stablished vascular networks formed by ECs cultured in complete medium. ConclusionHypoxia differentially affects CA-EVs in a cell line-dependent manner. The cellular environment (nutrient availability and extracellular matrix scaffold) influences the effect of CA-EV on the angiogenic potential of ECs.

Invitro germination and optimization of basal media for protocorm-like bodies proliferation in Dieniaophrydis(J. Koenig) Seidenf

Dienia ophrydis is an endangered terrestrial orchid used for its ornamental and therapeutic purposes. This orchid possesses an anti-malarial alkaloid known as Malaxin. There is ongoing exploitation in the wild due to illegitimate herbal collection and climate change. To control the potential risk of extinction, there are no conservational measures or in vitro protocols established in this genus. The study focuses on addressing a germination protocol and enhancement of protocorm-like bodies (PLB) proliferation by optimizing the basal media and vitamins. It also aims to improve the misinterpretation of tetrazolium viability results in orchid testa along with a permeability test. Further, the seeds were subjected to 1% TTC staining to perform embryo viability test and 0.4% trypan blue (TB) dye for testa permeability. Ten basal media such as Gamborg's medium, Mitra medium, Thomale GD medium, Vacin and Went medium, Knudson C medium, Knop's medium, MS medium and ½ MS medium, ¼ MS medium, and modified MS medium (MMS) were tested for seed germination. The Half-strength MS medium responded with 92% of the highest germination and T-GD medium is not suitable for seed germination. The seed viability and permeability result showed 93% of permeability, 72% viability, and 9% dead seeds. Protocorms were transferred to the modified mMS medium containing half-strength macronutrients, full micronutrients, and 10 ppm of thiamine in vitamin formulation. PLB proliferation was observed and 12 shoots emerged from a single protocorm explant. Subsequently, the seedlings were acclimatized with 8-0-24 ratios of nitrogen, phosphorus, and potassium (NPK) as determined by soil testing. The study contributes in experimenting with seed germination of terrestrial orchid seeds asymbiotically and to establishing a protocol for conservation programs, especially in this genus.

Long-term biomass dynamics of temperate forests in Europe after cessation of management

Forests can contribute to climate change mitigation by sequestering carbon when management intensity is reduced. However, there is high uncertainty regarding biomass dynamics in temperate forests after the cessation of management. We used forest inventory data from an extensive network of 224 plots in 37 natural forest reserves (NFR) covering a wide environmental gradient with mean annual temperatures ranging from 1 to 10.4 °C and mean annual precipitation ranging from 901 to 2317 mm. Inventories had been conducted approximately every 10 years during the last 60 years. We used mixed effect models to (i) analyse biomass development, (ii) assess the role of time since the cessation of management (TSCM) and (iii) disentangle the environmental and forest structural drivers of biomass change. After the cessation of management and in the absence of high-severity natural disturbances, biomass accumulated gradually along a saturation curve. There were large differences in biomass among reserves and plots, with values ranging from 101 Mg ha−1 to 851.2 Mg ha−1, with a median of 362.1 Mg ha−1 (SD = 122.5 Mg ha−1). The biomass curve did not yet tend towards an equilibrium, most likely because the majority of the NFRs do not exceed 100 years of TSCM. Compared to higher elevations, forests at lower, warmer sites showed a larger total biomass and higher rates of biomass accumulation. We found a reduction by 148 Mg ha−1 of biomass per 1000 m of elevation gain. The strongest positive rate of change (>8 Mg ha−1 year−1) was found in forests with high basal area (>60 m2 ha−1) and medium to high levels of tree density (1500 to 2000 stems ha−1). Overall, most reserves have not reached a biomass equilibrium yet and continue to act as carbon sinks in tree biomass. This highlights the carbon sequestration capacity of forest reserves and their role as carbon pools.

3D printed hybrid scaffolds do not induce adverse inflammation in mice and direct human BM-MSC chondrogenesis in vitro

Biomaterials that can improve the healing of articular cartilage lesions are needed. To address this unmet need, we developed novel 3D printed silica/poly(tetrahydrofuran)/poly(ε-caprolactone) (SiO2/PTHF/PCL-diCOOH) hybrid scaffolds. Our aim was to carry out essential studies to advance this medical device towards functional validation in pre-clinical trials. First, we show that the chemical composition, microarchitecture and mechanical properties of these scaffolds were not affected by sterilisation with gamma irradiation. To evaluate the systemic and local immunogenic reactivity of the sterilised 3D printed hybrid scaffolds, they were implanted subcutaneously into Balb/c mice. The scaffolds did not trigger a systemic inflammatory response over one week of implantation. The interaction between the host immune system and the implanted scaffold elicited a local physiological reaction with infiltration of mononuclear cells without any signs of a chronic inflammatory response.Then, we investigated how these 3D printed hybrid scaffolds direct chondrogenesis in vitro. Human bone marrow-derived mesenchymal stem/stromal cells (hBM-MSCs) seeded within the 3D printed hybrid scaffolds were cultured under normoxic or hypoxic conditions, with or without chondrogenic supplements. Chondrogenic differentiation assessed by both gene expression and protein production analyses showed that 3D printed hybrid scaffolds support hBM-MSC chondrogenesis. Articular cartilage-specific extracellular matrix deposition within these scaffolds was enhanced under hypoxic conditions (1.7 or 3.7 fold increase in the median of aggrecan production in basal or chondrogenic differentiation media).Our findings show that 3D printed SiO2/PTHF/PCL-diCOOH hybrid scaffolds have the potential to support the regeneration of cartilage tissue.

Bioaugmentation of halophilic consortium for degradation of 1,4 dioxane (1,4-DE) and treatment of cosmetic industrial wastewater in continuous stirred tank reactor under saline condition

The heterocyclic ether 1,4-Dioxane (1,4-DE) is a toxic by-product used widely in various industrial activities as a solvent or stabilizer. 1,4-DE was classified as a probable human carcinogen according to the United States Environmental Protection Agency (USEPA) due to its chemical and physical properties. Emerging pollutants in industrial wastewater is the major challenge for the operation of effluent treatment plant. The bioremediation of 1,4-DE is an effective approach to treat the industrial wastewater. The bacterial consortium was obtained from industrial sludge samples, Yanbu, Saudi Arabia and enriched in 1,4-DE supported basal salt saline medium. Biodegradation of 1,4-DE was studied at several concentrations ranging from 200 to 1200 mg/L. Complete mineralization of 1,4-DE from 200 to 800 mg/L was achieved in 4 to 8 days at 4% salinity. 1,4-DE utilization was studied at different salinity (4% and 6%), the results exhibited complete mineralization of 1,4-DE at 4 days and 6 days respectively. Increase in salinity to 8% displayed 92% degradation of 1,4-DE. Addition of yeast extract accelerated the biomass production correspondingly reduced the time required for biodegradation under saline condition. The bacterial consortium showed the ability to degrade 1,4-DE completely at various range of concentrations. Lab scale continuous stirred tank reactor (CSTR) was used to evaluate the industrial wastewater treatment efficacy of the bacterial consortium under saline condition. CSTR operated for 36 days displayed chemical oxygen demand (COD) removal and 1,4-DE degradation of 96% under saline condition. THF (Tetrahydrofuran) which accelerated the degradation process of 1,4-DE was completely degraded in 28 days. Bacterial analysis with high throughput sequencing technique confirmed the existence of bacterial strains like Ochrobactrum (45%), Marinobacter (21%), Stenotrophomonas (16%), Pseuodomonas (9%), Sphingomonas (4%), Bacillus (3.5%) and Sedimentibacter (1.5%).

Optimization of cold active amylase production by mesophilic Bacillus cereus RGUJS2023 under submerged fermentation

Aim: To produce the highest amount of cold-active alpha-amylase within a short time using mesophilic bacteria with optimized media to save the energy consumption cost and obtain higher enzyme production. Methodology: Amylase producing twenty-three strains were isolated on starch peptone agar plates. Among them, one strain, A5 was selected on the basis of highest clear (12 mm) zone on starch peptone agar plates. It was characterized and identified following Bergey's Manual of Systematic Bacteriology. Enzyme was characterized as Cold alpha amylase. All physico-chemical parameters (temperature, pH, Inoculum size) including carbon, nitrogen, metal ion and amino acid sources were optimized for maximum production of the enzyme. The optimized media was used for enhancing the cold active amylase production. Results: The strain, A5 was identified as Bacillus cereus RGUJS2023 by 16S rRNA sequencing analysis for further experiments. This strain showed the highest activity (9.922± 0.143 U ml-1) on the basal starch peptone media. Though, crude enzyme showed its activity at 4°C to 48°C temperature, but the temperature was 28°C. The highest cold active enzyme was produced (18.87±0.06 U ml-1) at 16 hr of bacterial growth at 35 °C with a pH 6.5 in the optimized media containing 0.5% starch, 0.1% peptone and 0.03% MgSO4.7H2O as a carbon, nitrogen and metal ion sources, respectively, with addition of 0.03% arginine. Interpretation: The cold active alpha amylase could be used commercially for the benefit of pharmaceutical and starch processing industries. Key words: Bacillus cereus RGUJS2023, Cold-active amylase, Starch hydrolysis

Standardization of in-vitro regeneration of Oryza sativa L.

When it comes to increasing rice’s agronomic characteristics at the cellular and molecular level, biotechnological methods offer a more effective and rapid manner than conventional breeding techniques. In the current study, the in-vitro standardization and regeneration of the seeds of Oryza sativa L. was carried out by employing tissue culture techniques. The morphogenetic response of the seed and leaf explants cultivated on media comprising MS basal medium supplemented with a variety of hormones, including BAP, Kinetin, Zeatin, and IAA in different combinations, varied. Explants were taken from in vitro seedlings that were seven days old. The induction of callus and the capacity for leaf and seed explant proliferation were investigated. The organogenesis success rates of the two explants are varied. Zeatin (0.5 mg/l) and IAA (0.1 mg/l) supplements added to MS medium produced 100% callus induction and proliferation. Thus, a rapid and highly effective genetic transformation strategy that can be applied to achieve multiple goals, such as CRISPR/Cas9 genome editing, was developed.

MICRORNAS 125B, 199A-5P, AND 214 AS MODULATORS OF BONE HOMEOSTASIS

Bone homeostasis is a highly regulated process involving pathways in bone as WNT, FGF or BMP, but also requiring support from surrounding tissues as vessels and nerves. In bone diseases, the bone-vessel-nerve triad is impacted. Recently, new players appeared as regulators of bone homeostasis: microRNAs (miRNA). Five miRNAs associated with osteoporotic fractures are already known, among which miR-125b is decreasing bone formation by downregulating human mesenchymal stem cells (hMSCs) differentiation. Other miRNAs, as miR-214 (in cluster with miR-199a), are secreted by osteoclasts to regulate osteoblasts and inhibit bone formation. This forms a very complex regulatory network.hMSCs and osteoblasts (n=3) were transfected with mimic/antagomiR of miR-125b, miR-199a-5p or miR-214, or with a scrambled miRNA (negative control) in osteogenic differentiation calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA expression by qPCR and protein by western blotting.Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. hMSCs transfected with miR-125b mimic in Ca++ presented less mineralization compared to scramble. This correlated with decreased levels of BMPR2 and RUNX2. hMSCs transfected with miR-125b inhibitor presented higher mineralization. Interestingly, hMSCs transfected with miR-214 mimic in Ca++ presented no mineralization while miR-214 inhibitor increased mineralization. No differences were observed in hMSCs transfected with miR-199a-5p modulators. On the contrary, osteoblasts transfected with miR-199a-5p mimic present less mineralization than scrambled-transfected and same was observed for miR-214 and miR-125b mimics.We highlight that miR-125b and miR-214 decrease mineralization of hMSCs in calcium-enriched medium. We noticed that miR-199a-5p is able to regulate mineralization in osteoblasts but not in hMSCs suggesting that this effect is cell-specific. Interestingly, the cluster miR-199a/214 is known as modulator of vascular function and could thus contribute to bone remodeling via different ways. With this work we slightly open the door to possible therapeutic approaches for bone diseases.

Improvements in in vitro spermatogenesis: oxygen concentration, antioxidants, tissue-form design, and space control.

Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMα, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10-15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species.

Induction of Protocorm-like Bodies (PLBs) and Plant Regeneration in Cymbidium chloranthum (Orchidaceae)

Cymbidium chloranthum Lindl. is an indigenous orchid native to Sabah and several tropical countries in Asia. The significant horticultural value of orchids has led to many wild orchid species being threatened with extinction due to overcollection and habitat destruction. In this study, an efficient protocol for the in vitro regeneration and mass propagation of this orchid was developed. Protocorms of C. chloranthum were cultured on Murashige and Skoog (MS) basal medium containing plant growth regulators (PGRs) such as meta-Topolin (mT) and 6-benzylaminopurine (BAP) at concentrations of 0.5 or 1.0 mg/L, and coconut water (CW) at 5 or 10% (v/v). The cultures were incubated at 25±2℃ under 12h of photoperiod. The MS basal medium devoid of any PGRs served as a control. After 90 days of culture, the combination of 0.5 mg/L mT and 0.5 mg/L BAP significantly promoted 2.97±0.53 shoots and 3.83±1.07 roots, respectively. The same treatment also promoted protocorms multiplication with an average production of 2.24±0.55 PLBs. During the acclimatization phase, 66% of the regenerated plants survived. The fully acclimatized plants were reintroduced into their natural habitat in Tenom Orchid Centre. The current approach offers a sustainable way to meet commercial demand while conserving the remarkable species in its natural habitat.

Biosynthesis of Bacitracin in Solid-state Fermentation using Different Substrates

A Bacillus isolate was obtained from soil sample. It was then studied for its cultural, morphological and biochemical characteristics. The isolate was tentatively identified as Bacillus subtilis. For solid-state fermentation of bacitracin, the isolate was grown on basal medium. Fermentation was carried out SSF using three different solid substrates, wheat bran, rice husk and defatted cotton seed oil cake. Bacitracin produced was detected by TLC with standard bacitracin. The product formed in wheat bran showed greater inhibitory activity against test organism i.e. Micrococcus luteus with 20mm diameter of zone of inhibition.

Micropropagação de Nolana intosa como ferramenta para sua domesticação e conservação

Abstract A complete protocol of Nolana intonsa I. M. Johnst, micropropagation was developed. The culture media used were WPM, DKW, and MS as control. DKW medium produced the best results over in vitro development of N. intonsa, generating a significant increase in the multiplication rate, lateral shoots formation, height, and general quality of the plantlets. In the case of fresh weight, dry weight, and water content, no differences were observed between MS and DKW, but a significant decrease in these parameters was produced when the WPM medium was used. Subsequently, when DKW was defined as basal medium, the effect of auxin supplementation was also evaluated. The addition of IBA generated a desirable effect over the in vitro plantlets. The addition of 0.05 mg L-1 of IBA improve the multiplication rate, the shoot's development, plant's height, and reduced in a significant way the damage to the plantlets. Fresh weight, dry weight, and water contents also showed a significant increase when IBA supplementation was added to the DKW medium. The plants where acclimatization of the plantlets was possible, obtaining finished pot plants capable of growth under greenhouse conditions. The development of a micropropagation protocol of N. intonsa could help the conservation and sustainable utilization of this endemic Chilean species.

Effects of Putrescine and Spermine on Pollen Germination Levels of Some Olive Cultivars

In this study, it is aimed to determine the effects of putrescine and spermine on pollen germination levels of 8 olive cultivars. Arbequina, Gemlik, Halhalı, Karamani, Kozan yerli, Saurani, Sayfi and Şami olive cultivars were used as material. In the study, 5 spermine doses (0,25mM, 0,125 mM, 0,025 mM, 0,05 mM and 0,005 mM) and 5 putrescine doses (0,20 mM, 0,10 mM, 0,05 mM, 0,025 mM and 0,1 mM) added to basal germination medium (15% sucrose + 1% agar + 100 ppm Boric acid) were tested for determining the effects on pollen germination of olive cultivars. Basal media was used for Control treatment. In terms of pollen germination media, pollen germination levels were increased with lower putrescine and higher spermine levels. In Sayfi olive cultivar, while pollen viability rate was 42,74%, pollen germination was 0,00% in control treatments. However, pollen germination was increased for Sayfi olive cultivar with 0,20 mM and 0,10 mM putrescine (3,64% and 3,40%, respectively) and 0,25 mM, 0,125 mM and 0,05 mM Spermine (12,56%, 4,73% and 3,40%, respectively). In conclusion, polyamine treatments were increased the low germinated cultivars. So, polyamine treatments reveal the potential of the pollen especially in low-germinated pollens.

Transplantation of neural stem cells improves recovery of stroke-affected mice and induces cell-specific changes in GSDMD and MLKL expression.

Stroke, the second leading cause of death and disability in Europe, is primarily caused by interrupted blood supply, leading to ischemia-reperfusion (IR) injury and subsequent neuronal death. Current treatment options are limited, highlighting the need for novel therapies. Neural stem cells (NSCs) have shown promise in treating various neurological disorders, including stroke. However, the underlying mechanisms of NSC-mediated recovery remain unclear. Eighty C57Bl/6-Tyrc-Brd mice underwent ischemic stroke induction and were divided into four groups: sham, stroke-affected, stroke-affected with basal cell medium injection, and stroke-affected with NSCs transplantation. NSCs, isolated from mouse embryos, were stereotaxically transplanted into the stroke-affected brains. Magnetic resonance imaging (MRI) and neurological scoring were used to assess recovery. Immunohistochemical analysis and gene expression assays were performed to evaluate pyroptosis and necroptosis markers. NSC transplantation significantly improved neurological recovery compared to control groups. In addition, although not statistically significant, NSCs reduced stroke volume. Immunohistochemical analysis revealed upregulation of Gasdermin D (GSDMD) expression post-stroke, predominantly in microglia and astrocytes. However, NSC transplantation led to a reduction in GSDMD signal intensity in astrocytes, suggesting an effect of NSCs on GSDMD activity. Furthermore, NSCs downregulated Mixed Lineage Kinase Domain-Like Protein (Mlkl) expression, indicating a reduction in necroptosis. Immunohistochemistry demonstrated decreased phosphorylated MLKL (pMLKL) signal intensity in neurons while stayed the same in astrocytes following NSC transplantation, along with increased distribution in microglia. NSC transplantation holds therapeutic potential in stroke recovery by targeting pyroptosis and necroptosis pathways. These findings shed light on the mechanisms underlying NSC-mediated neuroprotection and support their further exploration as a promising therapy for stroke patients.

Optimization of Tissue Culture Medium for Little-leaf Mockorange (Philadelphus microphyllus A. Gray) by Adjusting Cytokinin and Selected Mineral Components

Little-leaf mockorange is a native plant species with desirable characteristics for landscape use. The need to conserve specific genotypes and the difficulty of seed propagation and stem cutting propagation make axillary shoot micropropagation a good option for this species. A series of experiments were completed individually with the goal to improve in vitro propagation protocols by evaluating different types of cytokinins [benzylaminopurine (BA), kinetin (Kin), zeatin (Zea), meta-topolin (MT), and thidiazuron (TDZ)] at 0, 1.1, 2.2, 4.4, or 8.8 µM. Selected minerals (0 to 60 mM or 0 to 45 mM N, 0 to 500 µM or 0 to 100 µM Fe, 0 to 3 mM Ca, 0 to 1.5 mM Mg, or 0 to 1.25 mM P) were also tested separately in the tissue culture medium; the base medium was ½ strength MS in these mineral experiments. At the end of each experiment (8 or 12 weeks), plant growth characteristics including number of axillary shoots, shoot height, and dry weight were determined. Of the six cytokinins tested, Zea produced the largest increase in shoot growth. Supplementation with 1.1 µM Zea resulted in the most shoot dry weight, almost 2.5-fold more than control shoots. Shoots on 0.55, 1.1, or 2.2 µM Zea were at least 64% taller than control shoots. Shoots placed on regular ½ strength MS basal salts, described above, and/or media lacking the nutrient of interest, were used as a positive and negative control treatments. For each separate mineral tested, the best concentration for optimum shoot growth was the concentration of that mineral used in ½ strength MS medium. A medium containing mineral concentrations of 30 mM N, 50 µM Fe, 1.5 mM Ca, 0.75 mM Mg, and 0.625 mM P, and 1.1 µM Zea should be used to produce the optimum in vitro shoot growth of little-leaf mockorange.

Acetic Acid Production from Aspergillus terreus Isolated from Some Agricultural Soils Collected from Selected Locations within the North Gondar Zone, Amhara Region, Ethiopia.

Acetic acid, a substance with numerous uses as a bulk chemical, may be produced novelty by Aspergillus terreus. With the newfound understanding of Aspergillus species catabolism of glucose, fermentation techniques for the generation of secondary metabolites like acetic acid in the Ethiopian north Gondar zone can be developed with glucose feeding and pH feedback management. Previous works done on extracting organic acids including acetic acid from filamentous fungi in Ethiopia and at the global level are scanty. Therefore, this study aimed to produce acetic acid from A. terreus isolated from agricultural soils in the north Gondar zone of Ethiopia using barely straw as a substrate. In the current study, Aspergillus isolates were obtained in the samples taken from three different locations. The isolates were screened for acetic acid production. The optimum temperature and pH for the maximum production of acetic acid by the selected isolate were also undertaken. The potential isolates were further cultured using barley straw as a local substrate. Preliminary identification of the selected isolates was based on morphological methods. Molecular characterization (amplification and sequencing of the two intergenic spacers, ITS1 and ITS2, and the intervening 5.8S gene of the ribosomal RNA) was carried out to confirm the identity of the Aspergillus isolates. When the isolates were screened for the production of acetic acid, an isolate from low land (isolate LL2) had the highest yield (72.5 ± 1.65 g/l) on basal screening media. The optimum temperature and pH for the maximum production of acetic acid by this isolate were 30°C and pH 5.0. A sequence similarity of 98.5% to A. terreus isolate LL2 (KIA) was obtained by comparing the Aspergillus isolate to a reference sequence in the GenBank using the BLAST algorithm. It can be concluded from this study that A. terreus isolated from agricultural soil in the north Gondar zone of Ethiopia could produce more acetic acid using barely straw as a substrate.

Efficient plant regeneration through direct shoot organogenesis and two-step rooting in Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver (E. ulmoides Oliver), a multipurpose woody plant, holds great economic significance due to its expansive medicinal, food and industrial applications. The rapid advancement of E. ulmoides in various fields has resulted in the inadequacy of existing breeding methods to meet its growth and annual production demands. Consequently, there is an urgent need for innovative propagation strategies. This study introduces an optimized micropropagation protocol for E. ulmoides, facilitating direct shoot organogenesis from nodal segments with axillary buds. We systematically examined the impact of basal medium composition, plant growth regulators, photosynthetic photon flux density, and sucrose concentration on bud sprouting. Employing cuttings with axillary buds as propagation material, we achieved a shortened cultivation period of merely 4 weeks for bud elongation and proliferation, marking a substantial enhancement in propagation efficiency. Notably, the Driver Kuniyuki Walnut medium, supplemented with 20.0 g L-1 sucrose and 2.0 mg L-1 trans-zeatin, induced shoots sprouting with a 100% success rate and an average length of 5.18 cm per nodal segment, equating to a great bud propagation rate of approximately 500%. Furthermore, a light source with an intensity of 80 μmol m-2 s-1 was shown the most economical choice. To address the primary challenge of inducing roots in regenerated plants, we employed a refined two-step rooting technique. This method yielded the optimal rooting frequency of 93.02%, producing an average of 5.90 adventitious roots per plantlet, each with an average length of 2.77 cm. The micropropagation program developed in this work will be the cornerstone for the preservation of the germplasm of E. ulmoides and its long-term use in medicinal and industrial applications.

Land Treatment Polystyrene Bioremediation by Pseudomonas aeruginosa Bacteria

Products made from plastic material are commonly found in our life. Polystyrene is a type of plastic synthesises. In the nature, polystyrene is known to be recalcitrant due to its complex structural molecule. Number of studies reported that the existence of polystyrene plastic presents various threats for sea animals such as zebra fish, sea urchin’s early development gene, and lowered crustaceans’ life expectancy. This literature study was conducted by collecting various information on polystyrene pollution in the environment and the ability of Pseudomonas aeruginosa bacteria to degrade polystyrene. Pseudomonas aeruginosa (1 x 108 cell/ml) bacteria was cultured in 25 ml liquid carbon-free basal medium and 1 gram polystyrene on 25oC temperature for 15 days. The result shows that the compound was able to degrade 0.098% of polystyrene per day with yield constant of 1,111,111.11. This literature study will be continued on pilot scale by conducting experiment of polystyrene biodegradation on an area that is previously added with liquid carbon-free basal medium by land treatment method with dimensional size of 3m x 1.5m x 0.3m with additional 1.35 m3 LFCBM (liquid carbon-free basal medium).

Direct Shoot From Root and True-to-Type Micropropagation of Limonium "Misty Blue" in Partially Immersed Culture on an Aluminum Mesh Raft.

Commercial plant tissue culture now primarily serves the ornamental horticulture industry. The main pillars of the commercial tissue culture business are scalability of production, cost reduction, limited labor involvement, high quality, and genetic homogeneity of propagated plants. Based on these requirements, the current protocol employs a partially immersed liquid culture medium supported by a flexible aluminum mesh raft with a wire stand to facilitate shoot organogenesis from the horizontally placed root explants and hold the plants upright for shoot multiplication and rooting of Limonium Misty Blue. It is a florist crop that is in high demand as both dried and fresh flower fillers in various floral decorations. The majority of cultivated Limonium or statice cultivars are heterozygous in nature and propagate commercially through in vitro propagation to cater to the huge demand for planting materials needed for flower production. This is the first protocol to describe direct shoot organogenesis from the roots in a liquid half-component of Murashige and Skoog's (1962) (MS) basal medium supplemented with 1.6μM NAA and 1.1μM BA. The regenerated shoots are multiplied and rooted at the same time on the raft in a MS-based liquid culture medium that included 0.44μM BA and 1.07μM NAA. In comparison to agar-gelled medium, plants cultured in liquid medium grow more quickly without any signs of hyperhydricity. In liquid medium, a clump of 4-5 shoots is formed from a single shoot explant within 4weeks and are rooted simultaneously within 6weeks. On average, seven explants may fit on each raft, so on average, 25 healthy plants are produced from a single bottle. The regenerated plants are easily hardened in the greenhouse, and using ISSR-based molecular markers, the genetic homogeneity of the randomly selected hardened plants can be determined.