scholarly journals Effect of Cytokinins on Micropropagation of Moringa Oleifera Lam. and Genomic Stability Assessment Using Flow-Cytometry

2022 ◽  
Author(s):  
Rohit Bharati ◽  
Moses Okao ◽  
Katerina Hamouzová ◽  
Eloy Fernandez-Cusimamani
Keyword(s):  
Flow Cytometry ◽  
Moringa Oleifera ◽  
Genetic Fidelity ◽  
Limited Range ◽  
Surface Sterilization ◽  
Wide Range ◽  
The Family ◽  
Livestock Feeding ◽  
Planting Materials

Abstract Moringa oleifera Lam. is a multipurpose medicinal plant of the family Moringaceae which has been widely utilized as a pharmaceutical remedy to treat a wide range of diseases. In addition, the tree has several applications in human nutrition as well as livestock feeding. M. oleifera is easily multiplied through epigeal germination (recalcitrant) but seed propagated plants are heterogeneous and take longer to reach fruit-bearing age. As an alternative, branch cuttings have been used but their establishment is erratic and often leads to reduced growth of the mother plant. Thus, to produce superior planting materials, in-vitro propagation has become paramount. As a result, several studies using a limited range of cytokinin have been undertaken to multiply M. oleifera through tissue culture. Otherwise, a study was conducted to examine the effect of five different cytokinins on in-vitro regeneration of this tree species. Results showed that nodal explants cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 6-Benzylaminopurine (BAP) and subsequently rooted on basic MS media was the most optimal treatment. Furthermore, acclimatization of plantlets in sterile soil substrate and perlite (1:3;v/v) under transparent polythene sheet for 7 days resulted in survival rate of 100%. Assessment of genetic fidelity using flow cytometry revealed that surface sterilization alongside cytokinin treatments produced plantlets that were genetically stable regardless of the growth regulator used. Thus, the in-vitro protocol developed in this study can be utilized for in-vitro studies and mass propagation of this imperative plant species.

scholarly journals In Vitro Cellular Uptake Studies of Self-Assembled Fluorinated Nanoparticles Labelled with Antibodies

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1906
Author(s):  
Mona Atabakhshi-Kashi ◽  
Mónica Carril ◽  
Hossein Mahdavi ◽  
Wolfgang J. Parak ◽  
Carolina Carrillo-Carrion ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cellular Uptake ◽  
Hydrophobic Interactions ◽  
Intrinsic Properties ◽  
Wide Range ◽  
Self Assembled ◽  
Uptake Studies ◽  
Targeting Ability ◽  
Fluorinated Nanoparticles

Nanoparticles (NPs) functionalized with antibodies (Abs) on their surface are used in a wide range of bioapplications. Whereas the attachment of antibodies to single NPs to trigger the internalization in cells via receptor-mediated endocytosis has been widely studied, the conjugation of antibodies to larger NP assemblies has been much less explored. Taking into account that NP assemblies may be advantageous for some specific applications, the possibility of incorporating targeting ligands is quite important. Herein, we performed the effective conjugation of antibodies onto a fluorescent NP assembly, which consisted of fluorinated Quantum Dots (QD) self-assembled through fluorine–fluorine hydrophobic interactions. Cellular uptake studies by confocal microscopy and flow cytometry revealed that the NP assembly underwent the same uptake procedure as individual NPs; that is, the antibodies retained their targeting ability once attached to the nanoassembly, and the NP assembly preserved its intrinsic properties (i.e., fluorescence in the case of QD nanoassembly).

scholarly journals ASSESSMENT OF GENETIC STABILITY OF MICROPROPAGATED Eucalyptus globulus Labill HYBRID CLONES BY MEANS OF FLOW CYTOMETRY AND MICROSATELLITES MARKERS

2017 ◽  
Vol 41 (1) ◽  
Author(s):  
Leandro Silva Oliveira ◽  
Aloisio Xavier ◽  
Wagner Campos Otoni ◽  
José Marcello Salabert Campos ◽  
Lyderson Facio Viccini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Microsatellite Markers ◽  
Genetic Stability ◽  
Culture Medium ◽  
Eucalyptus Grandis ◽  
Genetic Fidelity ◽  
Shoot Multiplication ◽  
Micropropagated Plants ◽  
Hybrid Clones

ABSTRACT Flow cytometry and microsatellite markers were used to determine a genetic fidelity of micropropagated plants from the two Eucalyptus urophylla x E. globulus clones and a Eucalyptus grandis x E. globulus clone derived from adult material. Clones were repeatedly subcultured for 25 subcultures on MS medium supplemented with BA (2.22 µM) and ANA (0.05 µM) for in vitro shoot multiplication. The elongation was performed in MS culture medium supplemented with AIB (2.46 µM) and BA(0.22 µM). The ex vitro rooting and acclimatization phases were lead at the same time. The micropropagated clones showed genetic stability by flow cytometry and microsatellite markers. The results proved that micropropagation, for purposes of rejuvenation, can be a viable technique to generate genetically stable or identical E. globulus hybrid clones.

scholarly journals Effect of Isolation Conditions on Diversity of Endolichenic Fungal Communities from a Foliose Lichen, Parmotrema tinctorum

2021 ◽  
Vol 7 (5) ◽  
pp. 335
Author(s):  
Ji Ho Yang ◽  
Seung-Yoon Oh ◽  
Wonyong Kim ◽  
Jung-Jae Woo ◽  
Hyeonjae Kim ◽  
...  
Keyword(s):  
Biological Activities ◽  
Basal Medium ◽  
Limited Range ◽  
Isolation Method ◽  
Surface Sterilization ◽  
Wide Range ◽  
Lichen Thallus ◽  
Endolichenic Fungi ◽  
Parmotrema Tinctorum ◽  
Foliose Lichen

Endolichenic fungi (ELF) are emerging novel bioresources because their diverse secondary metabolites have a wide range of biological activities. Metagenomic analysis of lichen thalli demonstrated that the conventional isolation method of ELF covers a very limited range of ELF, and the development of an advanced isolation method is needed. The influence of four variables were investigated in this study to determine the suitable conditions for the isolation of more diverse ELF from a radially growing foliose lichen, Parmotrema tinctorum. Four variables were tested: age of the thallus, severity of surface-sterilization of the thallus, size of a thallus fragment for the inoculation, and nutrient requirement. In total, 104 species (1885 strains) of ELF were isolated from the five individual thalli of P. tinctorum collected at five different places. Most of the ELF isolates belong to Sordariomycetes. Because each part of lichen thallus (of different age) has unique ELF species, the whole thallus of the foliose lichen is needed to isolate diverse ELF. Moderate sterilization is appropriate for the isolation of diverse ELF. Inoculation of small fragment (1 mm2) of lichen thallus resulted in the isolation of highest diversity of ELF species compared to larger fragments (100 and 25 mm2). Moreover, ELF species isolated from the small thallus fragments covered all ELF taxa detected from the medium and the large fragments in this study. The use of two media—Bold’s basal medium (nutrient poor) and potato dextrose agar (nutrient rich)—supported the isolation of diverse ELF. Among the tested variables, size of thallus fragment more significantly influenced the isolation of diverse ELF than other three factors. Species composition and richness of ELF communities from different lichen thalli differed from each other in this study.

scholarly journals Characterization of a Novel Bacteriophage Henu2 and Evaluation of the Synergistic Antibacterial Activity of Phage-Antibiotics

Antibiotics ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 174
Author(s):  
Xianghui Li ◽  
Tongxin Hu ◽  
Jiacun Wei ◽  
Yuhua He ◽  
Abualgasim Elgaili Abdalla ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Burst Size ◽  
Open Reading Frames ◽  
Comparative Genomic ◽  
Sewage Sample ◽  
Bacterial Killing ◽  
Wide Range ◽  
The Family ◽  
Infected Cells

Staphylococcus aureus phage Henu2 was isolated from a sewage sample collected in Kaifeng, China, in 2017. In this study, Henu2, a linear double-stranded DNA virus, was sequenced and found to be 43,513 bp long with 35% G + C content and 63 putative open reading frames (ORFs). Phage Henu2 belongs to the family Siphoviridae and possesses an isometric head (63 nm in diameter). The latent time and burst size of Henu2 were approximately 20 min and 7.8 plaque forming unit (PFU)/infected cells. The Henu2 maintained infectivity over a wide range of temperature (10–60 °C) and pH values (4–12). Phylogenetic and comparative genomic analyses indicate that Staphylococcus aureus phage Henu2 should be a new member of the family of Siphoviridae class-II. In this paper, Phage Henu2 alone exhibited weak inhibitory activity on the growth of S. aureus. However, the combination of phage Henu2 and some antibiotics or oxides could effectively inhibit the growth of S. aureus, with a decrease of more than three logs within 24 h in vitro. These results provide useful information that phage Henu2 can be combined with antibiotics to increase the production of phage Henu2 and thus enhance the efficacy of bacterial killing.

A Non Mercuric Shoots Bud Sterilization Technique for Boesenbergia rotunda (L.) Mansf. Kulturpfl.

2013 ◽  
Vol 64 (2) ◽  
Author(s):  
Siti Ailla Md Afendi ◽  
Chew-Tin Lee ◽  
Marjan Najafi Disfani ◽  
Muhammad Arshad Javed
Keyword(s):  
Mercuric Chloride ◽  
Plant Growth Regulator ◽  
Soft Rot ◽  
Contamination Level ◽  
Surface Sterilization ◽  
Shoot Bud ◽  
Boesenbergia Rotunda ◽  
Planting Materials ◽  
Contamination Problem

Boesenbergia rotunda, a medicinal herb under the Zingiberaceae family, has been proven to be the most prominent anticancer remedies. The conventional breeding of this plant is inapplicable as it is susceptible to rhizome soft rot and leaf spot diseases. The yellow rhizome also produces limited buds. Therefore it is necessary to propagate this plant through in vitro propagation to obtain abundant uniform planting materials. Unfortunately, high cost is incurred due to the high shoot bud explants contamination level. Hence, it is addressed in the present study to solve the contamination problem. Mercuric Chloride is well known to solve this problem, but it is not advisable to use, because of its poisoning and other hazardous effects. Moreover, explant sterilization technique should also accelerate the shoot response. To find an alternative, 6 different surface sterilization methods (SSM) were designed and evaluated on the explants where different combinations of sodium hypochlorite and ethanol (instead of mercuric chloride) were applied. The sterilized shoot bud explants were then cultured on Murashige & Skoog (MS) media with no additional vitamins or plant growth regulator under the light below 25oC. The contamination was recorded for 3 consecutive weeks along with visible shoot responses. SSM 5 showed minimum contamination and maximum visible shoot response, compared to other SSMs. Therefore, it is suggested that SSM5 could be used to conduct surface sterilization to avoid contamination problem.

scholarly journals Failure of in vitro differentiation of Plasmodium falciparum gametocytes into ookinetes arises because of poor gamete fertilisation

2017 ◽  
Author(s):  
Michael J Delves ◽  
Sara R Marques ◽  
Andrea Ruecker ◽  
Ursula Straschil ◽  
Celia Miguel-Blanco ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Culture Conditions ◽  
Transmission Cycle ◽  
Potential Barriers ◽  
Wide Range ◽  
Species Specific ◽  
Transmission Biology ◽  
Avian Malaria Parasites

ABSTRACTA critical step towards malaria elimination will be the interruption of Plasmodium transmission from the human host to the mosquito. At the core of the transmission cycle lies Plasmodium sexual reproduction leading to zygote formation and mosquito midgut colonisation by ookinetes. Whilst in vitro ookinete culture from the murine and avian malaria parasites, Plasmodium berghei and P. gallinaceum, has greatly increased our knowledge of transmission biology; efforts to mimic the process in the human parasite P. falciparum have, to date, had only limited success. Using fluorescence microscopy and flow cytometry with antibodies specific to the male gametocyte and developing ookinetes, we sought to evaluate P. falciparum ookinete production using previously published in vitro protocols. We then compared in vitro versus in vivo ookinete production in both P. falciparum and P. berghei parasites, exploring potential barriers to complete development. Finally, we sought to test a wide range of literature-led culture conditions towards further optimisation of in vitro P. falciparum ookinete production. Despite extensive testing, our efforts to replicate published methods did not produce appreciable quantities of fully formed P. falciparum ookinetes in vitro. In parallel, however, gametocyte cultures that failed to differentiate fully in vitro successfully developed into ookinetes in vivo with an efficiency approximating that of P. berghei. Flow cytometry analysis showed that this disparity likely lies with the poor fertilization of P. falciparum gametes in vitro. Attempts to improve gametocyte fertility or define conditions more permissive to fertilisation/ookinete survival in vitro were also unsuccessful. Current in vitro conditions for P. falciparum ookinete production are not optimal for gamete fertilisation either due to the lack of parasite-species-specific mosquito factors missing from in vitro culture, or non-permissive cues contaminating culture preparations.

scholarly journals Isolation, cultivation, and in vitro susceptibility testing of Borrelia burgdorferi sensu lato: A review

2013 ◽  
Vol 65 (2) ◽  
pp. 533-547 ◽  
Author(s):  
Gorana Veinovic ◽  
Brankica Filipic ◽  
Jelena Stankovic
Keyword(s):  
Borrelia Burgdorferi ◽  
Lyme Borreliosis ◽  
Antimicrobial Agents ◽  
Borrelia Burgdorferi Sensu Lato ◽  
In Vitro Susceptibility ◽  
Wide Range ◽  
The Family ◽  
In Vitro Susceptibility Testing ◽  
Vector Borne

Lyme borreliosis is the most common vector-borne disease in the northern hemisphere. The agents of Lyme borreliosis are borrelia, bacteria of the family Spirochaetaceae, which are grouped in Borrelia burgdorferi sensu lato species complex. Borreliae are fastidious, slow-growing and biochemically inactive bacteria that need special attention and optimal conditions for cultivation. The isolation of Borrelia from clinical material and their cultivation is a time-consuming and demanding procedure. Cultivation lasts from 9 up to 12 weeks, which is much longer than is necessary to grow most other human bacterial pathogens. Although B. burgdorferi sensu lato is susceptible to a wide range of antimicrobial agents in vitro, up to now the susceptibility of individual Borrelia species to antibiotics is defined only partially.

scholarly journals Micropropagation of Feverfew (Tanacetum parthenium) and Quantification of Parthenolide Content in Its Micropropagated and Conventionally Grown Plants

Horticulturae ◽  
2022 ◽  
Vol 8 (1) ◽  
pp. 50
Author(s):  
Huda E. Mahood ◽  
Majeed Kadhem Abbas ◽  
Nisar Ahmad Zahid
Keyword(s):  
Callus Induction ◽  
High Performance ◽  
Peat Moss ◽  
Ms Medium ◽  
Surface Sterilization ◽  
Culture Techniques ◽  
Aseptic Culture ◽  
Tanacetum Parthenium ◽  
Planting Materials

Feverfew (Tanacetum parthenium) is a well-known multi-functional plant with anti-inflammatory, cardiotonic, antiangiogenic, and anticancer effects. The therapeutic value of this plant is due to its phytochemical constitutes, especially parthenolide. Tissue culture techniques have been applied to improve the bioactive components of many herbal plants. Hence, this study, was carried out to establish a protocol for micropropagation of the feverfew plant and to quantify parthenolide content in its micropropagated and conventionally grown plants. To establish an aseptic culture, different concentrations of sodium hypochlorite (NaOCl) were investigated for seed surface sterilization. Besides, the effects of plant growth regulators (PGRs) on the callus induction, shoot organogenesis from callus and in vitro rooting were evaluated. Additionally, the parthenolide yield of the micropropagated and conventionally grown plants was determined by using high-performance liquid chromatography (HPLC). The results showed that surface sterilization of feverfew seeds with 6% NaOCl for 15 min obtained 65.00 ± 2.69% aseptic seeds. Murashige and Skoog (MS) medium supplemented with 0.4 mg/L thidiazuron (TDZ) and 2 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) resulted in 86.00 ± 1.72% callus induction. The highest number of shoots (5.00 ± 0.15) per explant was obtained in the treatment of MS medium supplemented with 5 mg/L zeatin. MS medium fortified with 3 mg/L indole-3-butyric acid (IBA) produced the maximum number of roots per plantlet (8.90 ± 0.35). A total of 90% of the micropropagated plantlets survived when planted in perlite + peat moss (1:1 v/v); the micropropagated plantlets were successfully established in the ex vitro conditions. According to parthenolide analysis, its level was significantly higher in the micropropagated plants than conventionally grown plants. Among different solvents, ethanolic extraction obtained the highest parthenolide content of the feverfew plant. Hence, it can be concluded that micropropagation of feverfew could be applied to produce disease-free planting materials and to improve the parthenolide content of the feverfew plant.

Tetrahymena dimorpha sp.nov. (Hymenostomatida: Tetrahymenidae), a new ciliate parasite of Simuliidae (Diptera) with potential as a model for the study of ciliate morphogenesis

1983 ◽  
Vol 301 (1106) ◽  
pp. 345-363 ◽  
Keyword(s):  
Limited Range ◽  
Culture Conditions ◽  
Morphological Transformation ◽  
Sterile Culture ◽  
Proteose Peptone ◽  
Wide Range ◽  
Yeast Extract Medium ◽  
Insect Tissue Culture ◽  
Heavy Burden

A new species of hymenostome ciliate, Tetrahymena dimorpha sp.nov., is described. This ciliate occurs as a parasite in the haemocoel of larval, pupal and adult Simuliidae (Diptera). In larval hosts the total number of parasites never exceeds about 240 and the infection is benign. Within larval hosts the ciliates are large and broadly oval and possess an unusually wide range of somatic kineties, from 30 to 66; moreover a variable proportion of these kineties are characteristically disorganized, being incomplete, meandering or branched. Metamorphosis of the host to the adult fly is accompanied by a dramatic increase in the number of ciliates, which reach pathogenic intensity. Adult hosts may contain up to 19000 ciliates and the flies soon die from this heavy burden. Associated with ciliate population growth during host metamorphosis is a startling morphological transformation of the ciliates themselves. In adult hosts the ciliates are smaller and pyriform in shape and the cortex is greatly modified; the total number of somatic kineties is considerably reduced and has a limited range of 19—22. Most significantly, the kineties are ordered with typical tetrahymenine precision. By application of appropriate culture conditions to ciliates isolated from any host stage, either of the distinctive morphological forms of T. dimorpha may be selectively induced in vitro . In bacterized infusions, ciliates are produced that have the general form and cortical characteristics of those found naturally in adult hosts. Sterile culture in serum-supplemented Mitsuhashi and Maramorosch insect tissue culture medium produces a population showing features characteristic of ciliates from larval hosts. Sterile culture in proteose-peptone-yeast-extract medium results in populations exhibiting concurrent dimorphism, even after cloning. The extreme nature and multiple facets of the dimorphism together with the ease of its manipulation in vitro afford opportunities for the experimental investigation of many problems, particularly those related to cell surface patterning in ciliates, and these possibilities are discussed in relation to current concepts of ciliate morphogenesis.

scholarly journals Insight Into the Anti-staphylococcal Activity of JBC 1847 at Sub-Inhibitory Concentration

2022 ◽  
Vol 12 ◽  
Author(s):  
Troels Ronco ◽  
Line H. Kappel ◽  
Maria F. Aragao ◽  
Niccolo Biagi ◽  
Søren Svenningsen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Membrane ◽  
Single Gene ◽  
Antimicrobial Properties ◽  
Low Frequency ◽  
Stationary Growth Phase ◽  
Synthesis Rate ◽  
Compensatory Mechanisms ◽  
Wide Range

Multidrug-resistant pathogens constitute a serious global issue and, therefore, novel antimicrobials with new modes of action are urgently needed. Here, we investigated the effect of a phenothiazine derivative (JBC 1847) with high antimicrobial activity on Staphylococcus aureus, using a wide range of in vitro assays, flow cytometry, and RNA transcriptomics. The flow cytometry results showed that JBC 1847 rapidly caused depolarization of the cell membrane, while the macromolecule synthesis inhibition assay showed that the synthesis rates of DNA, RNA, cell wall, and proteins, respectively, were strongly decreased. Transcriptome analysis of S. aureus exposed to sub-inhibitory concentrations of JBC 1847 identified a total of 78 downregulated genes, whereas not a single gene was found to be significantly upregulated. Most importantly, there was downregulation of genes involved in adenosintrifosfat (ATP)-dependent pathways, including histidine biosynthesis, which is likely to correlate with the observed lower level of intracellular ATP in JBC 1847–treated cells. Furthermore, we showed that JBC 1847 is bactericidal against both exponentially growing cells and cells in a stationary growth phase. In conclusion, our results showed that the antimicrobial properties of JBC 1847 were primarily caused by depolarization of the cell membrane resulting in dissipation of the proton motive force (PMF), whereby many essential bacterial processes are affected. JBC 1847 resulted in lowered intracellular levels of ATP followed by decreased macromolecule synthesis rate and downregulation of genes essential for the amino acid metabolism in S. aureus. Bacterial compensatory mechanisms for this proposed multi-target activity of JBC 1847 seem to be limited based on the observed very low frequency of resistance toward the compound.

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