E30 led growth chamber

All plants were of the Col-0 ecotype and were grown at 22C. Prior to starting germination on plates or soil, seeds were cold-stratified in a 4C walk-in cooler in the dark for 2–3 days. Plants grown in cWL were grown in a Conviron ATC13 growth chamber or a custom walk-in growth chamber. Plants in single wavelength light experiments were grown in a Percival E30-LED growth chamber after initial exposures to 4 hours of white light to induce germination. Plants used in flowering-time experiments, rosette diameter measurements, and GUS staining were grown in a previously-described soil mixture [51 (link)]. All other plants were grown on 0.8–2% agar plates supplemented with Gamborg B-5 Basal Medium (PhytoTechnology Laboratories, product #G398). Nicotiana benthamiana plants were grown under long-day conditions (16h light/8h dark) at 22C in a Conviron ATC13 growth chamber. For UV experiments, plants were grown in cWL for 5 days under a Mylar filter (Professional Plastics, catalog #A736990500) or mock-filter.

All conidial inocula were harvested from glucose minimal medium (GMM) plates containing 1% glucose and ammonium tartrate as a nitrogen source, as described in reference 48 (link). RPMI 1640 agar contained, per liter, 10.4 g RPMI 1640 powder (Sigma; R6504-10L), 34.53 g morpholinepropanesulfonic acid (MOPS), 18 g glucose, and 15 g Bacto agar; pH was adjusted to 6.9 to 7.1. YG broth contained 2% glucose and 0.5% yeast extract.
All incubators were placed in a dark room, and “dark” samples were isolated with all lights off with the aid of a near-infrared imaging goggle. For white light-treated cultures, plates or flasks (as indicated) were irradiated under cool fluorescent light bulbs (General Electric F20T12-CW) emitting light over a broad spectrum from 400 to 700 nm; total light intensity was ~40 µmol/photon/m²/s. For blue and red light treatments, plates were incubated in an E-30LED growth chamber equipped with blue and red light-emitting diodes (Percival Scientific, Inc., Perry, IA). All incubations were performed at 37°C.

For DIC, roots were cleared by the chloral hydrate method as described by Inagaki et al. [41 (link)].
To measure root length and fresh weight of shoots, seedlings were germinated and grown on vertical MS plates under long day condition with different concentrations of tZ (Sigma, http://www.sigmaaldrich.com), thidiazuron (TDZ, Sigma), isopentenyladenine (iP, Sigma), 6-benzyladenine (6-BA, Sigma) or kinetin (KT, Sangon, http://www.sangon.com) at 7 DAG.
For hypocotyl inhibition assays, seedlings were germinated and grown on vertical MS plates in darkness with different concentrations of CKs, ACC (Sigma), IAA (Sigma) and GA3 (Sigma) at 5 DAG.
For adventitious root formation assays and callus induction assays, the methods were performed according to [42 (link)].
For light-induced analysis, all experiments involving blue, red, or far-red light illumination were performed under 16-hr L/8-hr D light conditions in an E-30 LED growth chamber (Percival, Boone, IA, http://www.percival-scientific.com) with the blue (k_max 469 nm, PPFD = 50 μmol m⁻² s⁻¹), red (k_max 680 nm, PPFD = 50 μmol m⁻² s⁻¹), or the far-red (k_max 730 nm, PPFD = 50 μmol m⁻² s⁻¹) diodes at 22 °C.
For Toluidine blue-O staining, plants were immersed for 5 min in 0.05% Toluidine blue-O (Sigma) and rinsed with water at room temperature.