Dry seeds were surface sterilized using 50% bleach with 0.01% (v/v) Triton X-100 for 7 mins and washed five times with sterile water. Then the sterilized seeds were placed on 1/2 MS medium plates containing 0.8% (w/v) Phytoblend Agar (Caisson Labs) supplemented with 0.01% (v/v) ethanol as mock or 2μM ABA (Sigma-Aldrich, dissolved in 100% ethanol) as treatment. In total, 180 seeds (60 per replicate) were used for each genotype. All plates with seeds were placed at 4°C in the dark for 3 days and then transferred to long day condition (16h light / 8h dark, 22°C) for further analysis. The germination event was defined as the emergence of the radicle, and germinated seeds were counted every 12 h until 120h after stratification.
Phytoblend agar
Manufactured by Caisson
Sourced in United States
Phytoblend agar is a solidifying agent commonly used in plant tissue culture media. It is a blend of agar and other gelling agents that provide a stable, transparent matrix for the growth and development of plant cells, tissues, and organs.
Automatically generated - may contain errors
Lab products found in correlation
Methyl ja meja, by Merck Group (2 mentions)
Linsmaier and skoog medium, by Caisson (1 mentions)
Sucrose, by Thermo Fisher Scientific (1 mentions)
Murashige and skoog basal salts, by Caisson (1 mentions)
Cell culture dishes, by Corning (1 mentions)
Li 250a light meter, by LI COR (1 mentions)
Petri plates, by Thermo Fisher Scientific (1 mentions)
Sucrose, by Caisson (1 mentions)
Vitamins, by PhytoTechnology Laboratories (1 mentions)
9 protocols using phytoblend agar
1
Seed Germination Assay with ABA
2
Brassinosteroid response of Arabidopsis T-DNA mutants
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T-DNA insertion mutants, rd26 (At4g27410, SALK_063576), anac019 (At1g52890, SALK_096295), anac055 (At3g15500, SALK_014331) and anac102 (At5g63790, SALK_030702) were obtained from ABRC (Arabidopsis Biological Resource Center). All plants were grown on 1/2MS plates and/or in soil under long day conditions (16 h light/8 h dark) at 22 °C. BRZ and BL response experiments were carried out as previous described59 (link). Briefly, seeds were sterilized with 70% ethanol and 0.1% Triton X-100 for 15 min and washed with 100% ethanol three times and dried in filter papers in a sterile hood. The seeds were sprinkled onto half Linsmaier and Skoog medium (Caisson Lab) with 0.7% Phytoblend agar (Caisson Lab) and various concentrations of BRZ (provided by Professor Tadao Asami) or BL (Wako Biochemical). Both BRZ and BL (1 mM stock in dimethylsulphoxide) were added to medium after autoclave and the plates with seeds were placed at 4 °C for 3 days. After exposing to light for 8 h, the plates were wrapped with three layers of aluminium foil and incubated in the dark at 25 °C for 5 days for BRZ response and in the constant light for 7 days for BL response experiments. Hypocotyls were scanned and measured using Image J (https://imagej.nih.gov/ij/ ). Ten to fifteen hypocotyls were measured, and averages and s.d. were calculated and plotted.
3
Sterilized Tomato Seed Cultivation
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Cultivated tomato S. lycopersicum cv. M82 seeds were obtained from the C.M. Rick Tomato Genetic Resource Center (https://tgrc.ucdavis.edu/ ; RRID:SCR_014954). Unless otherwise noted, plants were grown under sterile conditions. Tomato seeds were surface sterilized in 40% bleach for 20 min with gentle rocking, rinsed five times with sterile water, and then sown in a straight line on Falcon 150 mm–by–25 mm cell culture dishes (Corning Inc., Corning, NY, USA) containing half-strength Murashige and Skoog basal salts (Caisson Labs, Smithfield, UT, USA), supplemented with Gamborg’s B-5 vitamins (Caisson Labs, Smithfield, UT, USA), 3% sucrose (Thermo Fisher Scientific Inc., Waltham, MA, USA), and 0.7% Phytoblend agar (Caisson Labs, Smithfield, UT, USA) adjusted to pH 5.8. The plates were wrapped in Micropore surgical tape (3M Company Inc., Maplewood, MN, USA), then oriented vertically in steel alloy 4.25″ by 4.5″ by 13.5″ plate racks (Spectrum Diversified Designs LLC, Solon, OH, USA), and stratified in the dark at 25°C for 5 to 7 days to germinate seeds. Thereafter, vertically oriented plates were transferred to a growth chamber and grown under a 12-hour diurnal cycle, 25°C/18°C, 175 μE light intensity.
4
Seedling Growth of Transgenic BVR and Phytochrome Mutants
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Transgenic BVR lines, i.e., 35S::pBVR3 (Montgomery et al., 1999 (link)) and CAB3::pBVR2 (Warnasooriya and Montgomery, 2009 (link)), and T-DNA insertion mutants, i.e., phyA (Mayfield et al., 2007 (link); Ruckle et al., 2007 (link)), phyB (Mayfield et al., 2007 (link); Ruckle et al., 2007 (link)), and double mutant phyAphyB (Oh and Montgomery, 2013 (link)), were previously described. Sterilized seeds were planted and seedlings grown on MS medium containing 1% (w/v) sucrose and 0.7% (w/v) Phytoblend agar (Caisson Labs, UT) at 22°C for 7 days under the indicated light condition as previously described (Oh and Montgomery, 2013 (link)). Light sources utilized for far-red (FR; λ max ~735 nm), red (R; λmax ~670 nm), and white (W) light were described previously (Warnasooriya and Montgomery, 2009 (link)). Fluence rates of R, and W were measured using a LI-250A Light Meter (LI-COR) connected to a LI-COR quantum sensor and for FR using a StellarNet EPP2000 spectroradiometer (Apogee Instruments).
5
Root Growth Inhibition Assays
6
Root Growth Inhibition Assay Protocol
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Root growth inhibition assays (Shyu et al., 2012 ) were performed with seedlings grown on Petri plates (Thermo Fisher Scientific, Waltham, MA, USA) containing LS medium (0.5× Linsmaier and Skoog (Caisson Labs, Smithfield, UT, USA), 0.7% w/v phytoblend agar (Caisson Labs) and 0.8% w/v sucrose) supplemented with the concentration of methyl‐JA (MeJA; Sigma‐Aldrich) indicated in the legends to Figs 1 and 2 . Primary root length of WT and mutant lines (grown on the same plate) was determined in 8‐ to 11‐d‐old seedlings using imagej software (http://imagej.nih.gov/ij/ ). Growth parameters, including leaf dry weight, leaf area, petiole length, rosette diameter, and flowering time, were determined as described previously (Campos et al., 2016 ).
7
Arabidopsis Mutant Growth Assay
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Arabidopsis thaliana wild type (WT, ecotype Columbia-0) seedlings were used in the research. The T-DNA insertion mutants AT5G41330 (SALK_114523C), AT3G09030 (SALK_101331), and AT2G24240 (CS825825) were obtained from the Arabidopsis Biological Resource Center. The positions of T-DNA insertion sites are shown in supporting information Figure S2A . Homozygous mutant plants were screened and identified by PCR using the primers listed in Table S1 . Lines of double and triple mutants were constructed by genetic crosses.
For on-plate growth assays, the Arabidopsis seeds were sterilized with 75% ethanol for 5 min, washed three times with sterilized water and then sown on 1/2 MS supplement with 1% (w/v) sucrose and 0.8% (w/v) phytoblend agar (Caisson Labs, Smithfield, UT, USA). The pH was 5.8. The seeds were stratified at 4 °C for two days and were then placed in a growth chamber (16-h illumination of 150 μmol/m2/s, and 8-h dark cycle) at 22 °C. For soil culture, 10-day-old seedlings on 1/2 MS were transferred to nutrient-rich soil (Pindstrup Mosebrug, Denmark) and then grown in a greenhouse with a long-day cycle (16-h illumination of 150 μmol/m2/s, and 8-h dark cycle) at 22 °C.
For on-plate growth assays, the Arabidopsis seeds were sterilized with 75% ethanol for 5 min, washed three times with sterilized water and then sown on 1/2 MS supplement with 1% (w/v) sucrose and 0.8% (w/v) phytoblend agar (Caisson Labs, Smithfield, UT, USA). The pH was 5.8. The seeds were stratified at 4 °C for two days and were then placed in a growth chamber (16-h illumination of 150 μmol/m2/s, and 8-h dark cycle) at 22 °C. For soil culture, 10-day-old seedlings on 1/2 MS were transferred to nutrient-rich soil (Pindstrup Mosebrug, Denmark) and then grown in a greenhouse with a long-day cycle (16-h illumination of 150 μmol/m2/s, and 8-h dark cycle) at 22 °C.
8
Abiotic Stress Response in Arabidopsis
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Arabidopsis WT seeds were surface-sterilized as described in [43 (link)]. The sterilized seeds were germinated and grown on a nylon screen placed on top of ½ strength solid MS media with vitamins (PhytoTechnology Laboratories, Lenexa, KS, USA), supplemented with 0.8% (w/v) Phytoblend agar (Caisson Laboratories, Smithfield, UT, USA), and 1% (w/v) sucrose, in Petri dishes for two weeks. Arabidopsis seedlings were exposed to 100 mM D-mannitol, 100 mM NaCl, and 2.5 μM ABA in liquid culture. Plant shoot and root samples were harvested separately at 0, 6, 12, and 24 h time intervals; washed with deionized water, flash frozen in liquid nitrogen, and stored at −80 °C until RNA extraction and qRT-PCR analyses.
9
Moss Protonema Growth Conditions
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The Gransden WT strain [53] was used along with the Ppccd8 mutant [6] (link). Moss protonema were grown on PP-NO3 medium [54] (link) for phenotypic observation, and on PP-NO3 medium supplemented with 2.7 mM NH4-tartrate for propagation. Plants were cultivated either in 9 cm round (for the light experiments) or 12 cm square (for the dark experiments) Petri dishes, on medium solidified with 0.7% agar (Vitro Agar, KALYS SA, France) and overlaid with cellophane (Cannings, Bristol, UK). For dark conditions, 0.5% glucose was added to the medium, 1% Phytoblend agar (Caisson, USA) was used, and Petri dishes were positioned vertically for better observation of caulonema growth. For the light experiments, cultures were placed in growth chambers set at 60% humidity, and with 16 h of light (quantum irradiance of 80 µE m−2 s−1) at 24°C and 8 hours of dark at 22°C.
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