Effective medium for in vitro sprouting of the buds and multiplication of the plantlets, and identification of the fungal contaminant associated with the explants of Gnetum

Plant tissue culture requires the optimization of growth media. Gnetum, known locally in Cameroon as “Eru” is an indigenous gymnospermous vegetable with diverse medicinal, nutritional, cultural and socio-economic values. This resource is over-exploited and expected to neighboring countries, resulting to increased scarcity in the forest. Preliminary work on the in vitro culture of nodal cuttings was faced by the problem of fungal contamination. It was therefore necessary to isolate and identify the fungal contaminant, optimize the surface sterilization of field material and compose an appropriate medium for sprouting. Pure cultures of the fungus were obtained and grown on Potato Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA). The identification was based on the appearance of the fungal growth on plates and also on the microscopic view. This was affected by the use of keys. Gnetum explants were disinfected with the various concentrations of disinfectants, preceded in some instances by pre-treatments, as well as incorporating fungicides in the culture medium. Two different culture media were employed: the Woody Plant Medium (WPM) and the Murashige and Skoog (MS) based establishment medium (Y-1). Gnetum was found to live in association with a complex of Microsporum species. The level of contamination of cultures was reduced from 100% to 40% when pre-treated before disinfection and even lower to 10% by incorporating fungicides in the medium. Sprouting was observed in WPM. This study provides baseline information on the in vitro propagation of Gnetum and thus opens up avenues for more research to be carried out in this field.

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In vitro culture and non-invasive metabolic profiling of single bovine embryos

Reproduction Fertility and Development ◽

10.1071/rd17446 ◽

2019 ◽

Vol 31 (2) ◽

pp. 306

Author(s):

Monika Nõmm ◽

Rando Porosk ◽

Pille Pärn ◽

Kalle Kilk ◽

Ursel Soomets ◽

...

Keyword(s):

Molecular Weight ◽

Metabolic Profiling ◽

Culture Medium ◽

Culture Media ◽

Blastocyst Stage ◽

Growth Media ◽

Low Molecular Weight ◽

Bovine Embryos ◽

Non Invasive

Selecting high-quality embryos for transfer has been a difficult task when producing bovine embryos invitro. The most used non-invasive method is based on visual observation. Molecular characterisation of embryo growth media has been proposed as a complementary method. In this study we demonstrate a culture medium sampling method for identifying potential embryonic viability markers to predict normal or abnormal embryonic development. During single embryo culture, 20µL culture media was removed at Days 2, 5 and 8 after fertilisation from the same droplet (60µL). In all, 58 samples were analysed using liquid chromatography–mass spectrometry. We demonstrate that it is possible to remove samples from the same culture medium droplets and not significantly affect blastocyst rate (25.2%). Changes in any single low molecular weight compound were not predictive enough. Combining multiple low molecular weight signals made it possible to predict Day 2 and 5 embryo development to the blastocyst stage with an accuracy of 64%. Elevated concentrations of lysophosphatidylethanolamines (m/z=453, 566, 588) in the culture media of Day 8 well-developing embryos were observed. Choline (104m/z) and citrate (215m/z) concentrations were increased in embryos in which development was retarded. Metabolic profiling provides possibilities to identify well-developing embryos before transfer, thus improving pregnancy rates and the number of calves born.

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Very Low Ethanol Concentrations Affect the Viability and Growth Recovery in Post-Stationary-Phase Staphylococcus aureus Populations

Applied and Environmental Microbiology ◽

10.1128/aem.72.4.2627-2636.2006 ◽

2006 ◽

Vol 72 (4) ◽

pp. 2627-2636 ◽

Cited By ~ 20

Author(s):

Indranil Chatterjee ◽

Greg A. Somerville ◽

Christine Heilmann ◽

Hans-Georg Sahl ◽

Hans H. Maurer ◽

...

Keyword(s):

Amino Acids ◽

Staphylococcus Aureus ◽

Amino Acid ◽

Stationary Phase ◽

Culture Medium ◽

Culture Media ◽

Growth Media ◽

Cell Integrity ◽

Amino Acid Depletion

ABSTRACT Pharmaceuticals, culture media used for in vitro diagnostics and research, human body fluids, and environments can retain very low ethanol concentrations (VLEC) (≤0.1%, vol/vol). In contrast to the well-established effects of elevated ethanol concentrations on bacteria, little is known about the consequences of exposure to VLEC. We supplemented growth media for Staphylococcus aureus strain DSM20231 with VLEC (VLEC+ conditions) and determined ultramorphology, growth, and viability compared to those with unsupplemented media (VLEC− conditions) for prolonged culture times (up to 8 days). VLEC+-grown late-stationary-phase S. aureus displayed extensive alterations of cell integrity as shown by scanning electron microscopy. Surprisingly, while ethanol in the medium was completely metabolized during exponential phase, a profound delay of S. aureus post-stationary-phase recovery (>48 h) was observed. Concomitantly, under VLEC+ conditions, the concentration of acetate in the culture medium remained elevated while that of ammonia was reduced, contributing to an acidic culture medium and suggesting decreased amino acid catabolism. Interestingly, amino acid depletion was not uniformly affected: under VLEC+ conditions, glutamic acid, ornithine, and proline remained in the culture medium while the uptake of other amino acids was not affected. Supplementation with arginine, but not with other amino acids, was able to restore post-stationary-phase growth and viability. Taken together, these data demonstrate that VLEC have profound effects on the recovery of S. aureus even after ethanol depletion and delay the transition from primary to secondary metabolite catabolism. These data also suggest that the concentration of ethanol needed for bacteriostatic control of S. aureus is lower than that previously reported.

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Pertumbuhan Dan Perkembangan Anggrek Dendrobium anosmum Pada Media Kultur In Vitro Dengan Beberapa Konsentrasi Air Kelapa

Agrologia ◽

10.30598/a.v1i1.293 ◽

2018 ◽

Vol 1 (1) ◽

Author(s):

S. Tuhuteru ◽

Meity L Hehanussa ◽

Simon H.T Raharjo

Keyword(s):

Culture Medium ◽

Culture Media ◽

Water Concentration ◽

Medium Composition ◽

Coconut Water ◽

Orchid Species ◽

Randomized Design ◽

Wet Weight ◽

Completely Randomized Design

Dendrobium anosmum is one of natural orchids in Indonesia. Optimization of medium composition for orchid propagation through in vitro culture is necessary to enhance propagule multiplication capabilities and quality. This study was aimed to study the influence of concentration of coconut water in culture medium on in vitro growth and development of D. anosmum orchid species and to determine the optimal coconut water concentration in culture media. The experiment were arranged in a Completely Randomized Design with four treatments and eight replications. The treatments consisted of the addition of coconut water with concentrations: 0 ml•l -1 (control), 50 ml•l-1, 100 ml•l-1 and 150 ml•l-1. The results showed that addition of coconut water in culture medium gave different effect on shoot growth and multiplication of D. anosmum orchids. Coconut water concentration of 100 ml•l-1 was the best concentration for growth and multiplication of D. anosmum orchids, based on both shoots and roots growth, plantlet height and wet weight.

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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

Polysaccharides ◽

10.3390/polysaccharides2020032 ◽

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.

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Biochemical markers for pregnancy in the spent culture medium of in vitro produced bovine embryos

Biology of Reproduction ◽

10.1093/biolre/ioab095 ◽

2021 ◽

Author(s):

Gabriela de Oliveira Fernandes ◽

Marcella Pecora Milazzotto ◽

Andrei Antonioni Guedes Fidelis ◽

Taynan Stonoga Kawamoto ◽

Ligiane de Oliveira Leme ◽

...

Keyword(s):

Mass Spectrometry ◽

Biochemical Markers ◽

Culture Medium ◽

Culture Media ◽

Ionization Mass ◽

Metabolic Profiles ◽

Bovine Embryos ◽

The Media

Abstract The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]+, 444.33 Da [M + H]+, and 305.97 Da [M + H]+ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.

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In vitro culture as an aid to conservation of indigenous ferns: Diplazium proliferum

International Journal of Plant Biology ◽

10.4081/pb.2015.6020 ◽

2015 ◽

Vol 6 (1) ◽

Author(s):

Zubeir M. Golamaully ◽

Vishwakalyan Bhoyroo ◽

Nadeem Nazurally ◽

Vineshwar Gopal

Keyword(s):

Growth Rate ◽

In Vitro Culture ◽

Mercuric Chloride ◽

Rare Species ◽

Vascular Plants ◽

Culture Media ◽

Fungal Contamination ◽

Increase Growth Rate ◽

Growing Population

With the ever growing population and economic needs of Mauritius, the flora of Mauritius has never been in more danger and one group of vascular plants is even more in peril; ferns. Diplazium proliferum is indigenous to the Mascarene region and is considered as a rare species in Mauritius. The need to develop a tested in vitro propagation protocol is a must to protect the biodiversity of Mauritius. This experiment was geared towards the establishment of a proper sterilization technique and the effect of 6-benzylaminopurine (BAP) and light on in vitro culture of this fern. Sterilization with 0.05% Mercuric chloride was effective to eliminate fungal contamination and allow germination of spores. Culture media supplemented with BAP did not significantly increase growth rate of both gametophytes and sporophytes of D. proliferum. Present results suggest efficient sterilization methods to be a crucial stage for successful in vitro regeneration of ferns. The established protocol will be used as an optimized baseline protocol for the propagation of other indigenous ferns.

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MODIFICAÇÕES NO MEIO DE CULTURA, FOTOPERÍODO E TEMPO DE CULTIVO AFETAM O ALONGAMENTO E ENRAIZAMENTO IN VITRO DE BANANEIRA CV. PACOVAN

Nativa ◽

10.31413/nativa.v6i1.4731 ◽

2018 ◽

Vol 6 (1) ◽

pp. 27

Author(s):

Marcos Vinícius Marques Pinheiro ◽

Ana Cristina Portugal Pinto De Carvalho ◽

Fabrina Bolzan Martins

Keyword(s):

Plant Height ◽

Culture Medium ◽

Culture Media ◽

Dry Weight ◽

Salt Mixture ◽

Musa Spp ◽

Fresh Biomass ◽

Shoot Dry Weight ◽

Number Of Leaves

No intuito de elevar as taxas de sobrevivência durante a etapa de aclimatização e posterior plantio a campo, avaliou-se o enraizamento in vitro de bananeira cv. Pacovan, em diferentes concentrações de sais MS e de sacarose. Utilizou-se DIC, esquema fatorial (6x2x3), com seis meios de cultura [sendo três concentrações de nutrientes do meio MS (100%; 50% de macronutrientes; e 50% dos sais macro e micronutrientes), e duas concentrações de sacarose (1,5/3,0%)], dois fotoperíodos (12/16 h) e três tempos de cultivo (21, 28 ou 35 dias) e seis repetições/tratamento. Analisaram-se: altura da planta, número de folhas/planta, massas frescas e secas das partes aérea e radicular. Para altura da planta, massa fresca da parte aérea e radicular, o meio MS 50% dos sais + sacarose (1,5%) com fotoperíodo de 16 h e tempo de cultivo de 35 dias foi satisfatório. Para massa seca da parte aérea foi MS 50% de sais + sacarose (3%), e para massa seca da parte radicular, MS 100% + sacarose (3%) (em 12hs/28 dias e 16hs/21 dias). Para o alongamento/enraizamento in vitro da bananeira cv. Pacovan sugere-se MS 50% de sais (macro e micronutrientes), redução ou manutenção de sacarose (1,5 ou 3%) em 16h/35 dias de cultivo.Palavra-chave: Musa spp., propagação in vitro, sistema radicular. CHANGES IN CULTURE MEDIUM, PHOTOPERIOD AND TIME OF CULTIVATION AFFECT THE IN VITRO ELONGATION AND ROOTING OF BANANA CV. PACOVAN ABSTRACT:In order to achieve high rates of survival during the acclimatization and later planting in the field, was evaluated the in vitro of banana cv. Pacovan plants under different concentrations of sucrose and MS basal salt mixture. The experiment was assembled in a DIC, in 6x2x3, six different culture media [three different MS salt mixture concentrations (100%; 50% of macronutrients; and 50% of macro/micronutrients) and two sucrose concentrations (1.5/3%)], two photoperiods (12/16 hours) and three cultivation times (21, 28 or 35 days). Each treatment was composed by 6 replicates. Plant height, number of leaves/plant, fresh and dry weight of roots and shoots, were analyzed. Satisfactory results for plant height and shoot and root fresh biomass were observed in MS with macro/micronutrients (50%) + sucrose (3%), 16 hours/35 days. The highest values of shoot dry weight were observed in MS with macro/micronutrients (50%) + sucrose (3%); the highest root dry weight was achieved with MS 100% + sucrose (3%) (12hs/28 and 16hs/21 days). The suggested medium for the in vitro elongation and rooting stage of banana cv. Pacovan is the MS with 50% of salts (macro and micronutrients), reduction or maintenance of sucrose (1.5 or 3%) in 16h/35 days of cultivation.Keywords: Musa spp., in vitro propagation, root system. DOI:

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In vitro answer of Bulgarian pepper (Capsicum annuum L.)

Genetika ◽

10.2298/gensr0602129r ◽

2006 ◽

Vol 38 (2) ◽

pp. 129-136

Author(s):

Velichka Rodeva ◽

Stanislava Grozeva ◽

Velichka Todorova

Keyword(s):

Culture Medium ◽

Culture Media ◽

Casein Hydrolysate ◽

Basal Medium ◽

Regeneration Ability ◽

Stem Explants ◽

Media Composition ◽

Breeding Lines ◽

High Level

Callusogenesis and regeneration ability of cotyledon and hypocotyl explants from three Bulgarian pepper varieties in MS basal medium supplemented with l-3mg/l BAP. l.0mg/1 IAA and 0.5mg/l GA3 was studied. In the different variants of culture medium was registered high level of callusogenesis and organogenesis in both type of explants from the all varieties. The highest percentage of plant-regenerants is established in cotyledon explants (from 3.3 to 18.3) in variant 3 of the culture medium containing 3mg/l BA. In the process of micropropagation by stem explants of the same studied pepper varieties the addition of the vitamins C. B12. Casein hydrolysate and Ferulic acid had a stimulation effect on the plant growth in height and rooting. In result of anther cultivation from three pepper varieties and four breeding lines the highest percentage of embryo structure formation was registered in varieties Albena and Strjama (12.0 and 13.8 respectively). The Bulgarian peppers are recalcitrant and their in vitro answer is different depending from the explants type, genotype and the culture media composition.

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Selection of filamentous fungi that are resistant to the herbicides atrazine, glyphosate and pendimethalin

Acta Scientiarum Agronomy ◽

10.4025/actasciagron.v43i1.51656 ◽

2021 ◽

Vol 43 ◽

pp. e51656

Author(s):

Nara Priscila Barbosa Bravim ◽

Anatércia Ferreira Alves ◽

José Fábio França Orlanda ◽

Patricia Barbosa Rodrigues Silva

Keyword(s):

Filamentous Fungi ◽

Mycelial Growth ◽

Culture Medium ◽

Agricultural Soils ◽

Culture Media ◽

Fusarium Verticillioides ◽

Fungal Growth ◽

Relative Growth ◽

Maximum Inhibition ◽

Selection Of

The objective of the present study was to isolate fungi from agricultural soils and evaluate fungal growth in culture medium contaminated with atrazine, glyphosate and pendimethalin. Filamentous fungi were isolated from agricultural soils and cultured in a modified culture medium containing 0, 10, 20, 50, and 100 μg mL-1 atrazine, glyphosate and pendimethalin for 14 days at 28°C. The fungi that presented optimal and satisfactory growth were plated in Sabouraud culture medium with 4% dextrose and containing the herbicides at concentrations of 0, 10, 20, 50, and 100 μg mL-1 for seven days at 28°C. The mean mycelial growth values were submitted to analysis of variance and the Tukey test (p < 0.05%) for comparison and relative growth determination, and maximum inhibition rates were calculated. The isolated fungi Aspergillus fumigatus, Fusarium verticillioides and Penicillium citrinum were shown to be resistant to atrazine, glyphosate and pendimethalin. F. verticillioides showed higher mean mycelial growth in the culture media contaminated with atrazine and glyphosate than the other two fungi. In the culture medium contaminated with pendimethalin, F. verticillioides, and A. fumigatus presented the highest mean mycelial growth values.

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In vitro growth of Cattleya nobilior Rchb. f.: culture media, sealing systems and irradiance

Pesquisa Agropecuária Tropical ◽

10.1590/1983-40632021v5167131 ◽

2021 ◽

Vol 51 ◽

Author(s):

Kaliana Gottschalk de Freitas ◽

José Carlos Sorgato ◽

Jackeline Schultz Soares ◽

Luan Marlon Ribeiro

Keyword(s):

Plant Height ◽

Natural Ventilation ◽

Culture Medium ◽

Culture Media ◽

Ventilation System ◽

Light Condition ◽

Leaf Length ◽

In Vitro Growth ◽

Randomized Design

ABSTRACT Some factors may influence the in vitro culture of native orchids, especially the culture medium, sealing system and light condition. This study aimed to evaluate the addition of banana pulp and peel in the formulation of culture media, sealing system and irradiance, in the in vitro growth of Cattleya nobilior Rchb. f. A completely randomized design was used in a factorial scheme with five culture media [Murashige & Skoog supplemented or not with banana pulp or peel (100 or 200g L-1)], two light conditions [3,000 K LED lamps (43 µmol m-2 s-1 or 86 µmol m-2 s-1)] and two sealing systems (natural and conventional ventilation), with six replicates of one vial each. The plant height, diameter of the largest pseudobulb, length of the largest root and of the largest leaf, fresh weight and number of leaves, roots and shoots were evaluated. The use of both the banana pulp and peel as additives in the culture medium formulation promoted the C. nobilior growth. The natural ventilation system increased the plant height, pseudobulb diameter and leaf length, while the conventional system promoted tillering.

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