Arabidopsis thaliana ecotype Columbia (Col), Wassilewskija (Ws), and qrt1-2 (CS8846; Col-3) were used as the wild type. The gex1-1 (CS817261; Col-3) and gex1-2 (FLAG_484E09; Ws) mutant alleles have been described by Alandete-Saez et al. (2011) (link). The seeds were surface sterilized and sown on soil or Murashige and Skoog (MS) medium (Fuji Film Wako, Osaka, Japan) containing 0.7% agar and 1% sucrose. The plants were grown at 22°C under continuous light.
Ms medium
Manufactured by Fujifilm
Sourced in Japan
MS medium is a type of tissue culture media developed by Toshio Murashige and Folke Skoog in 1962. It is a commonly used growth medium for plant cell and tissue cultures, providing essential nutrients for plant cell proliferation and differentiation.
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7 protocols using ms medium
1
Arabidopsis thaliana Ecotypes and Mutants
2
Rice Cultivar Disease Resistance Screening
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Rice (Oryza sativa L.) wild-type (WT) cultivar ‘Nipponbare’, Xoo-resistant cultivar ‘Asominori’, RSV-resistant cultivar ‘Sainokagayaki’ and two transgenic plant lines (BSR1-OX-5 and -9) were grown under greenhouse conditions at 27°C to 30°C. BSR1-OX-5 and BSR1-OX-9 correspond to the previously reported AK070024:OX-5 and AK070024:OX-9 (Dubouzet et al. 2011 (link)), respectively.
For disease resistance tests, except for B. glumae, dehusked seeds were surface sterilized, sown on one-half-strength MS medium (Wako Pure Chemicals, Osaka, Japan), containing 3% (w/v) sucrose and 0.4% (w/v) Gelrite (Wako Pure Chemicals), in Agripots and grown in the growth chamber at 28°C in the dark for 3 days, then at 25°C under long-day conditions (16 h light [60–70 μmol m−2 s−1]/8 h dark) for 4–7 days. For transgenic seeds, Hygromycin B (30–50 μg/ml; Wako Pure Chemicals) was added to the medium. WT seedlings and hygromycin-resistant transgenic seedlings were transplanted into soil (Bonsol No. 2, Sumitomo Kagaku Kougyo, Osaka, Japan) and used for disease resistance tests.
For disease resistance tests, except for B. glumae, dehusked seeds were surface sterilized, sown on one-half-strength MS medium (Wako Pure Chemicals, Osaka, Japan), containing 3% (w/v) sucrose and 0.4% (w/v) Gelrite (Wako Pure Chemicals), in Agripots and grown in the growth chamber at 28°C in the dark for 3 days, then at 25°C under long-day conditions (16 h light [60–70 μmol m−2 s−1]/8 h dark) for 4–7 days. For transgenic seeds, Hygromycin B (30–50 μg/ml; Wako Pure Chemicals) was added to the medium. WT seedlings and hygromycin-resistant transgenic seedlings were transplanted into soil (Bonsol No. 2, Sumitomo Kagaku Kougyo, Osaka, Japan) and used for disease resistance tests.
3
Establishing Transgenic Rice Seedlings for Functional Analysis
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Rice (Oryza sativa L. cv. Nipponbare) was used as the WT control. Seeds from T1 to T4 of the POsUbi7-BSR1 and PPR1b-BSR1 lines were sown on half-strength MS medium (Wako, Osaka, Japan) containing 3% sucrose, 0.4% Gelrite (Wako), and hygromycin B (30 μg/mL; Wako), and the hygromycin-resistant seedlings were selected on this medium. WT seeds were sown and grown on the same medium without hygromycin B. WT and hygromycin-resistant transgenic seedlings were then transplanted into pots containing soil (Bonsol no. 2; Sumitomo Kagaku Kougyo, Osaka, Japan) and grown in a greenhouse at 27–30 °C, as previously described [19 (link)].
4
Arabidopsis Seed Germination and Growth
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The WT plant used in this study was Columbia-0 (Col-0), and all of the other mutants were based on the Col-0 background. Seeds of ibr1–2, ibr3–1, ibr10–1, pen3–4, and ech2–1 were a gift from Professor Bonnie Bartel (Rice University). icl–2, mls–2, and pck1–2 mutants seeds were a gift from Professor Ian Graham (The University of York).
Seeds were sown on rockwool (Nippon Rockwool Corporation), watered daily with 0.5 g L-1 Hyponex solution and grown under a 16/8 h light/dark cycle with white light from fluorescent lamps at approximately 50 μmol m-2 s-1 at 22°C.
Sterilized seeds were sown on MS medium (Wako Pure Chemical) or on MS medium with 2% (w/v) Suc where indicated, and solidified using 0.2–0.4% (w/v) gellan gum to determine the effects of medium composition on plant phenotype. After sowing the seeds, the MS plates were stored at 4°C in the dark for 3 d. After cold treatment, the seedlings were grown either in the light (for the cellular phenotype analyses) or in the dark (for Suc or TAG quantification) for the designated periods of time.
Seeds were sown on rockwool (Nippon Rockwool Corporation), watered daily with 0.5 g L-1 Hyponex solution and grown under a 16/8 h light/dark cycle with white light from fluorescent lamps at approximately 50 μmol m-2 s-1 at 22°C.
Sterilized seeds were sown on MS medium (Wako Pure Chemical) or on MS medium with 2% (w/v) Suc where indicated, and solidified using 0.2–0.4% (w/v) gellan gum to determine the effects of medium composition on plant phenotype. After sowing the seeds, the MS plates were stored at 4°C in the dark for 3 d. After cold treatment, the seedlings were grown either in the light (for the cellular phenotype analyses) or in the dark (for Suc or TAG quantification) for the designated periods of time.
5
Arabidopsis Growth and Phenotyping Protocol
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The WT used in this study was Arabidopsis Columbia-0 (Col-0) and all mutants had the Col-0 background. The SALK T-DNA homozygous knockout line (SALK_005342c) for At1g75730 was ordered from the Arabidopsis Biological Resource Center (Columbus, OH, USA). ech2-1 mutant allele seeds were a kind gift from Prof. Bonnie Bartel (Department of Biochemistry and Cell Biology, Rice University). Seeds were sown on rockwool (Nitto Boseki, Tokyo, Japan), watered daily with 0.5 g L–1 Hyponex solution (Hyponex Tokyo, Japan), and grown under a 16/8 h light/dark cycle with white light fluorescent lamps at approximately 50 μmol m–2 s–1 at 22°C. Sterilized seeds were sown on MS medium (Wako Pure Chemical, Osaka, Japan) or MS medium with 2% (w/v) sucrose where indicated and solidified using 0.2–0.5% (w/v) gellan gum (Murashige and Skoog, 1962 (link)) to determine the effect of medium composition on growth. After sowing the seeds, the MS plates were stored at 4°C in the dark for 3 days. After cold treatment, the seedlings were grown either under light (for cellular phenotype analysis) or in the dark (for analysis of etiolated seedlings) for designated periods of time.
6
Growth Conditions of Arabidopsis thaliana
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Arabidopsis thaliana ecotype Columbia was used in this study. The atg2-1 (SALK_076727) mutant was obtained from the ABRC. Seeds were sown on one-half MS medium (Wako Pure Chemical Industries, Ltd., Japan) supplemented with 1.5% (w/v) sucrose and stored at 4 °C for more than 2 days. After vernalization, plants were grown for 2 weeks on one-half MS agar medium and were then transferred to soil. Plants were cultured for 40–50 days. All growth occurred under a 16:8-h light/dark cycle at 23 °C in a growth chamber.
7
Growth of Arabidopsis thaliana ecotype C24
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Plants of Arabidopsis thaliana ecotype C24 obtained from the Arabidopsis Biological Resource Center (ABRC; https://abrc.osu.edu/) were used as the wild type (WT).
AlSRK-b+AlSCR-b A. thaliana transformants have been described previously [34] . Seeds were sterilized with 20% bleach and sown on Murashige and Skoog (MS) medium (Wako, Osaka, Japan) containing 0.7% (w/v) agar and 1% (w/v) sucrose. All A. thaliana plants were grown at a constant temperature of 23°C under long-day photoperiod (16 h light/8 h dark)
and 100 µmol photons m -2 s -1 light intensity.
AlSRK-b+AlSCR-b A. thaliana transformants have been described previously [34] . Seeds were sterilized with 20% bleach and sown on Murashige and Skoog (MS) medium (Wako, Osaka, Japan) containing 0.7% (w/v) agar and 1% (w/v) sucrose. All A. thaliana plants were grown at a constant temperature of 23°C under long-day photoperiod (16 h light/8 h dark)
and 100 µmol photons m -2 s -1 light intensity.
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