scholarly journals Cultivar and Phenological Stage Effects on the Success of In Vitro Meristem Culture and GLRaV-3 Elimination of Croatian Autochthonous Grapevine Cultivars

Agronomy ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1395
Author(s):  
Zvjezdana Marković ◽  
Anđela Zrilić ◽  
Iva Šikuten ◽  
Petra Štambuk ◽  
Ivana Tomaz ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Viral Infections ◽  
Meristem Culture ◽  
Phenological Stages ◽  
Planting Material ◽  
In Vitro Establishment ◽  
Meristem Explants ◽  
High Degree

The population of Croatian autochthonous cultivars has a high degree of infection with economically important viruses, so it is necessary to carry out the elimination of the viruses in some cultivars to obtain healthy planting material. In this research, we tested in vitro meristem culture establishment on 18 autochthonous cultivars with different viral infections and the possibility of GLRaV-3 elimination through in vitro meristem culture. Plant material was sampled in a vineyard in two phenological stages, 10 days before flowering and 10 days after flowering of the grapevine. Apical meristem explants (1 mm) were placed into the MS culture medium supplemented with 0.5 mg/L benzyl adenine (BA) and 0.05 mg/L indol-3-acetic acid (IAA), and their survival, regeneration, and rooting were monitored. The results showed that the cultivar and the growth phase have a significant impact on the success of in vitro culture. In all cultivars studied higher success of in vitro culture establishment parameters (survival, regeneration, and rooting) was obtained in the case of explants sampled after flowering, with the exception of one cultivar for explants survival. Contrary to expectations, genotypes infected with three viruses (GLRaV-1, GLRaV-3, and GFLV) showed better results than genotypes infected with one or two viruses. The results showed successful in vitro establishment of Croatian autochthonous cultivar and GRLaV-3 elimination in one cultivar. However, due to the significant effect of cultivar, for routine application of this in vitro protocol on more than 100 autochthonous cultivars in need of sanitation, further studies should be conducted.

scholarly journals Rheological and Microstructural Features of Plant Culture Media Doped with Biopolymers: Influence on the Growth and Physiological Responses of In Vitro-Grown Shoots of Thymus lotocephalus

2021 ◽  
Vol 2 (2) ◽  
pp. 538-553
Author(s):  
Natacha Coelho ◽  
Alexandra Filipe ◽  
Bruno Medronho ◽  
Solange Magalhães ◽  
Carla Vitorino ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Average Pore Size ◽  
Physiological Responses ◽  
Gum Arabic ◽  
Shoot Elongation ◽  
Hydroxyethyl Cellulose ◽  
Plant Culture

In vitro culture is an important biotechnological tool in plant research and an appropriate culture media is a key for a successful plant development under in vitro conditions. The use of natural compounds to improve culture media has been growing and biopolymers are interesting alternatives to synthetic compounds due to their low toxicity, biodegradability, renewability, and availability. In the present study, different culture media containing one biopolymer (chitosan, gum arabic) or a biopolymer derivative [hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC)], at 100 or 1000 mg L−1, were tested regarding their influence on the growth and physiological responses of Thymus lotocephalus in vitro culture. Cellulose-based biopolymers (HEC and CMC) and gum arabic were used for the first time in plant culture media. The results showed that CMC at 100 mg L−1 significantly improved shoot elongation while chitosan, at the highest concentration, was detrimental to T. lotocephalus. Concerning only the evaluated physiological parameters, all tested biopolymers and biopolymer derivatives are safe to plants as there was no evidence of stress-induced changes on T. lotocephalus. The rheological and microstructural features of the culture media were assessed to understand how the biopolymers and biopolymer derivatives added to the culture medium could influence shoot growth. As expected, all media presented a gel-like behaviour with minor differences in the complex viscosity at the beginning of the culture period. Most media showed increased viscosity overtime. The surface area increased with the addition of biopolymers and biopolymer derivatives to the culture media and the average pore size was considerably lower for CMC at 100 mg L−1. The smaller pores of this medium might be related to a more efficient nutrients and water uptake by T. lotocephalus shoots, leading to a significant improvement in shoot elongation. In short, this study demonstrated that the different types of biopolymers and biopolymer derivatives added to culture medium can modify their microstructure and at the right concentrations, are harmless to T. lotocephalus shoots growing in vitro, and that CMC improves shoot length.

P–218 Analysis of the occurrence of microbial contamination in IVF culture system and the effect of microorganisms on embryo development and clinical outcomes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
F Du ◽  
R Li ◽  
Q Zhang ◽  
W Wang
Keyword(s):  
Escherichia Coli ◽  
In Vitro Culture ◽  
Clinical Outcomes ◽  
Embryo Transfer ◽  
Follicular Fluid ◽  
Culture System ◽  
Microbial Contamination ◽  
Culture Medium ◽  
Ivf Et

Abstract Study question what is the source, prevalence, and influence of microbial contamination on in vitro fertilization (IVF) and embryo transfer (ET) cycles? Summary answer Microbial contamination mainly occurs on Day 2, most caused by Escherichia coli carried with semen. ICSI could prevent contamination effectively and get good clinical outcomes. What is known already Microbial contamination occurs in IVF-ET system occasionally, which is hard to stop happening. The IVF culture system and laboratory environment, the patients’ follicular fluid and semen are not absolutely sterile, while the antibiotics in culture medium isn’t effective for all microbe types, and the artificial operations may bring in microbes. Generally, microbial contamination leads to degradation of embryos, reduction the number of embryos available, and infection of female reproductive tract, which would increase the cost of patients’ time, money, and bring psychological damages. A better understanding of embryo contamination in IVF culture system is of added value. Study design, size, duration A total of 29583 IVF-ET cycles were enrolled in this prospective observational study, from January 2010 to December 2020, included 70 microbial contamination cycles discovered in Day1-Day3 (D1-D3) of in vitro culture. Follicular fluid and semen saved on oocyte retrieval day, and culture medium contaminated were examined and identified for microorganisms at each contamination cycle. Participants/materials, setting, methods Compared the contamination rate of different insemination methods (IVF/ICSI/IVF+ICSI), different in vitro culture days (D1-D3), and different samples examination (follicular fluid, semen, culture medium) respectively, identified the source of microorganism types, compared the IVF culture outcomes and clinical outcomes between total contamination group (TC group, 42 cases) and partial contamination group (PC group, 28 cases). Main results and the role of chance A total of 70 microbial contamination cases occurred in 29583 oocyte retrieving cycles (0.24%), and it was observed only in IVF embryos but never in ICSI (Intracytoplasmic sperm injection) embryos. 38 contamination cases occurred on D2 with a highest ratio (54.3%) compared to D1 (32.9%) and D3(12.9%); Compared with follicular fluid, semen was the main cause inducing contamination from D1 to D3, and Escherichia coli in semen and culture medium, Enterococcus faecalis in follicular fluid proved to be the most common sources. Compared with TC group, the PC group showed a lower rate of No-available embryos (21.4% vs 81.0%) and a higher rate of blastocyst formation (41.2% vs 28.6%), In addition, the clinical pregnancy rate of PC group was higher than that of TC group in both fresh and frozen-thawed embryo transfer cycles (31.3% vs 16.7%, 38.5% vs 0.0%). Limitations, reasons for caution Further study is still necessary to better understand the sources that induce microbial contamination embryos, and more efficient methods are required to remove the microbes on these contaminated embryos so as better develop and manage a sterile micro-environment for successful embryo growth. Wider implications of the findings: The differential embryonic microbe types associated to different IVF culture and clinical outcomes in patients undergoing IVF-ET might have profound implications for understanding the microbial sources and making a better management of IVF culture system. Trial registration number Not applicable

Synthesis of RNA in oocytes of Xenopus laevis during culture in vitro

Development ◽  
1974 ◽  
Vol 32 (2) ◽  
pp. 515-532
Author(s):  
A. Colman
Keyword(s):  
Xenopus Laevis ◽  
In Vitro Culture ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Salt Medium ◽  
Complex Media ◽  
18S Ribosomal Rna ◽  
Rna Precursor

RNA synthesis can be maintained in large oocytes of Xenopus laevis during periods of in vitro culture of at least 10 days. A simple salt medium, modified Barth's solution, is found to be as effective a culture medium for these oocytes as several other complex media. The newly synthesized RNA is characterized electrophoretically and shown to consist predominantly of ribosomal RNA precursor, 28S and 18S ribosomal RNA, and 4S RNA. The distribution of this RNA within the oocyte is detected autoradiographically, where it is found to be greatly concentrated over the nucleoli. No qualitative alterations in either of these parameters are found during culture, within the limits of sensitivity of the assay procedures.

scholarly journals A new experimental in vitro culture medium for cultivation of Leishmania species.

1997 ◽  
Vol 35 (9) ◽  
pp. 2430-2431 ◽  
Author(s):  
M E Limoncu ◽  
I C Balcioğlu ◽  
K Yereli ◽  
Y Ozbel ◽  
A Ozbilgin
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Leishmania Species

scholarly journals Morphophysiological responses of Billbergia zebrina Lindl. (Bromeliaceae) in function of types and concentrations of carbohydrates during conventional in vitro culture

2020 ◽  
Vol 26 (1) ◽  
pp. 18-34 ◽  
Author(s):  
Elizangela Rodrigues Santos ◽  
João Paulo Rodrigues Martins ◽  
Luiz Carlos de Almeida Rodrigues ◽  
Andreia Barcelos Passos Lima Gontijo ◽  
Antelmo Ralph Falqueto
Keyword(s):  
Photosystem Ii ◽  
In Vitro Culture ◽  
Leaf Anatomy ◽  
Culture Medium ◽  
Stomatal Density ◽  
Photosynthetic Apparatus ◽  
Fluorescence Transient ◽  
Chlorophyll A Fluorescence Transient ◽  
Concentration Interval

Abstract When propagated in vitro, explants receive all the nutrients needed for their growth, including carbohydrates, from the culture medium. However, it is not well understood how the type and concentration of carbohydrates can affect the functioning of the photosynthetic apparatus (particularly photosystem II) of these plants. The aim was to assess the morphophysiological responses of Billbergia zebrina plants in function of sources and concentrations of carbohydrates during in vitro culture. Side shoots of plants previously established in vitro were individualized and transferred to a culture medium containing fructose, glucose or sucrose in four concentrations (0, 15, 30 or 45 g L−1). After growth for 55 days, the chlorophyll a fluorescence transient, leaf anatomy and growth were analyzed. The concentration and type of carbohydrate employed during in vitro culture did not decrease the photosynthetic apparatus performance. However, concentrations above 30 g L−1 led to anatomical modifications, revealing some degree of stress suffered by the plants. When grown in concentrations of 15 and 30 g L−1, irrespective of the carbohydrate used, the plants presented greater stomatal density. The supplementation of the culture medium with monosaccharides caused alterations in the development of the xylem vessels, such as increased number and diameter, allowing adjustment to the microenvironmental conditions. The in vitro conditions influenced the photosynthetic and anatomical responses of plants. The concentration interval from 15 to 30 g L−1 sucrose had a better effect by not causing large changes in the performance of the photosynthetic apparatus and anatomy of plants.

scholarly journals Clonal bamboo production based on in vitro culture

2020 ◽  
Vol 36 (4) ◽  
Author(s):  
Anatálya dos Santos Ribeiro ◽  
Alexssandra Jéssica Rondon de Figueiredo ◽  
Gabriela Cristina Rech Tormen ◽  
André Luís Lopes da Silva ◽  
Wellington Ferreira Campos ◽  
...  
Keyword(s):  
Activated Carbon ◽  
Large Scale ◽  
Culture Medium ◽  
Clonal Plant ◽  
Adventitious Rooting ◽  
Clonal Plants ◽  
Ex Vitro ◽  
Bambusa Vulgaris ◽  
In Vitro Establishment

Bamboo species are an alternative for the composition of forest plantations. However, their potential has not been explored due to the hard time in producing large-scale clonal plants. Thus, the aim this work was to evaluate the in vitro establishment, bud multiplication and ex vitro rooting of Bambusa vulgaris. The first experiment tested different systemic and contact fungicide solutions, based on exposure time, during the establishment phase. Established explants were subjected to evaluation of residual fungicide effect on subcultures during the multiplication and elongation phases. The second experiment evaluated the influence of activated carbon on ex vitro survival and on adventitious rooting. Explant immersion in liquid culture medium added with 1.0 mL of fungicide for 120 hours has favored the in vitro establishment and reduced fungal contamination. On the other hand, it favored the shoot emission of shoots per explant during the multiplication phase. Both rooting induction culture medium and mini-incubator system use were effective in enabling adventitious root formation. The presence of activated carbon in the rooting induction culture medium resulted in a higher clonal plant survival rate.  

160 COMPARATIVE STUDY OF IN VITRO CULTURE MEDIA AND ASSISTED HATCHING TECHNIQUES ON MOUSE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 188
Author(s):  
N. C. Negota ◽  
L. P. Nethenzheni ◽  
M. L. Mphaphathi ◽  
D. M. Barry ◽  
T. L. Nedambale
Keyword(s):  
In Vitro Culture ◽  
Chorionic Gonadotropin ◽  
Culture Medium ◽  
Culture Media ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Significant Difference ◽  
Equine Chorionic Gonadotropin ◽  
F1 Generation

The in vitro culture media and assisted hatching techniques remain challenging obstacles to be utilised widely. Mechanical, chemical, enzymatic thinning, and laser-assisted techniques have been used previously but information is still lacking on its application in livestock. The aim of this study was to compare the effect of 2 in vitro culture media (Hamster F10 and TMC-199) and 4 (mechanical, chemical, enzymatic, and laser) assisted hatching techniques on blastocyst formation and hatching rate using murine embryos as a model. The C57/b and Balb/c breeds were raised until they reached maturity and bred naturally to produce F1 generation. The light in the breeding house was controlled at 14 h light and 10 h dark. Feed and water were provided ad libitum for the mice. Superovulation of females were stimulated using equine chorionic gonadotropin and human chorionic gonadotropin. The F1 generation was used for the collection of the 400 blastocysts and randomly allocated into 4 assisted hatching techniques. Blastocysts were paired into a group of 10 and replicated 4 times for each assisted hatching technique. The general linear model of SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) was used to analyse the data. Assisted hatching techniques of laser, mechanical, enzymatic, and chemical yielded 46.9 ± 37.1, 51.1 ± 40.2, 39.1 ± 35.8, and 33.3 ± 4.5%, respectively, under in vitro culture of Hamster F10. The TCM-199, laser, mechanical, enzymatic, and chemical assisted hatching techniques yielded 56.3 ± 43.3, 52.6 ± 35.5, 49.2 ± 37.5, and 33.9 ± 35.5%, respectively, with a significant difference. There was no significant difference observed in assisted hatching techniques and Hamster F10 culture medium. However, the hatching rate of embryos for all techniques was higher when in vitro cultured in TCM than cultured in Hamster F10. Hatching rate of blastocysts increased from chemical, enzymatic, mechanical, and laser with response to Hamster F10 and TCM; thus, laser is a suitable assisted hatching technique with TCM-199.

scholarly journals In vitro establishment of Bambusa oldhamii Munro from field-grown matrices and molecular identification of endophytic bactéria

2019 ◽  
Vol 49 ◽  
Author(s):  
Ana Paula de Azevedo Pasqualini ◽  
Gabriela Xavier Schneider ◽  
Hugo Pacheco de Freitas Fraga ◽  
Luiz Antonio Biasi ◽  
Marguerite Quoirin
Keyword(s):  
Molecular Identification ◽  
Culture Medium ◽  
Bacterial Isolate ◽  
Fungal Growth ◽  
Endophytic Bacterium ◽  
Liquid Culture Medium ◽  
Rdna Sequencing ◽  
In Vitro Establishment ◽  
Bambusa Oldhamii

ABSTRACT In plant micropropagation, the establishment stage is difficult, due to the presence of microorganisms in tissues from field-grown matrices, especially for bamboo. This study aimed to establish an efficient asepsis protocol for Bambusa oldhamii explants from field plants, as well as to carry out the molecular identification of a possible endophytic bacterial isolate. The explants were exposed to 70 % alcohol, 1 % sodium hypochlorite (NaOCl), 0.1 % mercuric chloride (HgCl2), thiophanate-methyl (Cercobin®) and chlorhexidine digluconate (2 % Riohex®) in different combinations, and introduced into Murashige and Skoog culture medium (solid or liquid), supplemented or not with 4 mL L-1 of Plant Preservative Mixture (PPMTM), totaling seven treatments. The asepsis and immersion of the explants in the liquid culture medium containing 4 mL L-1 of PPMTM visually inhibited the bacterial and fungal growth, allowed the development of shoots with a mean length of 2.2 cm and posterior subcultures, being the best treatment used for the in vitro establishment of B. oldhamii. The molecular identification of an endophytic bacterium performed by 16S rDNA sequencing allowed to identify the bacterial isolate as Ralstonia sp., with 100 % of similarity, and the phylogenetic analysis grouped it with Ralstonia pickettii. In addition, the bacterial isolate showed to be sensitive to 4 mL L-1 of PPMTM by the minimum inhibitory concentration test.

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