Establishing Split-Root Systems in Barley

Square 120 × 15 mm Petri dishes (Corning) were lined with two sheets of autoclaved Whatman filter papers and moistened using half-strength Hoagland’s solution (Hoagland's No. 2 Basal Salt Mixture, Sigma-Aldrich) with a pH of 5.8. A row of five BK (variety: Lileja) seeds were placed in each Petri dish and covered with half-sized filter papers. They were sealed using parafilm and placed on a stand at a 90° angle in a phytotron (Aralab, Clitec) set at 24 °C for 16 h and 18 °C for 8 h with a relative humidity of 70% and kept in the dark for 3 days.
Emerging BK roots were carefully cut a few millimetres above the root apex using a sterilised scalpel (Fig. 1a) to induce the development of secondary roots^{90 (link)} facilitating the subsequent splitting of the root system (Fig. 1c). Root cutting is a stress-inducing procedure that requires a recovery process. Partial root cutting with 2 weeks of recovery was chosen because it induces less stress than de-rooting^{91 (link)}. The Petri dishes were again sealed with parafilm and transferred to the phytotron set at, 16:8-h light/dark photoperiod, 24/18 °C and a relative humidity of 70% for 5 days. The 7-day-old BK seedlings were transferred to split-root systems (Fig. 1b).