MOLECULAR-GENETIC ANALYSIS OF PROPAGATED IN VITRO CLONES OF POPULUS ALBA L. AND POPULUS TREMULA L. USING MICROSATELLITE MARKERS

Use of planting material of forest trees with improved hereditary characteristics is one of the ways to increase the productivity and biological stability of forest stands. It requires taking measures to develop and improve selection base using modern approaches and methods of genetics and biotechnology. A molecular genetics assessment of clone plants of aspen (Populus tremula L.) and white poplar (Populus alba L.) from a long-term in vitro collection (up to 24 years), planted in a greenhouse and field conditions (nursery), was carried out. SSR loci of the PTR series (PTR5, PTR7, PTR8, PTR12, PTR14) were used as DNA markers. Evaluation of clones' ploidy was carried out on the basis of the diagnosis of "loss of heterozygosity" (LOH) effect. Analysis of 5 microsatellite loci of the specimens showed their high intraclonal genotypic stability and homogeneity in vitro and ex vitro. For the first time, data on the results of a comparative determination of ploidy using karyological and microsatellite analysis were presented. Based on the results of the SSR analysis, it can be concluded that the structure of molecular markers is stable among the samples of one clone that are in long-term cultivation. The ratio of the representation (dose) of electrophoretic variants of PCR products serves as an indirect sign of determining ploidy, but for its reliable assessment it is necessary to study the number of loci that are three times larger than the main set of chromosomes. The specimen also requires information on the amplification coefficient of the markers under study. Thus, it is necessary to use both chromosomal and microsatellite analyzes for reliable assessment of intraclonal homogeneity of various specimens, the development of understanding of clone genotypes formation and determination of their ploidy

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Extending and maintaining the in vitro collection of (inter)national hop varieties in Ukraine

Agricultural science and practice ◽

10.15407/agrisp7.03.061 ◽

2020 ◽

Vol 7 (3) ◽

pp. 61-71

Author(s):

V. B. Kovalev ◽

T. I. Kozlik ◽

L. V. Protsenko ◽

A. V. Bober ◽

B. F. Kormiltsev

Keyword(s):

Culture Medium ◽

Molecular Genetic ◽

Molecular Genetic Analysis ◽

Medium Composition ◽

Biologically Active ◽

Planting Material ◽

Genetic Pool

Despite the decline in the national hop production, a part of hop products, made of Ukrainian aroma hop, is highly estimated in the international market and exported. Therefore, a relevant prerequisite of ensuring a suffi cient level of competitiveness of domestic hop production is the expansion of its assortment. To satisfy this need the breeders face the task of creating varieties with unique characteristics, which requires searching for new forms and strains of hop with increased content of different biologically active compounds in cones. In this regard national research program aimed to investigate genetic stab ility of hop varieties during multiple planting and storing of explants in in vitro conditions while introducing them into the collection, adapting the composition of Murashige and Skoog culture medium for specifi c varieties is discussed. Studies conducted included the analysis of plants by morphological and variety-specifi c traits, the identifi cation of varieties by biochemical criteria, improvement of hop regenerants using ELISA, molecular-genetic analysis based on PCR (polymerase chain reaction) for identifi cation of hop genotypes and determination of genetic stability, and the improvement of method of microclonal reproduction of hop. As a result of the perennial research of cultivating hop planting material using the Murashige and Skoog complex of nutrients, the foundations of creating and maintaining the in vitro collection of hop varieties were fi rst elaborated in Ukraine which allow for the possibility of decreasing the concentration of nutrients in the culture medium by 50 %, replacing expensive gel-forming preparation for the maintenance of plants in the culture medium - agar-agar, the share of whose cost in the medium composition is up to 70 %, with a cheaper substance - modifi ed starch DDKamod or agroperlite, and reducing the expenses for the maintenance of genetic pool in the in vitro collection. It was determined that the spectra of microsatellite loci of the amplifi ed DNA of the explants, cultivated in vitro, during the study period did not differ from the spectra of plant DNA prior to cloning, which demonstrated DNA stability and allowed cultivating hop varieties in the in vitro culture without any changes in the genome. The composition of media for cultivation and long- term storing of hop varieties in in vitro conditions was selected.

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Determination of two fluoroquinolones and their combinations with hyaluronan effect in in vitro canine cartilage explants

PeerJ ◽

10.7717/peerj.6553 ◽

2019 ◽

Vol 7 ◽

pp. e6553

Author(s):

Puntita Siengdee ◽

Waranee Pradit ◽

Siriwadee Chomdej ◽

Korakot Nganvongpanit

Keyword(s):

Adverse Effects ◽

Interleukin 1 ◽

Expression Patterns ◽

Cartilage Explants ◽

Beneficial Effects ◽

Regulated Expression ◽

Safranin O

Background Previous studies reported the effect of enrofloxacin (Enro) and marbofloxacin (Mar) on cell death and alteration of the key genes involved in catabolic and anabolic processes and demonstrated the beneficial effects of hyaluronan (HA) combined with fluoroquinolones (FQs) on primary canine chondrocytes. This study further determines the effects of these treatments on canine cartilage explants in both normal and interleukin-1 beta (IL-1β)-stimulated conditions. Methods We examined sulfate glycosaminoglycan (s-GAG) release, uronic acid (UA) content, and safranin-O staining, as well as the expression patterns of inflammatory, extracellular matrix (ECM) component and enzymes. Results Enro treatment alone effectively stimulated proteoglycan anabolism by increasing UA content and glycosaminoglycans (GAGs) in normal and pre-IL-1β-stimulated explant, whereas Mar showed opposite results. The combination of HA and FQs increased s-GAG release and UA content in normal explants in addition to effective down-regulated expression of MMP3. HA reduced the adverse effects of Mar by enhancing UA and GAG contents in both normal and pre-IL-1β-explants. Moreover, HA effectively induced HAS1and ACANup-regulation and reduced MMP9, TNF, PTGS2,and NFKB1 expression for a long term. Discussion Our results suggest the direct effects of Enro and Mar may selectively stimulate the conditioned explants to express MMP-codinggenes and promote gene expression involved in matrix production, pro-inflammatory cytokines, and cell degradation in different directions. HA successfully reduced the adverse effects of FQs by enhancing s-GAG and UA contents and down-regulated expression of MMPs.

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Determination of content of indole alkaloids in cell biomass of Rauwolfia serpentina Benth. ex Kurz tissue culture

Current issues in pharmacy and medicine science and practice ◽

10.14739/2409-2932.2021.1.226810 ◽

2021 ◽

Vol 14 (1) ◽

pp. 73-78

Author(s):

O. A. Bieda ◽

I. I. Konvaliuk ◽

L. P. Mozhylevska ◽

S. S. Lukashov ◽

V. A. Kunakh ◽

...

Keyword(s):

Tissue Culture ◽

Indole Alkaloids ◽

Natural Plant ◽

Rauwolfia Serpentina ◽

Cell Biomass ◽

Total Alkaloids ◽

Dry Cell

Cardiovascular diseases are the most common human diseases, hence, the production of cardiological (in particular, anti-arrhythmic) medications from the natural sources is an ever-actual task. Rauwolfia serpentina Benth. is a tropical fruticose plant that is able to produce and concentrate indole alkaloids, especially ajmaline and its derivatives, which are the most effective medications against ventricular arrhythmia with low side effects. Aim of the study. Determination of the qualitative and quantitative content of indole alkaloids in cell biomass of Rauwolfia serpentina tissue culture, obtained by the prolonged in vitro growth. Materials and methods. Object: cell biomass of Rauwolfia serpentina tissue culture (K-27 strain), obtained by methods of long-term cell selection in vitro. Alkaloids content determination: TSQ Vantage LC-MS (ThermoFischer Scientific). Results. 20 indole alkaloids are found in cell biomass of Rauwolfia serpentina tissue culture (K-27 strain). The highest content is registered for ajmaline and its derivatives (0.690 % mass. for ajmaline). The contents of reserpine and yohimbine were found to be as low as 0.009 % and 0.020 %, respectively. Conclusions. It is established that the content of indole alkaloids is higher in K-27 strain in comparison to natural plant and is stable over more than 30 years of its growth. Total alkaloids content was found to be 2.8 % of dry cell biomass, and total ajmaline-type alkaloids content (including ajmaline) was found to be 1.6 % of dry cell biomass. In contrast, the total alkaloid contents in the natural plant material is reported to be in the range of 0.8–1.3 %.

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Impact of Plant-Associated Bacteria on the In Vitro Growth and Pathogenic Resistance against Phellinus tremulae of Different Aspen (Populus) Genotypes

Microorganisms ◽

10.3390/microorganisms9091901 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1901

Author(s):

Greta Striganavičiūtė ◽

Jonas Žiauka ◽

Vaida Sirgedaitė-Šėžienė ◽

Dorotėja Vaitiekūnaitė

Keyword(s):

Populus Tremula ◽

Subsequent Stage ◽

Pseudomonas Sp ◽

Growth Responses ◽

Associated Bacteria ◽

Paenibacillus Sp ◽

Development In Vitro ◽

Potential Tool

Aspens (Populus tremula and its hybrids), economically and ecologically important fast-growing trees, are often damaged by Phellinus tremulae, a rot-causing fungus. Plant-associated bacteria can be used to increase plant growth and resistance; however, no systematic studies relating the activity of symbiotic bacteria to aspen resistance against Phellinus tremulae have been conducted so far. The present pioneer study investigated the responses of two Populus tremula and two P. tremula × P. tremuloides genotypes to in vitro inoculations with, first, either Pseudomonas sp. or Paenibacillus sp. bacteria (isolated originally from hybrid aspen tissue cultures and being most closely related to Pseudomonas oryzihabitans and Paenibacillus tundrae, respectively) and, in the subsequent stage, with Phellinus tremulae. Both morphological parameters of in vitro-grown plants and biochemical content of their leaves, including photosynthesis pigments and secondary metabolites, were analyzed. It was found that both Populus tremula × P. tremuloides genotypes, whose development in vitro was significantly damaged by Phellinus tremulae, were characterized by certain responses to the studied bacteria: decreased shoot development by both Paenibacillus sp. and Pseudomonas sp. and increased phenol content by Pseudomonas sp. In turn, these responses were lacking in both Populus tremula genotypes that showed in vitro resistance to the fungus. Moreover, these genotypes showed positive long-term growth responses to bacterial inoculation, even synergistic with the subsequent fungal inoculation. Hence, the studied bacteria were demonstrated as a potential tool for the improved in vitro propagation of fungus-resistant aspen genotypes.

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Study of the amount of oxidative damage to mitochondrial and chloroplast DNA in clones of white poplar (Populus alba L.) during long-term in vitro cultivation for 26 years

Plant Molecular Biology ◽

10.1007/s11103-021-01157-5 ◽

2021 ◽

Author(s):

Artem P. Gureev ◽

Olga S. Mashkina ◽

Ekaterina A. Shabanova ◽

Inna Yu. Vitkalova ◽

Vadim V. Sitnikov ◽

...

Keyword(s):

Oxidative Damage ◽

Chloroplast Dna ◽

In Vitro Cultivation ◽

Populus Alba ◽

White Poplar

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Cytogenetic determination of sex among individually identified humpback whales (Megaptera novaeangliae)

Canadian Journal of Zoology ◽

10.1139/z88-181 ◽

1988 ◽

Vol 66 (6) ◽

pp. 1243-1248 ◽

Cited By ~ 10

Author(s):

Richard H. Lambertsen ◽

C. Scott Baker ◽

Deborah A. Duffield ◽

Jan Chamberlin-Lea

Keyword(s):

Population Biology ◽

Marine Species ◽

Megaptera Novaeangliae ◽

Humpback Whales ◽

Dermal Tissue ◽

Behavioral Observations ◽

The Social

Using a small biopsy dart, samples of living dermal tissue were collected from individually identified humpback whales (Megaptera novaeangliae) in southeastern Alaskan waters. In vitro culture of these samples provided enough chromosomal material to karyotype 10 individuals. Examination of the sex chromosomes in the karyotypes demonstrated the sex of each of the 10 whales. In two cases, cytogenetic findings verified previous inferences of sex based on behavioral observations. In another two cases, karyotyping allowed the determination of sex when even long-term sighting histories and behavioral observations had been insufficient. For the rest of the whales, sex determination by cytogenetic findings has preceded long-term sightings and will facilitate the interpretation of future behavioral observations. We conclude that in vitro culturing of dermal tissue collected from naturally marked humpback whales, coupled with karyology, promises to be a powerful technique in future studies of the social behavior and population biology of this marine species.

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Long-term Cytotoxicity Testing of the First Twenty MEIC Chemicals by the Determination of the Protein Content in Human Embryonic Lung Cells

Alternatives to Laboratory Animals ◽

10.1177/026119299202000220 ◽

1992 ◽

Vol 20 (2) ◽

pp. 285-289

Author(s):

Paul J. Dierickx ◽

Björn Ekwall

Keyword(s):

Protein Content ◽

Good Alternative ◽

In Vitro Cytotoxicity ◽

Cell Protein ◽

Cytotoxicity Test ◽

Hep G2 ◽

Human Embryonic Lung

Within the framework of the MEIC (the multicentre evaluation of in vitro cytotoxicity) programme, the long-term cytotoxicity of the first twenty MEIC chemicals was investigated on MRC-5 cells. After treatment of the cells with each chemical for six weeks, the PI50 value was determined. The PI50 is the concentration (mM) of test compound required to induce a 50% reduction in total cell protein content. The results were compared with the acute PI50 values previously measured after treatment for 24 hours with the same compounds in Hep G2 cells. A good correlation between these two sets of data (r2 = 0.94) was observed, with the exception of digoxin. Together with other preliminary MEIC validation results, this study indicates that a good alternative cytotoxicity test may be found for the prediction of human long-term toxicity.

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Preparation And elemental analysis of ancient fibers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100126846 ◽

1987 ◽

Vol 45 ◽

pp. 410-411

Author(s):

Allen Angel ◽

Kathryn A. Jakes

Keyword(s):

Cross Sections ◽

Physical Structure ◽

Energy Dispersive ◽

Archaeological Sites ◽

X Ray ◽

Long Term Storage ◽

Backscattered Electron ◽

Electron Imaging

Fabrics recovered from archaeological sites often are so badly degraded that fiber identification based on physical morphology is difficult. Although diagenetic changes may be viewed as destructive to factors necessary for the discernment of fiber information, changes occurring during any stage of a fiber's lifetime leave a record within the fiber's chemical and physical structure. These alterations may offer valuable clues to understanding the conditions of the fiber's growth, fiber preparation and fabric processing technology and conditions of burial or long term storage (1).Energy dispersive spectrometry has been reported to be suitable for determination of mordant treatment on historic fibers (2,3) and has been used to characterize metal wrapping of combination yarns (4,5). In this study, a technique is developed which provides fractured cross sections of fibers for x-ray analysis and elemental mapping. In addition, backscattered electron imaging (BSI) and energy dispersive x-ray microanalysis (EDS) are utilized to correlate elements to their distribution in fibers.

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Ultrastructural investigations of MDL 19,660-induced effects on the canine platelet and megakaryocyte

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100159412 ◽

1990 ◽

Vol 48 (3) ◽

pp. 374-375

Author(s):

D.E. Loudy ◽

J. Sprinkle-Cavallo ◽

J.T. Yarrington ◽

F.Y. Thompson ◽

J.P. Gibson

Keyword(s):

Antidepressant Drug ◽

Beagle Dogs ◽

Long Term Effects ◽

Short Term ◽

Platelet Counts ◽

Toxicological Studies ◽

Induced Aggregation ◽

Internal Architecture

Previous short term toxicological studies of one to two weeks duration have demonstrated that MDL 19,660 (5-(4-chlorophenyl)-2,4-dihydro-2,4-dimethyl-3Hl, 2,4-triazole-3-thione), an antidepressant drug, causes a dose-related thrombocytopenia in dogs. Platelet counts started to decline after two days of dosing with 30 mg/kg/day and continued to decrease to their lowest levels by 5-7 days. The loss in platelets was primarily of the small discoid subpopulation. In vitro studies have also indicated that MDL 19,660: does not spontaneously aggregate canine platelets and has moderate antiaggregating properties by inhibiting ADP-induced aggregation. The objectives of the present investigation of MDL 19,660 were to evaluate ultrastructurally long term effects on platelet internal architecture and changes in subpopulations of platelets and megakaryocytes.Nine male and nine female beagle dogs were divided equally into three groups and were administered orally 0, 15, or 30 mg/kg/day of MDL 19,660 for three months. Compared to a control platelet range of 353,000- 452,000/μl, a doserelated thrombocytopenia reached a maximum severity of an average of 135,000/μl for the 15 mg/kg/day dogs after two weeks and 81,000/μl for the 30 mg/kg/day dogs after one week.

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