Arabidopsis thaliana ecotype Columbia-0 was used for all experiments. Seeds were surface-sterilized (20% bleach, 0.5%SDS), rinsed with sterile water and sown on half-strength Murashige & Skoog medium including vitamins (Duchefa Biochemie), containing 0,1% MES monohydrate buffer (Duchefa Biochemie) and 1% v/w Daishin agar (Duchefa Biochemie). Salts were added from a 1 mM stock in the desired concentration before autoclaving. The pH of all media was set at 5.8 using 0.1 N KOH to prevent any harmful acidification effects as described by Babayigit et al. (Babayigit et al., 2016a (link)) Seeds were sown on petri dishes (9 cm diameter) containing 20 mL medium, stratified (4°C, dark) for 4 days to synchronize germination, and afterwards placed in a climate chamber (16 hours light period, 22°C, photosynthetic active radiation 150 μmol photons/m2/s).
Murashige skoog medium
Manufactured by Duchefa Biochemie
Sourced in Netherlands
Murashige & Skoog medium is a formulation of inorganic salts, vitamins, and organic compounds designed to support the growth and development of plant tissue cultures. It provides a balanced nutrient solution for optimal plant cell, tissue, and organ cultivation.
Automatically generated - may contain errors
Lab products found in correlation
Sucrose, by Duchefa Biochemie (2 mentions)
Daishin agar, by Duchefa Biochemie (1 mentions)
Gelrite, by Duchefa Biochemie (1 mentions)
Inositol, by Merck Group (1 mentions)
1 naphthaleneacetic acid, by Merck Group (1 mentions)
2 4 dichlorophenoxyacetic acid, by Merck Group (1 mentions)
Kinetin, by Merck Group (1 mentions)
Organic solvents, by Merck Group (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (1 mentions)
Plant agar, by Duchefa Biochemie (1 mentions)
Cd3od, by Deutero (1 mentions)
Elx800, by Agilent Technologies (1 mentions)
Eclipse e200 light microscope, by Nikon (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
6 benzyladenine, by Duchefa Biochemie (1 mentions)
Zebularine, by Abcam (1 mentions)
Sucrose, by Solarbio (1 mentions)
13 protocols using murashige skoog medium
1
Arabidopsis Growth Conditions Optimization
2
Arabidopsis Genetic Manipulation Protocol
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All experiments were carried out with Arabidopsis thaliana ecotype Col-0. The T-DNA insertion lines tpst-1 (SALK_009847) and the double knock out line pskr1-3 pskr2-1 were described previously (Komori et al., 2009; Kutschmar et al., 2009; Stührwohldt et al., 2011) . The triple knockout line tpst-1 pskr1-3 pskr2-1 was generated by crossing tpst-1 with pskr1-3 pskr2-1. Loss of all three transcripts was verified by semi-quantitative RT-PCR. Seeds were surface-sterilized in 2% (v/v) sodium hypochlorite for 15 min, washed five times with autoclaved water and subsequently laid out on 0.5 x Murashige-Skoog medium (Duchefa, Harlem, Netherlands), 1.5% (w/v) sucrose, solidified with 0.4% (w/v) Gelrite (Duchefa Harlem, Netherlands). If indicated, media were supplemented with 1 µM PSK (Pepscan, Lelystad, Netherlands). For growth on soil, plants were grown in a 2:3 sand:humus mixture that (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. was frozen at -80°C for 2 days to avoid insect contamination and watered regularly with tap water. After two days of stratification at 4°C in darkness, plants were transferred to long-day conditions (16 h light with 70 µM photons m -2 s -1 , 8 h dark) at 21-22°C and 60% humidity for the times indicated.
3
Cultivation of Arabidopsis Dark Cell Cultures
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Plant System Biology Dark type (PSB-D) cell suspension cultures (A. thaliana ecotype Landsberg erecta) were maintained in 50 mL Murashige & Skoog medium (4.43 g/L; Duchefa Biochemie, Netherlands), 30 g/L sucrose, 0.5 mg/L naphthalene acetic acid, 0.05 mg/L kinetin; adjusted to pH 5.7 with 1 M KOH) at 27 °C under gentle agitation (130 rpm) in the dark. Cells were subcultured in fresh medium at a 1/10 dilution every seven days.
4
Arabidopsis thaliana Root Growth Analysis
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For analysis of root growth, Arabidopsis thaliana seeds were grown in axenic conditions on 12x12cm square plates containing 60 ml agar-solidified medium. Seed were surface sterilized either by vapour sterilization, or by washing with 1 ml of 70% (v/v) ethanol and 0.05% (v/v) Triton X-100 with gentle mixing by inversion for 6 minutes at room temperature, followed by 1 wash with 96% ethanol and 5 washes with sterile distilled water. For primary root length and lateral root density plants were grown in Cambridge and Leeds on plates containing ATS medium [68 (link)] supplemented with 1% sucrose (w/v) and solidified with 0.8% ATS. For measurements of skewing, waving, cell length, root diameter, root hair density and root hair length, seedlings were grown in Munich on plates containing 0.5X Murashige & Skoog medium, pH5.8 (½ MS) (Duchefa, Netherlands), supplemented with 1% sucrose and solidified with 1.5% agar. Plates were stratified at 4°C for 2–3 days in the dark, and then transferred to a growth cabinet under controlled conditions at 22°C, 16-h/8-h light/dark cycle (intensity ~120 μmol m-2 s-1). Unless otherwise indicated, the plates were placed vertically.
5
Cell Culture Protocols for Arabidopsis and Tobacco
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Arabidopsis (Arabidopsis thaliana cv. Landsberg erecta) cell line (Ath-Ler) [61 (link)] was grown in sterile Murashige-Skoog medium (4.4 g·L−1, pH 5.8; Duchefa Biochemie, Haarlem, The Netherlands) supplemented with vitamins, 3% sucrose, 0.232 µM kinetin and 5.37 µM 1-naphthaleneacetic acid (Merck Life Science, Darmstadt, Germany). Tobacco (Nicotiana tabacum cv. Bright Yellow 2) cell line (BY-2) [62 (link)] was grown in sterile Murashige-Skoog medium (4.3 g·L−1 pH 5.8) supplemented with 3% sucrose, 4 µM thiamin, 555 µM inositol, 1.47 mM KH2PO4, and 0.9 µM 2,4-dichlorophenoxyacetic acid (Merck Life Science, Darmstadt, Germany). Both cell lines were subcultured weekly into fresh media in a volume ratio of 1:10. The cells were cultivated at 23 °C in the dark and shaken at 120 rpm. Five-day-old cells were used for all experiments. As a stock material, cell calli of both cell lines were cultivated on the same solidified media (1.2% agar) and subcultured monthly.
6
Germination and Growth of Arabidopsis Mutants
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The T-DNA mutant srr1–1 in the Col-7 background has been described [1 (link), 37 (link)]. All seeds were stratified for 3 d at 4 °C before putting on soil. Seeds grown on plates were surface sterilized and stratified for 3 d at 4 °C before plating on agar-solidified half-strength MS (Murashige & Skoog) medium (Duchefa) supplemented with 0.5% sucrose and 0.5 g MES. Plants were grown in Percival incubators AR66-L3 (CLF Laboratories) in 100 μmol m− 2 s− 1 light intensity, with the light-dark and temperature conditions as indicated.
7
Arabidopsis thaliana MM2d Cell Culture
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Arabidopsis thaliana MM2d cell line (17 (link)) was grown at 26 °C and 120 rpm, in the absence of light. The cells were subcultured every 7 days into fresh Murashige & Skoog medium (MS, pH 5.8, Duchefa) supplemented with 3% sucrose (Duchefa), 0.5 μg/ml 1-naphthaleneacetic acid (Duchefa), 0.1 μg/ml kinetin (Sigma) and 0.103 μg/ml vitamins (Duchefa).
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Arabidopsis thaliana MM2d cell line (17 (link)) was grown at 26 °C and 120 rpm, in the absence of light. The cells were subcultured every 7 days into fresh Murashige & Skoog medium (MS, pH 5.8, Duchefa) supplemented with 3% sucrose (Duchefa), 0.5 μg/ml 1-naphthaleneacetic acid (Duchefa), 0.1 μg/ml kinetin (Sigma) and 0.103 μg/ml vitamins (Duchefa).
8
In Vitro Cultivation of Plant Cells
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Murashige & Skoog medium, plant agar, and sucrose used for in vitro cultivation were purchased from Duchefa Biochemie (Haarlem, The Netherlands). The organic solvents used for extraction, the reagents, and the standards used for the determination of total phenols and flavonoids, as well as the DPPH, ABTS, and FRAP free radicals were purchased from Sigma-Aldrich (Madrid, Spain). CD3OD and D2O came from Deutero GmbH (Kastellaun, Germany). The sensitized red sheep erythrocytes, hemolysin and the guinea pig serum were purchased from BulBio (Sofia, Bulgaria).
9
Quantifying Tomato Cell Viability
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Tomato cell suspensions were grown in Murashige-Skoog medium (Duchefa, Haarlem, The Netherlands) supplemented with 5.4 M naphthalene acetic acid, 1 M 6-benzyladenine, and vitamins (Duchefa) at 24°C with continuous agitation in darkness as described by Laxalt et al. (2007) (link). Tomato cell viability was assayed by Evans blue staining (Sukenik et al., 2018 ). The cells were incubated with 10 and 100 μg/ml of NMPC at 30°C for 24 h in darkness. As a positive control, cells were treated with 1% Triton X-100 (v/v). Twenty-five μl of 1% Evans blue solution were added to 50 μl of treated-suspension cells, incubated at room temperature for 5 min, and observed under Eclipse E200 light microscope (Nikon). The extent of dye uptake by dead cells was quantified spectrophotometrically by incubating 250 μl of each suspension with 150 μl 1% Evans blue for 5 min at room temperature. Unbound Evans blue stain was removed by washing four times with 0.1 M Tris-HCl pH 7.5. Cells were collected by centrifuging at 800 rpm for 15 s and lysed with 250 μl 100% dimethyl sulfoxide (Sigma-Aldrich) at 100°C for 15 min. The absorbance at 595 nm was measured by using a microplate reader ELx800 (BioTek, Winooski, VT, USA).
10
Arabidopsis Growth and Phenotypic Analysis
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The growth of Arabidopsis plants on media has been described previously [44 (link)]. In brief, the seeds were surface sterilized in two steps: 0.1% (v/v) Triton X-100 for 1 h followed by 70% ethanol for 1 min. The excess ethanol was removed by 5X washing with sterile distilled water. The seeds were spread on MS (Murashige-Skoog) medium (Duchefa Biochemie) supplemented with 1% (w/v) sucrose and 0.8% (w/v) phytoagar and stratified for 3 d at 4°C. The plates were maintained in long-day growth conditions of 16 h/8 h light-dark cycle at 22°C. Plants in soil were maintained using similar growth conditions in a walk-in growth chamber.
The phenotypic analysis was performed by photographing seedlings grown vertically on MS containing 1% (w/v) sucrose and adult plants grown in soil. A Canon EOS 7D camera was used for photography, while the series of images were processed in ImageJ (NIH, USA) for the root length and rosette diameter measurements.
The phenotypic analysis was performed by photographing seedlings grown vertically on MS containing 1% (w/v) sucrose and adult plants grown in soil. A Canon EOS 7D camera was used for photography, while the series of images were processed in ImageJ (NIH, USA) for the root length and rosette diameter measurements.
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