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

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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Effects of a commercial biocide, kasugamycin and consistency of the culture medium on the in vitro establishment of bamboo1

Pesquisa Agropecuária Tropical ◽

10.1590/1983-40632019v4955435 ◽

2019 ◽

Vol 49 ◽

Author(s):

Daniela Werner Ribeiro dos Santos ◽

Théo Piucco Rocker ◽

Thiago Sanches Ornellas ◽

Miguel Pedro Guerra

Keyword(s):

Liquid Culture ◽

Culture Medium ◽

Liquid Culture Medium ◽

Bambusa Vulgaris ◽

Phyllostachys Bambusoides ◽

In Vitro Establishment ◽

Dendrocalamus Asper

ABSTRACT The contamination by microorganisms and oxidation of explants in the in vitro establishment of bamboo are recurrent problems for its micropropagation. In the present study, effects of the biocide Plant Preservative Mixture (PPM™), the antibiotic kasugamycin and the consistency of the culture medium were evaluated in the in vitro establishment of Bambusa vulgaris,Phyllostachys bambusoides and Dendrocalamus asper. The presence of PPM™ in the culture medium had a significant effect using 2 mL L-1 or 4 mL L-1 concentrations, as well as in the liquid culture medium, increasing the plants established in the autumn. Kasugamycin promoted variable responses for all the three species, depending on the season. There was interaction among the factors, demonstrating that higher rates of viable plants can be obtained by combining different strategies to reduce the oxidation and contamination. For the in vitro establishment of B. vulgaris,P. bambusoides and D. asper, it is recommended to add 2 mL L-1 or 4 mL L-1 of PPM™ to the liquid culture medium.

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In vitro culture and diversity of endophytic fungi in Bambusa oldhamii

Pesquisa Agropecuária Tropical ◽

10.1590/1983-40632019v4953760 ◽

2019 ◽

Vol 49 ◽

Cited By ~ 1

Author(s):

Ana Paula de Azevedo Pasqualini ◽

Mariely Cristine dos Santos ◽

Bruno Francisco Sant´Anna-Santos ◽

Hugo Pacheco de Freitas Fraga ◽

Marguerite Quoirin

Keyword(s):

In Vitro Culture ◽

Molecular Identification ◽

Endophytic Fungi ◽

Large Scale ◽

Culture Medium ◽

Current Knowledge ◽

Nodal Segments ◽

Scale Production ◽

Bambusa Oldhamii

ABSTRACT Bambusa oldhamii Munro is a fast-growing species of woody bamboo with strong commercial appeal. In Brazil, the use of this species is limited, mainly due to the low availability of seedlings for commercial plantations. Micropropagation is a technique used for the large scale production of seedlings, but protocols for the establishment of aseptic cultures are hampered by the presence of endophytic contamination. This study aimed to develop an in vitro establishment protocol for B. oldhamii, as well as to make the molecular identification of fungi associated with the explants used. Nodal segments of adult plants grown in the field were used as explants. This material was submitted to two experiments carried out to evaluate the effect of 6-benzylaminopurine (BAP) on multiplication and of Plant Preservative Mixture (PPMTM) as a disinfectant. In the first one, 10 µM, 15 µM or 20 µM of BAP were combined with 1 mL L-1, 2 mL L-1 or 3 mL L-1 of PPMTM; while the second one used 0 µM, 2.5 µM, 5 µM or 7.5 µM of BAP with 4 mL L-1 of PPMTM, both added to MS culture medium. After 21 days of culture, the use of 4 mL L-1 of PPMTM inhibited the bacterial growth and reduced fungal contamination. The addition of BAP to the culture medium above 10 µM inhibited the formation and growth of new shoots, while additions of less than 7,5 µM had no effect. The molecular identification of the endophytic fungi isolated during the in vitro culture indicated the presence of numerous fungal species, increasing the current knowledge about the diversity of fungi associated with bamboo.

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Clonal bamboo production based on in vitro culture

Bioscience Journal ◽

10.14393/bj-v36n4a2020-48169 ◽

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.

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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

GSC Biological and Pharmaceutical Sciences ◽

10.30574/gscbps.2021.16.2.0221 ◽

2021 ◽

Vol 16 (2) ◽

pp. 001-013

Author(s):

Abwe Mercy Ngone ◽

Lawrence Monah Ndam ◽

Rita Mungfu Njilar ◽

Doungous Oumar ◽

Thomas Eku Njock

Keyword(s):

Culture Medium ◽

Culture Media ◽

Fungal Growth ◽

Growth Media ◽

Fungal Contamination ◽

Surface Sterilization ◽

Pure Cultures ◽

Nodal Cuttings ◽

Baseline Information

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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Fate of Safening Agent L-2-Oxothiazolidine-4-Carboxylic Acid in Sorghum (Sorghum bicolor)

Weed Science ◽

10.1017/s004317450005640x ◽

1990 ◽

Vol 38 (3) ◽

pp. 201-205 ◽

Cited By ~ 6

Author(s):

James L. Hilton ◽

Parthasarathy Pillai ◽

Helen A. Norman

Keyword(s):

Carboxylic Acid ◽

Sorghum Bicolor ◽

Culture Medium ◽

In Vitro Assay ◽

Thiol Compounds ◽

Vitro Assay ◽

Herbicide Safener ◽

Liquid Culture Medium ◽

Germinating Seeds

The herbicide safener OTC (L-2-oxothiazolidine-4-carboxylic acid) increased the amount of reduced thiol compounds in sorghum [Sorghum bicolor(L.) Moench. ‘DK 427′] seedlings. When seedlings were grown in liquid culture medium containing35S-OTC, the compound was metabolized to radiolabeled cysteine and glutathione. The addition of tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane] increased conversion of35S-OTC to cysteine and resulted in the formation of one additional35S-labeled compound. When35S-glutathione was injected into germinating seeds it was converted to35S-cysteine and both thiols were subsequently found in roots and shoots. Seeds injected with35S-OTC both translocated the compound to developing roots and shoots and metabolized35S-OTC to cysteine and glutathione. Excised roots and shoots also metabolized35S-OTC to the thiols. In an in vitro assay the enzyme 5-oxoprolinase converted OTC to cysteine.

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EFFECT OF 6- BENZYLAMINOPURINE AND THE CULTURE MEDIUM MS (1962) ON THE IN VITRO ESTABLISHMENT OF Prosopis pallida (WILLD.) KUNTH

REBIOL ◽

10.17268/rebiol.2019.39.02.03 ◽

2019 ◽

Vol 39 (2) ◽

pp. 30-40

Author(s):

Diego Campos ◽

Claudia Chávez ◽

Segundo Lopéz ◽

Armando Gil- Rivero ◽

Angélica Lopéz -Zavaleta ◽

...

Keyword(s):

Culture Medium ◽

In Vitro Establishment

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Influence of genotype on protein and oil concentration of soybean seeds

Brazilian Archives of Biology and Technology ◽

10.1590/s1516-89132010000400007 ◽

2010 ◽

Vol 53 (4) ◽

pp. 793-799 ◽

Cited By ~ 3

Author(s):

Esmael Lopes dos Santos ◽

Antonio Eduardo Pípolo ◽

Ricardo Tadeu de Faria ◽

Cássio Egidio Cavenaghi Prete

Keyword(s):

Protein Concentration ◽

Culture Medium ◽

Seed Oil ◽

Soybean Seeds ◽

Glutamine Concentration ◽

Liquid Culture Medium ◽

Immature Seeds ◽

Oil Concentration

Aiming at evaluating genotype influence on the concentration of protein and oil, immature seeds of cultivars CD 202 and CD 206 were removed from the mother-plant, in the stage R5, and were grown in vitro, in a liquid culture medium which contained 20, 40 and 60 mM of glutamine, during eight days. Afterwards, the concentrations of oil and protein were compared to the contents of the seeds cultivated in vivo. With a higher availability of glutamine for the seed, there was an increase of protein content. The genotypes were statistically different as far as the protein concentration was concerned,which confirmed that the genotype had influence on the concentration of protein in the seed. Oil and protein concentrations were inversely related when a variation of glutamine concentration occurred.

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Early studies on the effect of peptide growth factor phytosulfokine-α on Brassica oleracea var. capitata L. protoplasts

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.3558 ◽

2017 ◽

Vol 86 (3) ◽

Cited By ~ 3

Author(s):

Agnieszka Kiełkowska ◽

Adela Adamus

Keyword(s):

Brassica Oleracea ◽

Shoot Regeneration ◽

Liquid Culture ◽

Culture Medium ◽

Breeding Lines ◽

Liquid Culture Medium ◽

Peptide Growth Factor ◽

The Media ◽

Cells Cultured

Phytosulfokines (PSK) are peptidyl growth factors with the potential of inducing cell proliferation. We examined the effect of supplementation of liquid culture medium with 0.1 µM phytosulfokine-α (PSK-α) on protoplast viability and division frequencies in seven accessions of Brassica oleracea var. capitata L., including cultivars and breeding lines. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants and immobilized in calcium-alginate layers. Cabbage protoplast-derived cells cultured in medium supplemented with 0.1 µM of PSK-α had higher viability and division frequencies compared to cells cultured in PSK-α-free control medium. The effect of PSK-α was more pronounced in low-responding accessions (‘Sława z Gołębiewa’, ‘Ramkila F1’, LM, and LM98); however, in two cultivars with very low response (‘Badger Shipper’ and ‘Oregon 123’), although the division frequencies in the media supplemented with PSK-α were increased over the control, the differences were not significant. Obtained callus colonies were subjected to regeneration. PSK-α supplemented into the liquid culture medium had an indirect effect on shoot regeneration by inducing sustained cell divisions leading to an increase in shoot regeneration in Sława z Gołębiewa and both breeding lines.

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Evaluation of different antioxidants during in vitro establishment of allspice (Pimenta dioica L. Merrill): a recalcitrant species

10.21203/rs.3.rs-21190/v1 ◽

2020 ◽

Author(s):

Sugey Vásquez-Hernández ◽

Carlos Alberto Cruz-Cruz ◽

Maricela Santiago-Santiago ◽

Jericó Jabín Bello-Bello

Keyword(s):

Lipid Peroxidation ◽

Methylene Blue ◽

Cell Wall ◽

Antioxidant Capacity ◽

Culture Medium ◽

Control Treatment ◽

Soluble Phenols ◽

In Vitro Establishment ◽

Pimenta Dioica

Abstract Background: Pimenta dioica L. Merrill is a tree whose fruits are used as a spice due to their culinary and therapeutic uses. Conventional propagation techniques using seeds and cuttings do not guarantee the phytosanitary quality of this crop. Therefore, the use of Plant Tissue Culture techniques are an option for in vitro establishment. The aim of this study was to evaluate the effect of different antioxidant agents (Methylene blue, L-cysteine and silver nanoparticles) added to MS (Murashige and Skoog) culture medium at different concentrations (0, 50, 100 and 200 mg L-1) during axenic establishment of buds used as explants of P. dioica. Results: The percentage of survival, oxidation and contamination was determined, as well as the content of soluble phenols, cell wall-linked phenols, antioxidant capacity and lipid peroxidation. Results showed significant differences among the different antioxidants for the evaluated variables; the highest survival occurred in the treatments with the addition of L-cysteine with percentages greater than 40 %, while the lowest survival occurred in the control treatment, 100 and 200 mg L-1 methylene blue, with 0, 3.3 and 0% survival, respectively. The highest percentage of oxidation was observed in the control treatments, 100 and 200 mg L-1 methylene blue with 96.67% oxidation, while the lowest percentages were observed in explants treated with L-cysteine, with 30% oxidation. Treatments with 100 and 200 mg L-1 AgNPs had the lowest contamination values, with 20%. Biochemical determinations showed that L-cysteine and 50 and 100 mgL-1 AgNPs resulted in an increase in the content of soluble phenols. The highest contents of cell wall-linked phenols were obtained in treatments with 200 mg L-1 methylene blue, L-cysteine, and 200 mg L-1 AgNPs. Analysis of antioxidant capacity revealed that all treatments had a reaction of scavenging / reduction mechanisms free radicals. Regarding lipid peroxidation, the highest content of malondialdehydes was observed in the control treatment and 200 mgL-1 methylene blue. Conclusion: the addition of L-cysteine to the culture medium showed a higher survival rate, decreased oxidation, greater production of phenolic compounds, increased antioxidant capacity and decreased lipid peroxidation, this amino acid being an alternative to reduce oxidation during in vitro introduction of allspice and other species that exhibit recalcitrance in vitro during establishment.

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In vitro pollen culture and the regeneration of Brassica campestris L. plants

Agricultural and Food Science ◽

10.23986/afsci.72627 ◽

1995 ◽

Vol 4 (5-6) ◽

pp. 513-518

Author(s):

Yang-Dong Guo ◽

Seppo Pulli

Keyword(s):

Culture Medium ◽

Brassica Campestris ◽

Higher Plants ◽

Oilseed Crop ◽

Pollen Culture ◽

Culture Techniques ◽

Liquid Culture Medium ◽

Embryo Yield ◽

Colchicine Solution

Brassica campestris (Brassica rapa L. ssp. oleifera) is an important oilseed crop, particularly in Finland. Pollen culture techniques for haploid production have been developed, but B. campestris is relatively recalcitrant in pollen culture. Twenty eight genotypes of B. campestris were included in this study. The donor plants were grown in the greenhouse and transferred to the growth cabinet before bolting. Buds (2-4 mm long) were selected, macerated in B 5 medium, then NLN liquid culture medium was added. The microspores were incubated in the dark at 32°C for 72 h, then at 25°C for a further three weeks. Nineteen genotypes produced microspore-derived embryos. The highest yield was more than 300 embryos per 100 buds. Activated charcoal (150 mg/L) promoted embryogenesis, pollen development was faster and the embryo yield was higher. Plants were regenerated after transferring embryos to a solid B 5 medium. Colchicine solution was used to double the chromosome complements. About 100 regenerate plants have been obtained in our laboratory, and these haploids will be useful for the oilcrop breeding.

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