Agar type e

The control medium (MS-0) consisted of Murashige & Skoog [23 (link)] (MS; M524 Phytotechnology Laboratories, KS, USA) nutrients, 3% sucrose, 0.8% (w/v) type E agar (Sigma Aldrich, St. Louis, MO), and pH adjusted to 5.7 using 1 M NaOH and 1 M HCl [12 (link)]. The callus induction medium (hereafter referred to as LT-C) consisted of MS (M524; Phytotechnology Laboratories) nutrients, 3% sucrose, 0.8% (w/v) type E agar (Sigma Aldrich), 0.5 μM NAA (Sigma Aldrich), and 1.0 μM TDZ (Caisson Laboratories, Inc., Smithfield, UT) adjusted to a pH of 5.7 [12 (link)]. The shoot induction medium (hereafter referred to as LT-S) consisted of MS (M524; Phytotechnology Laboratories) nutrients, 3% sucrose, 0.8% (w/v) type E agar (Sigma Aldrich), 0.5 μM TDZ (Caisson) and adjusted to a pH of 5.7 [12 (link)]. Each glass culture vessel (9.05 cm high × 5.8 cm diameter baby food jars; Phytotechnology Laboratories) contained 25 mL of media which were sterilized by autoclaving for 20 minutes at 121°C and 18 PSIG.

For DILS assays, seeds were surface sterilized and stratified at 4 °C for 3 d. Seedlings were then grown for 2 wk in large square Petri dishes containing 0.5× Murashige Skoog Basal Salts (Sigma #M6899), 0.5% (w/v) agar type E (Sigma #A4675), 0.05% (w/v) MES pH 5.7 (Sigma #M8250), at 16 h light/8 h dark, average lighting of 120 μmol m⁻² s⁻¹, 22 °C day/20 °C night, 55% humidity. Plates were covered with aluminium foil to create complete darkness for 3 or 6 d. For DILS in pots, 4-wk-old adult plants grown in jiffy pots were covered with a lid and completely wrapped in aluminium foil to create constitutive darkness.

toc120-3 (SALK_017374) and ppi3-1 were provided by Paul Jarvis (Constan et al., 2004 (link); Kubis et al., 2004 (link)), and toc120-3 was backcrossed to WS wild type before use. mar2-1 was previously described as a mutation in the TOC132 gene (AT2G16640) (Stanga et al., 2009 (link)). To comply with the Arabidopsis nomenclature, we have renamed this allele toc132-4^mar2−1 in this manuscript. The arg1-2 and arg1-2 toc132-4^mar2−1 mutants have also been described previously (Sedbrook et al., 1999 (link); Stanga et al., 2009 (link)).
The seeds were sterilized by washing with 95% ethanol. They were plated on half-strength buffered Linsmaier and Skoog medium containing macro- and micro-nutrients, vitamins, and 1.5% sucrose (Caisson Laboratories, North Logan, UT) supplemented with 1.5% agar type E (Sigma-Aldrich, St. Louis, MO) unless otherwise indicated. The seedlings were grown in a Conviron (Asheville, NC) TC16 growth chamber set at 22°C and a 16 h light/8 h dark cycle. The light intensity was 50–70 μmol m⁻²s⁻¹ and was provided by cool white fluorescent bulbs (Grainger, Lake Forest, IL).