scholarly journals Viability Assessment ofGenipa americanaL. (Rubiaceae) Embryonic Axes after Cryopreservation UsingIn VitroCulture

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Izulmé Rita Imaculada Santos ◽  
Antonieta Nassif Salomão
Keyword(s):  
Moisture Content ◽  
Liquid Nitrogen ◽  
Culture Medium ◽  
Nitrogen Control ◽  
Viability Assessment ◽  
Embryonic Axes ◽  
High Germination ◽  
Water Contents

Embryonic axes excised from seeds ofGenipa americanaL. desiccated to different water contents were successfully cryopreserved by rapidly plunging seed samples directly into liquid nitrogen. Control and cryopreserved embryonic axes were excised and grown in WPM culture medium for viability assessment. All control embryonic axes (−LN2) excised from fully hydrated seeds (43.89% moisture content) germinated after 21 days of culturein vitro. These high germination percentages persisted even after the water content of the seeds was as low as 6.79%. After freezing in liquid nitrogen high germination percentages, 93%, 96%, and 93%, were observed for embryonic axes excised from seeds dehydrated to 13.26%, 9.57%, and 6.79 moisture content, respectively. The cryopreservation technique described here is recommended for long term conservation ofG. americanagermplasm.

Short-term storage of seeds and cryopreservation of embryonic axes of Lepisanthes fruticosa

2021 ◽  
Author(s):  
Suryanti Bustam ◽  
Mohd Shukri Mat Ali ◽  
Uma Rani Sinniah ◽  
Nur Atisha Shamsuddin ◽  
Abdul Muhaimin Abdul Kadir
Keyword(s):  
Moisture Content ◽  
Sodium Alginate ◽  
Liquid Nitrogen ◽  
Short Term ◽  
Embryonic Axes ◽  
Short Term Storage ◽  
Vitrification Solution ◽  
Term Storage

Abstract This work highlights short-term storage of recalcitrant Lepisanthes fruticosa seeds and long-term conservation attempts of its embryonic axes (EAs) through cryopreservation. Short-term storage was carried out using fresh seeds at 54 % moisture content and stored at 8 ±1 °C and 25 ±2 °C for 7 weeks. Three variations to sterilization were attempted to optimize survival while keeping contamination low for cryopreservation. Cryopreservation using two different methods were tested, namely vitrification and the encapsulation vitrification method. Vitrification technique involved the pre-culturing of EAs overnight in different sucrose pre-culture concentrations (0, 0.2, 0.4 and 0.6 M) prior to, loading, dehydration with plant vitrification solution (PVS2), rapid immersion into liquid nitrogen (-196 °C), rapid warming, unloading and recovery. While, encapsulation vitrification involved encapsulation of the EAs using 3 % sodium alginate followed by exposure to different duration (0, 10, 20, 30, 40 and 50 minutes) of PVS2 prior to cryopreservation. L. fruticosa seeds can be safely stored for short-term with no loss in germination up to 7 weeks of storage either at 8 ±1 °C or 25 ±2 °C. This study also showed that EA of L. fruticosa was amenable to cryopreservation, 13.0 – 66.67% of viability was obtained when the EAs were cryopreserved using the vitrification technique while the best result was obtained (66.67 % viability) when the EAs were pre-cultured with 0.4 M sucrose prior to exposure to PVS2 and liquid nitrogen. Cryopreservation of EAs using the encapsulation-vitrification method was unsuccessful.

scholarly journals Temperature-induced reorganisation of Schistocephalus solidus (Cestoda) proteome during the transition to the warm-blooded host

Biology Open ◽  
2021 ◽  
Vol 10 (11) ◽  
Author(s):  
Ekaterina V. Borvinskaya ◽  
Albina A. Kochneva ◽  
Polina B. Drozdova ◽  
Olga V. Balan ◽  
Victor G. Zgoda
Keyword(s):  
Model Experiment ◽  
Protein Production ◽  
Culture Medium ◽  
Gasterosteus Aculeatus ◽  
Protein Composition ◽  
Tegument Protein ◽  
Schistocephalus Solidus ◽  
Induced Genes

ABSTRACT The protein composition of the cestode Schistocephalus solidus was measured in an experiment simulating the trophic transmission of the parasite from a cold-blooded to a warm-blooded host. The first hour of host colonisation was studied in a model experiment, in which sticklebacks Gasterosteus aculeatus infected with S. solidus were heated at 40°C for 1 h. As a result, a decrease in the content of one tegument protein was detected in the plerocercoids of S. solidus. Sexual maturation of the parasites was initiated in an experiment where S. solidus larvae were taken from fish and cultured in vitro at 40°C for 48 h. Temperature-independent changes in the parasite proteome were investigated by incubating plerocercoids at 22°C for 48 h in culture medium. Analysis of the proteome allowed us to distinguish the temperature-induced genes of S. solidus, as well as to specify the molecular markers of the plerocercoid and adult worms. The main conclusion of the study is that the key enzymes of long-term metabolic changes (glycogen consumption, protein production, etc.) in parasites during colonisation of a warm-blooded host are induced by temperature.

scholarly journals Long-term in Vitro Regeneration of Hamelia patens

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 873G-874
Author(s):  
D. Sankhla ◽  
T.D. Davis ◽  
N. Sankhla ◽  
A. Upadhyaya
Keyword(s):  
Culture Medium ◽  
In Vitro Regeneration ◽  
Shoot Formation ◽  
Shoot Tips ◽  
Shoot Cultures ◽  
Ms Medium ◽  
Prolific Shoot ◽  
Ornamental Shrub

This report describes an efficient in vitro regeneration protocol for H. patens (firebush), a heat-tolerant ornamental shrub native to tropical and subtropical America. Shoot cultures were initially established using shoot tips placed on MS-revised medium containing 2.3 μM 2,4-D, 2.3 μM kinetin, and 0.25% polyvinylpyrrolidone. Other types of explants (nodal and internodal segments, leaf pieces, floral buds) did not regenerate shoots when placed on this medium. Two-month-old plantlets derived from the shoot tips were subcultured on MS medium supplemented with 0.5 μM thidiazuron (TDZ), and within 3 to 4 weeks, some callus was produced at the root–shoot junction. When this callus, with a small portion of the root and shoots, was placed on MS medium with 0.05 μM TDZ and 0.01 μM ABA, prolific shoot formation occurred within 3 to 4 weeks followed by root formation. By regular subculturing every 5 to 6 weeks, hundreds of plantlets have been obtained over the past 3 years with no apparent decline in regeneration potential. Addition of activated charcoal (0.5%) to the culture medium has greatly improved growth of the plantlets.

scholarly journals Expression of Otx Genes in Müller Cells Using an In Vitro Experimental Model of Retinal Hypoxia

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Claudio Azzolini ◽  
Simone Donati ◽  
Giovanni Micheloni ◽  
Vittoria Moretti ◽  
Roberto Valli ◽  
...  
Keyword(s):  
Recovery Time ◽  
Culture Medium ◽  
Cobalt Chloride ◽  
Retinal Diseases ◽  
Cellular Events ◽  
In Vitro Response ◽  
Retinal Pathologies ◽  
Eye Morphogenesis

Introduction. Müller glial cells typically activate to react to hypoxic tissue damage in several retinal diseases. We evaluated the in vitro response to a hypoxia-mimicking stimulus on the expression of a set of genes, known to contribute to eye morphogenesis and cell differentiation. Materials and Methods. A MIO-M1 Müller cell line was cultured in a hypoxia-mimicking environment by the addition of cobalt chloride to the culture medium, followed by a recovery time in which we mimic restoration from the hypoxic insult. The HIF-1α protein and VEGF-A gene expression were quantified to verify the induction of a hypoxia-like state. Results. Among the genes under study, we did not observe any difference in the expression levels of Otx1 and Otx2 during treatment; conversely, Otx1 was overexpressed during recovery steps. The VEGF-A gene was strongly upregulated at both the CoCl2 and recovery time points. The transactivated isoform (TA) of the TP73 gene showed an overexpression in long-term exposure to the hypoxic stimulus with a further increase after recovery. Discussion. Our molecular analysis is able to describe the activation of a set of genes, never before described, that can drive the response to a hypoxia-like status. The improved comprehension of these cellular events will be useful for designing new therapeutical approaches for retinal pathologies.

113 EVALUATION OF DIFFERENT CRYOPROTECTANT AND FORSKOLIN IN THE CULTURE MEDIUM FOR IMPROVING THE EFFICACY OF VITRIFICATION OF BOS INDICUS IN VITRO-DERIVED EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 215 ◽  
Author(s):  
B. V. Sanches ◽  
B. D. O. Filho ◽  
J. H. F. Pontes ◽  
A. C. Basso ◽  
M. L. G. Meirinhos ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Lipid Droplets ◽  
Culture Medium ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Calf Serum ◽  
Genetic Material ◽  
Embryo Cryopreservation ◽  
Hatching Rate

Embryo cryopreservation is an essential method for the biotechnology of reproduction. This is the safest option for interchange of genetic material for research and commercial purposes. For cattle, Brazil has become the leading country in the world for the number of in vitro-produced embryos, using mostly Bos indicus animals. However, considering the in vitro method of embryo production, field results have shown a lower resistance to cryopreservation for B. indicus when compared with Bos taurus embryos. A possible explanation for this is a great concentration of lipid droplets in the cytoplasm of cells fromB. indicus embryos. The objective of this study was to compare 2 cryoprotectants (Propanediol and DMSO) to vitrification and evaluate the effect of adding 10 μM forskolin to the SOF medium for embryo culture before cryopreservation. For all the experiments, ovaries from slaughtered Nelore Bos indicus donors were recovered and maintained at 30 to 35°C in NaCl solution until recovery of the COC. Embryos submmited to vitrification were expanded blastocysts at Day 7 of in vitro culture. In the first experiment embryos were first incubated in 10% ethylene glycol (EG) plus 10% DMSO dissolved in holding medium (TCM-HEPES with 20% calf serum) for 1 min and then transferred to droplet of 20% EG plus 20% DMSO in holding medium and 0.5 M sucrose for 20 s before immersing in liquid nitrogen (n = 107; group EG + DMSO). For the group EG + Propanediol (EG + PRO; n = 96), blastocysts were placed in 10% EG plus 10% PRO in holding medium for 1 min and then transferred to a droplet of 20% EG plus 20% PRO in holding medium and 0.5 M sucrose for 20 s before immersing in liquid nitrogen. Both treatments were performed using the Cryotop system. Results were compared with embryos (n = 118) not submitted to cryopreservation. The evaluation was done by the hatching rate of blastocysts at Day 9, being higher (86.4%) for embryos not cryopreserved, when comparing with 77.1% for group EG + PRO and 72.9% for group EG + DMSO (P < 0.05). In the second experiment, Day 5 embryos obtained in vitro from Nelore donors were cultured using SOF medium with 10 μM forskolin (n = 112) or not (control; n = 101), being all submitted to cryopreservation using Cryotop and the same vitrification method for group EG + DMSO. Results were compared with embryos cultured with SOF medium and not submitted to cryopreservation (n = 96). The evaluation was performed by considering hatching rate at Day 9, being higher (85.4%) for not cryopreserved, when compared with 63.3% for control and 70.5% for forskolin group (P < 0.05). Considering embryos submitted to cryopreservation, the hatching rate was higher (P < 0.05) for the forskolin group.

scholarly journals Extract of Amburana cearensis maintains the survival of ovine preantral follicles during long-term ovarian tissue transport and promotes primordial follicle activation after in vitro culture

2018 ◽  
Vol 39 (5) ◽  
pp. 2001
Author(s):  
Vanúzia Gonçalves Menezes ◽  
Ricássio De Sousa Barberino ◽  
Bruna Bortoloni Gouveia ◽  
Rodrigo José de Souza Gonçalves ◽  
Jackson Roberto Guedes da Silva Almeida ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Ovarian Tissue ◽  
Primordial Follicle ◽  
Chi Square ◽  
Preantral Follicles ◽  
Primordial Follicle Activation ◽  
Chi Square Test

This study evaluated the effect of Amburana cearensis extract as a preservation or culture medium for ovine ovarian tissue. Ovarian fragments were fixed in 4% buffered formaldehyde for 18 h (fresh control), stored in Minimal Essential Medium (MEM) or in A. cearensis extract (0.1; 0.2 or 0.4 mg/mL) at a temperature of 4ºC for 6, 12 or 24 h (preservation - experiment 1) or cultured for 7 days in ?-MEM+ or in A. cearensis extract without (0.1; 0.2 or 0.4 mg/mL) or with supplements (0.1+ ; 0.2+ or 0.4+ mg/ mL; experiment 2). The percentages of morphologically normal follicles and follicular activation were submitted to analysis of variance (ANOVA) and Tukey´s test. The values of TUNEL-positive cells were submitted to Chi-square test (P < 0.05). The storage of fragments for 6 h in MEM showed higher percentages of normal follicles (62%) and a lower rate of TUNEL positive cells (36.17%) compared to other treatments (normal follicles: 46%; 43% and 52%; TUNEL positive cells: 58.57%; 55.30% and 55.63% for Amb 0.1; Amb 0.2 and Amb 0.4 mg/mL, respectively). However, after 12 or 24 h, MEM (12 h: 48%; 24 h: 45%) and Amb 0.2 mg/mL (12 h: 37%; 24 h: 39%) showed similar percentages of normal follicles and TUNEL positive cells (MEM - 12 h: 43.26%; 24 h: 58%; Amb 0.2 mg/mL - 12 h: 50%; 24 h: 61%). After culture, ?-MEM+ recorded a higher percentage of normal follicles (58.25%) than A. cearensis treatments (32.8%; 25.4% and 34.2% for Amb 0.1; Amb 0.2 and Amb 0.4 mg/mL, and 22.25%; 20.0% and 36.6% for Amb 0.1+ ; Amb 0.2+ and Amb 0.4+ mg/mL, respectively) (P < 0.05). Follicular activation increased in all treatments (52.5%; 36.73%; 54.05%; 47.5% and 58.19% for ?-MEM+ ; Amb 0.1; Amb 0.1+ ; Amb 0.2+ and Amb 0.4+ mg/mL, respectively) compared to the fresh control (11.65%), except for Amb 0.2 mg/mL (23.69%) and Amb 0.4 mg/mL (28.85%) (P > 0.05). Moreover, after in vitro culture, A. cearensis at a concentration of 0.1 mg/mL maintained the percentage of TUNEL positive cells (30.0%) in a way that is similar to that observed in the fresh control (22%) (P > 0.05). In conclusion, ovine preantral follicles can be preserved at 4°C in MEM for 6 h. For longer periods of preservation (24 h), MEM and 0.2 mg/mL A. cearensis are recommended. Moreover, after in vitro culture, A. cearensis extract (0.1 mg/mL) showed higher activation and lower DNA fragmentation in ovine preantral follicles.

scholarly journals Effect of the Duration of Liquid Nitrogen Storage on the Regrowth of Blackberry Cryopreserved by Droplet Vitrification

2017 ◽  
Vol 66 (1-2) ◽  
pp. 44-50
Author(s):  
Tatjana Vujović ◽  
Đurđina Ružić ◽  
Radosav Cerović
Keyword(s):  
Liquid Nitrogen ◽  
Room Temperature ◽  
Shoot Tips ◽  
Lower Survival Rate ◽  
Droplet Vitrification ◽  
Vitrification Solution ◽  
Long Term Preservation ◽  
Direct Immersion

SummaryIn vitro shoot tips of the blackberry cultivar ‘Čačanska Bestrna’ were cryopreserved using the droplet vitrification technique. Upon loading (30 min) in a solution of 1.9 M glycerol and 0.5 M sucrose, the explants were dehydrated for 40 min on ice with the PVS A3 vitrification solution (glycerol 37.5%, dimethyl sulfoxide 15%, ethylene glycol 15% and sucrose 22.5%) and for 40 min at room temperature with the PVS3 solution (glycerol 50% and sucrose 50%). They were subsequently frozen in individual microdroplets of vitrification solution, by direct immersion in liquid nitrogen (LN), and kept therein for 2, 4, 8 and 24 h. The explant rewarming was performed in an unloading solution (0.8 M sucrose) for 30 min at room temperature. The duration of LN exposure did not exert significant effects on the survival and regrowth of explants in both types of vitrification solutions. The survival and regrowth of cryopreserved shoot tips dehydrated with PVS3 solution ranged between 90–95% and 80–90%, respectively. However, dehydration with PVS A3 resulted in a lower survival rate (80–90%) and a considerably lower regrowth rate (55–65%) of explants. Monitoring the shoots regenerated in the in vitro culture revealed their normal capacity for multiplication and rooting in comparison with the controls, which fully confirms the purpose of cryopreservation in the long-term preservation of plant material.

scholarly journals Effects of Exposure to Tobacco Cigarette, Electronic Cigarette and Heated Tobacco Product on Adipocyte Survival and Differentiation In Vitro

Toxics ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 9 ◽  
Author(s):  
Zoi Zagoriti ◽  
Mohamed A. El Mubarak ◽  
Konstantinos Farsalinos ◽  
Stavros Topouzis
Keyword(s):  
Culture Medium ◽  
Electronic Cigarettes ◽  
Body Weight Loss ◽  
Tobacco Product ◽  
Dependent Manner ◽  
Ppar Γ ◽  
Beige Adipocytes ◽  
Thermogenic Adipocytes

Cigarette smoking (CS) causes significant morbidity worldwide, attributed to the numerous toxicants generated by tobacco combustion. Electronic cigarettes (ECIG) and heated tobacco products (HTP) are considered alternative smoking/vaping products that deliver nicotine through an inhaled aerosol and emit fewer harmful constituents than CS. However, their long-term impacts on human health are not well established. Nicotine exposure has been linked to lipolysis and body weight loss, while smoking has been associated with insulin resistance and hyperinsulinemia. Enhanced function of beige (thermogenic) adipocytes has been proposed as a means to reduce obesity and metabolic disorders. In this study, we compared the effect of extract-enriched media via exposure of culture medium to CS, HTP aerosol, and ECIG aerosol on the viability and the differentiation of 3T3-L1 pre-adipocytes to beige adipocytes. Only CS extract caused a decrease in cell viability in a dose- and time-dependent manner. Furthermore, relative lipid accumulation and expression levels of the adipocyte markers Pgc-1α, Ppar-γ and Resistin were significantly decreased in cells exposed to CS extract. Our results demonstrate that CS extract, in contrast to HTP and ECIG extracts, significantly impairs differentiation of pre-adipocytes to beige adipocytes and may therefore impact significantly adipose tissue metabolic function.

scholarly journals Cryopreservation of Pistacia vera embryonic axes

2016 ◽  
Vol 61 (No. 4) ◽  
pp. 182-187 ◽  
Author(s):  
B. Benmahioul ◽  
F. Daguin ◽  
M. Kaïd-Harche
Keyword(s):  
Germination Rate ◽  
Dry Weight ◽  
Pistacia Vera ◽  
Ambient Conditions ◽  
Embryo Viability ◽  
Embryonic Axes ◽  
Simple Protocol ◽  
Set Up

This preliminary study investigated the conservation of Pistacia vera genetic resources using seeds and isolated embryonic axes. First, the effect of storing seeds in ambient conditions on embryo viability was evaluated by in vitro culture. The germination rate of P. vera embryonic axes gradually decreased from 100% to 31% after 30-month storage of seeds. Cryopreservation may thus be necessary for the long-term conservation of embryos. A simple protocol was set up using embryonic axes. It included a single dehydration step with silica gel prior to direct freezing in liquid nitrogen (&ndash;196&deg;C). The optimal germination rate was obtained after 60 min dehydration (water content of 0.2 grams of water per gram of dry weight [g&middot;g<sup>&ndash;1</sup> DW]). However, 90 minutes of dehydration (0.14 g&middot;g<sup>&ndash;1</sup> DW) were necessary to obtain seedlings whose qualitative development was equivalent to that of the control embryonic axes.

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