scholarly journals CYTOLOGICAL ASSESSMENT OF DOUBLED HAPLOIDS IN SUMMER SQUASH (CUCURBITA PEPO L.)

2018 ◽  
pp. 3-7 ◽  
Author(s):  
E. A. Domblides ◽  
L. Yu. Kan ◽  
G. A. Khimich ◽  
I. B. Korotseva ◽  
A. S. Domblides
Keyword(s):  
Doubled Haploids ◽  
Hybrid Breeding ◽  
Mitotic Chromosomes ◽  
Optimal Method ◽  
Regenerated Plants ◽  
Summer Squash ◽  
Doubled Haploid Plants ◽  
Chromosome Staining ◽  
Winter Squash

147 new forms were obtained only from nine responsive unpollinated ovules of summer squash in 2015-2017. The cytological analysis is used to estimate the level of ploidy in regenerated plants R0. The small size of mitotic chromosomes and large number in Cucurbita genus make it difficult to count them even though they are well separated. As a result of analysis the optimal method of chromosome staining has been chosen with the use of modified propiono- lacmoid cytological technique. The rootlet tips and apical meristems were used to make smear preparations. As it was shown the summer squash was a difficult species as a cytological object because of low mitosis frequency and low number of metaphase plates with well scattered chromosomes. The photos of chromosomes in squash C. pepo subsp. brevicaulis var. giraumons Duch; distant hybrid (breeding accession N37) between variety ‘Cornishon’ and winter squash C. pepo subsp. longicaulis Greb. var. pepo and doubled haploid plants produced from them were made. In spite of the small size 2 μm the chromosomes observed were clearly seen. Nearly all regenerated plants that had been analyzed passed well the adaptation in vivo and occurred to be a doubled haploids (2n = 2x=40). The seed progeny was then obtained through self-pollination. About 20% of plants R0 analyzed were mixploids and supposedly only 7% were haploids.

scholarly journals Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shojiro Katoh ◽  
Atsuki Fujimaru ◽  
Masaru Iwasaki ◽  
Hiroshi Yoshioka ◽  
Rajappa Senthilkumar ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Replicative Senescence ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Human Chondrocytes ◽  
Cartilage Tissue ◽  
Optimal Method ◽  
Beta Galactosidase

AbstractRegenerative medicine applications require cells that are not inflicted with senescence after in vitro culture for an optimal in vivo outcome. Methods to overcome replicative senescence include genomic modifications which have their own disadvantages. We have evaluated a three-dimensional (3D) thermo-reversible gelation polymer (TGP) matrix environment for its capabilities to reverse cellular senescence. The expression of senescence-associated beta-galactosidase (SA-βgal) by human chondrocytes from osteoarthritis-affected cartilage tissue, grown in a conventional two-dimensional (2D) monolayer culture versus in 3D-TGP were compared. In 2D, the cells de-differentiated into fibroblasts, expressed higher SA-βgal and started degenerating at 25 days. SA-βgal levels decreased when the chondrocytes were transferred from the 2D to the 3D-TGP culture, with cells exhibiting a tissue-like growth until 42–45 days. Other senescence associated markers such as p16INK4a and p21 were also expressed only in 2D cultured cells but not in 3D-TGP tissue engineered cartilage. This is a first-of-its-kind report of a chemically synthesized and reproducible in vitro environment yielding an advantageous reversal of aging of human chondrocytes without any genomic modifications. The method is worth consideration as an optimal method for growing cells for regenerative medicine applications.

scholarly journals Triple-Helical DNA in Drosophila Heterochromatin

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 227 ◽  
Author(s):  
Eduardo Gorab
Keyword(s):  
Nucleic Acids ◽  
Dna Sequences ◽  
Detection Methods ◽  
Triplex Dna ◽  
Thiazole Orange ◽  
Chromosomal Dna ◽  
Mitotic Chromosomes ◽  
Satellite Repeats

Polynucleotide chains obeying Watson-Crick pairing are apt to form non-canonical complexes such as triple-helical nucleic acids. From early characterization in vitro, their occurrence in vivo has been strengthened by increasing evidence, although most remain circumstantial particularly for triplex DNA. Here, different approaches were employed to specify triple-stranded DNA sequences in the Drosophila melanogaster chromosomes. Antibodies to triplex nucleic acids, previously characterized, bind to centromeric regions of mitotic chromosomes and also to the polytene section 59E of mutant strains carrying the brown dominant allele, indicating that AAGAG tandem satellite repeats are triplex-forming sequences. The satellite probe hybridized to AAGAG-containing regions omitting chromosomal DNA denaturation, as expected, for the intra-molecular triplex DNA formation model in which single-stranded DNA coexists with triplexes. In addition, Thiazole Orange, previously described as capable of reproducing results obtained by antibodies to triple-helical DNA, binds to AAGAG repeats in situ thus validating both detection methods. Unusual phenotype and nuclear structure exhibited by Drosophila correlate with the non-canonical conformation of tandem satellite arrays. From the approaches that lead to the identification of triple-helical DNA in chromosomes, facilities particularly provided by Thiazole Orange use may broaden the investigation on the occurrence of triplex DNA in eukaryotic genomes.

scholarly journals Double Haploid Development and Assessment of Androgenic Competence of Balkan Pepper Core Collection in Bulgaria

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2414
Author(s):  
Stanislava Grozeva ◽  
Gancho Pasev ◽  
Vesela Radeva-Ivanova ◽  
Velichka Todorova ◽  
Valentina Ivanova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Double Haploid ◽  
Genetic Improvement ◽  
Core Collection ◽  
Culture Medium ◽  
Doubled Haploids ◽  
Caps Marker ◽  
Regenerated Plants ◽  
Direct Embryogenesis ◽  
Pepper Breeding

This study was designed to assess the androgenic potential of 180 pepper accessions and 11 progenies (four F1 and seven BC) possessing PMMoV resistance in order to complement an ongoing pepper breeding program. The experiment was carried out in 10 replications with 20 anthers for each accession in two different induction mediums from 2017 to 2019. The highest androgenic response was observed in culture medium 17-2 but differences between two mediums were nonsignificant. From a total of 191 genotypes, 102 genotypes expressed a potential for direct embryogenesis. Embryo induction was seen to be genotype-dependent and decreased in the following order: Pumpkin > Conical > Bell or blocky > Round > Elongate as the most responsive genotypes with over 10% reacted anthers being observed in CAPS-23, CAPS-29, CAPS-127, CAPS-157, CAPS-169, F1 and BC 887 derived from CAPS-23. The number of regenerated plants was higher in the conical group and least in the round varietal group. Regenerated plants were examined visually and by flow cytometry for identification of spontaneous doubled haploids (DH) and haploids. Those originating from F1 and BC progenies were additionally evaluated by a CAPS marker targeting L4 allele for resistance against PMMoV. Obtained results revealed two groups consisting of homozygous susceptible and resistant plants. Therefore, use of anther culture in ongoing breeding will greatly facilitate the pepper genetic improvement.

Haploidy and plant breeding

1981 ◽  
Vol 292 (1062) ◽  
pp. 499-507 ◽  
Keyword(s):  
Plant Breeding ◽  
Doubled Haploids ◽  
Chromosome Complement ◽  
Basic Research ◽  
Breeding Value ◽  
Additive Variance ◽  
Ploidy Levels ◽  
Breeding Programmes ◽  
Dh Lines

A haploid is an organism that looks like a sporophyte, but has the chromosome complement of a reduced gamete. There are several ways in which haploids can occur or be induced in vivo : spontaneously, mostly associated with polyembryony, and through abnormal processes after crosses, like pseudogamy, semigamy, preferential elimination of the chromosomes of one parental species, and androgenesis. In the crops described, haploids are or are near to being used in basic research and plant breeding. The application of haploids in breeding self-pollinated crops is based on their potential for producing fully homozygous lines in one generation, which can be assessed directly in the field. Early generation testing of segregating populations is possible through haploids, because doubled haploids (DH) possess additive variance only. Haploids can also be applied in classical breeding programmes to make these more efficient through improved reliability of selection. The application of haploids in cross-pollinated crops is also based on a rapid production of DH-lines, which can be used as inbred lines for the production of hybrid varieties. By means of haploids all natural barriers to repeated selfing are bypassed. In autotetraploid crops there are two types of haploid. One cycle of haploidization leads to dihaploids; a second cycle produces monohaploids. The significance of dihaploids is in their greatly simplified genetics and breeding and in the possibility of estimation of the breeding value of tetraploid cultivars by assessing their dihaploids. The main drawback of dihaploids is their restriction to two alleles per locus. Also, after doubling, it is impossible to achieve tetra-allelism at many loci, the requirement for maximal performance of autotetraploid cultivars. Tetra-allelism can be obtained when improved dihaploids have a genetically controlled mechanism of forming highly heterozygous restitution gametes with the unreduced number of chromosomes. Monohaploids, after doubling or twice doubling, may lead to fully homozygous diploids and tetraploids. These are important for basic research, but not yet for practical application. Meiotic data of potato homozygotes at three ploidy levels are presented.

scholarly journals Human Papillomavirus 16 Capsids Mediate Nuclear Entry during Infection

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Patricia M. Day ◽  
Andrea S. Weisberg ◽  
Cynthia D. Thompson ◽  
Michelle M. Hughes ◽  
Yuk Ying Pang ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Viral Entry ◽  
Large Family ◽  
Viral Capsid ◽  
Mitotic Chromosomes ◽  
Nuclear Entry ◽  
Human Papillomavirus 16 ◽  
Transport Vesicle ◽  
Causative Agents

ABSTRACTInfectious human papillomavirus 16 (HPV16) L1/L2 pseudovirions were found to remain largely intact during vesicular transport to the nucleus. By electron microscopy, capsids with a diameter of 50 nm were clearly visible within small vesicles attached to mitotic chromosomes and to a lesser extent within interphase nuclei, implying nuclear disassembly. By confocal analysis, it was determined that nuclear entry of assembled L1 is dependent upon the presence of the minor capsid protein, L2, but independent of encapsidated DNA. We also demonstrate that L1 nuclear localization and mitotic chromosome association can occurin vivoin the murine cervicovaginal challenge model of HPV16 infection. These findings challenge the prevailing concepts of PV uncoating and disassembly. More generally, they document that a largely intact viral capsid can enter the nucleus within a transport vesicle, establishing a novel mechanism by which a virus accesses the nuclear cellular machinery.IMPORTANCEPapillomaviruses (PVs) comprise a large family of nonenveloped DNA viruses that include HPV16, among other oncogenic types, the causative agents of cervical cancer. Delivery of the viral DNA into the host cell nucleus is necessary for establishment of infection. This was thought to occur via a subviral complex following uncoating of the larger viral capsid. In this study, we demonstrate that little disassembly of the PV capsid occurs prior to nuclear delivery. These surprising data reveal a previously unrecognized viral strategy to access the nuclear replication machinery. Understanding viral entry mechanisms not only increases our appreciation of basic cell biological pathways but also may lead to more effective antiviral interventions.

scholarly journals Increased expression of the MALE STERILITY1 transcription factor gene results in temperature-sensitive male sterility in barley

2020 ◽  
Vol 71 (20) ◽  
pp. 6328-6339
Author(s):  
José Fernández-Gómez ◽  
Behzad Talle ◽  
Zoe A Wilson
Keyword(s):  
Transcription Factor ◽  
Male Sterility ◽  
Pollen Development ◽  
Breeding Systems ◽  
Hybrid Breeding ◽  
Temperature Sensitive ◽  
Hybrid Vigour ◽  
Male Sterile ◽  
The Impact

Abstract Understanding the control of fertility is critical for crop yield and breeding; this is particularly important for hybrid breeding to capitalize upon the resultant hybrid vigour. Different hybrid breeding systems have been adopted; however, these are challenging and crop specific. Mutants with environmentally reversible fertility offer valuable opportunities for hybrid breeding. The barley HvMS1 gene encodes a PHD-finger transcription factor that is expressed in the anther tapetum, which is essential for pollen development and causes complete male sterility when overexpressed in barley. This male sterility is due at least in part to indehiscent anthers resulting from incomplete tapetum degeneration, failure of anther opening, and sticky pollen under normal growth conditions (15 °C). However, dehiscence and fertility are restored when plants are grown at temperatures >20 °C, or when transferred to >20 °C during flowering prior to pollen mitosis I, with transfer at later stages unable to rescue fertility in vivo. As far as we are aware, this is the first report of thermosensitive male sterility in barley. This offers opportunities to understand the impact of temperature on pollen development and potential applications for environmentally switchable hybrid breeding systems; it also provides a ‘female’ male-sterile breeding tool that does not need emasculation to facilitate backcrossing.

In vitro Propagation and Chemical and Biological Studies of the Essential Oil of Salvia przewalskii Maxim

2007 ◽  
Vol 62 (11-12) ◽  
pp. 839-848 ◽  
Author(s):  
Ewa Skała ◽  
Danuta Kalemba ◽  
Anna Wajs ◽  
Marek Róźalski ◽  
Urszula Krajewska ◽  
...  
Keyword(s):  
Essential Oil ◽  
Shoot Multiplication ◽  
Quantitative Composition ◽  
Human Leukemia ◽  
Qualitative And Quantitative ◽  
H Nmr ◽  
Regenerated Plants ◽  
Biological Studies

The procedure of Salvia przewalskii shoot multiplication and the ability of regenerated plants to produce essential oil is reported. The essential oil was obtained by hydrodistillation from leaves and flowering stems of field-grown plants, and their chemical composition was examined by GC, GC-MS and 1H NMR. The differences in yield as well as qualitative and quantitative composition between the oils isolated from in vitro and in vivo plants were observed. S. przewalskii essential oil was tested for its antimicrobial and cytotoxic properties. It was found that cytotoxicity against human leukemia HL-60 cells and antimicrobial activity (especially, against Staphylococcus aureus and S. epidermidis strains) of oils isolated from in vitro plants were higher than those for oils from in vivo S. przewalskii plants.

scholarly journals Doubled haploids of interspecific hybrids between Brassica napus and Brassica rapa for canola production with valuable breeding traits

OCL ◽  
2020 ◽  
Vol 27 ◽  
pp. 45
Author(s):  
Ainash Daurova ◽  
Dias Daurov ◽  
Dmitriy Volkov ◽  
Kuanysh Zhapar ◽  
Daniyar Raimbek ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Seed Germination ◽  
Brassica Rapa ◽  
Doubled Haploid ◽  
Interspecific Hybrids ◽  
Nutritional Value ◽  
Doubled Haploids ◽  
Doubled Haploid Plants ◽  
Haploid Plants

Doubled haploids (DH) were obtained from two interspecific hybrids between Brassica napus and Brassica rapa. Seeds of doubled haploid plants differed in colour and size. The hybridity of the obtained doubled haploid is shown using genomic in situ hybridization (GISH) analysis. Evaluation of drought tolerance during seed germination on PEG-6000 showed the advantage of doubled haploid plants of interspecific hybrids over the parent cultivars. The oil from seeds of doubled haploid plants showed good nutritional value.

Newmouse models of melanoma metastasis and differences in brain tropism and metastatic growth pattern.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e19015-e19015
Author(s):  
Amr M. Morsi ◽  
Avital Gazial-Sovran ◽  
Hana Baig ◽  
Robert S. Kerbel ◽  
John Golfinos ◽  
...  
Keyword(s):  
Brain Metastases ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Melanoma Cells ◽  
Melanoma Metastasis ◽  
Optimal Method ◽  
In Vivo Models ◽  
Dismal Prognosis ◽  
Intracardiac Injection

e19015 Background: 75% of patients with metastatic melanoma develop brain metastases (B-mets). Such patients show dismal prognosis with a median survival of < 6 months. Scarcity of clinically relevant in vivo models has hindered melanoma B-met studies. We compared the in vivo dissemination upon ultrasound (u/s) guided intracardiac injection of B16F10 cells to 131/4-5B1 (hereafter 5B1), a WM239A subclone with enhanced brain tropism. We also implemented an ex vivo MRI protocol as a high throughput three dimensional approach for characterizing B-mets penetrance and growth. Methods: B16-F10 or 5B1 melanoma cells were injected in C57BL/6J mice (n=40) or athymic/nude mice (n=40) respectively using u/s-guided intracardiac injection. Upon weight loss, mice were euthanized, and heads prepared for ex vivo imaging. All µMRI experiments were performed with a 7T Bruker Avance II console. The protocol consisted of (110-mm)3 isotropic T1-, T2- and T2*-weighted sequences. Results: Our ex vivo MRI recapitulates the clinical radiological T1 and T2 brightening as well as susceptibility-induced T2* darkening effect of melanoma. The B16F10 model revealed exclusive ventricular and leptomeningeal spread while the 5B1 model showed parenchymal lesions. In addition, 90% of the 5B1 mice with brain tumors showed multiple lesions (3-16) vs. 18% in the B16F10 model (1- 3). Finally, 3D volume studies revealed a higher B-met penetrance (68% vs. 18%), delayed onset of tumor detection (earliest-day 27 vs. day 15) post-injection and a slower growth rate of 5B1 brain metastases compared to B16F10 tumors. Conclusions: Our results suggest that u/s-guided intracardiac injection of melanoma cells is an optimal method to capture the cells’ spontaneous dissemination pattern (or site-specific tropism) and that the 5B1 model is a more clinically relevant model of melanoma B-met for preclinical studies.

Indium (111In)-Labelled Human Platelets: Optimal Method

1980 ◽  
Vol 58 (3) ◽  
pp. 243-248 ◽  
Author(s):  
R. J. Hawker ◽  
Linda M. Hawker ◽  
A. R. Wilkinson
Keyword(s):  
Healthy Volunteer ◽  
Survival Time ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Laboratory Method ◽  
Clinical Use ◽  
Optimal Method ◽  
Normal Platelet

1. A detailed laboratory method is described for the labelling of human platelets with [111In]indium oxine. The 45 min method is simple, requires only 26 ml of blood and is suitable for routine clinical use. 2. After the labelling and resuspension of the platelets in plasma, aggregation responses to both adenosine diphosphate and collagen were similar to those of normal platelet-rich plasma. Less than 5% of the [111In]indium oxine was released by secretory functions of platelets. 3. Labelling efficiencies of 90·1 ± 4·29% (n = 28) were achieved in 60 s by normal concentrations of plasma-free platelet suspensions. 4. Platelet survival in vivo in healthy volunteer subjects follows a linear function with a survival time of 8·44 ± 0·18 days.

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