Plant agar

Seeds were surface sterilized with 50% (vol/vol) hypochlorous acid for 5 min and then washed three times with sterile deionized water. Plant seeds were plated on 0.5 strength Murashige and Skoog medium (2.17 g salts per 1 l), at pH 5.8 and solidified with 1% plant agar (Duchefa, Haarlem, The Netherlands). Seeds were stratified at 4°C for 48 h in the dark to synchronize germination, and then incubated vertically in a culture room under 12-h light at 22°C and 12-h dark at 22°C (light: Philips TL-D 58W/840, 120–150 μmol m⁻² s⁻¹). The Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as the wild-type in all experiments. Mutant lines were in the Col-0 background and were obtained from the ABRC and NASC seed repositories for crossing and imaging, lines were genotyped as described in references: we used DII-VENUS (Brunoud et al., 2012 (link)), b1-100 (SALK_083649, Lin and Wang, 2005 (link)) abcb4: mdr4-1 (SALK_072020, Lewis et al., 2007 (link)), and b19-3 (SALK_033455, Lewis et al., 2007 (link)).

The study involved the cultivation of callus (Fig. 1A) on MS agar medium with the inclusion of 0.5 mg/L BA and 2 mg/L IBA (indole-3-butyric acid, Sigma-Aldrich), 3% (w/v) sucrose, and 0.72% (w/v) agar (plant agar, Duchefa, Haarlem, the Netherlands). The cultures were maintained at 25 ± 2 °C, under continuous artificial illumination with white LED light, with a photosynthetic photon flux density (PPFD) of 40 μmol m⁻² s⁻¹ and cultivated over the 30-day breeding cycles. The agar callus culture was carried out in glass containers intended for plant tissues (No. V8630, Sigma-Aldrich, Algés, Portugal). The 0.5 g of inoculum per one container was used for the experiment (three series, n = 10).Fig. 1

Morphological appearance of tested types in vitro cultures of S. henryi: A callus culture; B suspension culture; C agar microshoot culture; D microshoot agitated culture; E microshoot culture in PlantForm bioreactor

Plants were grown in soil (16/8 h light/dark; 24°C/19°C; 150 µE/m2/s; 70% humidity) or in vitro on 2.2 g/l Murashige and Skoog nutrient mix (Duchefa), 0.7% (w/v) plant agar (Duchefa), 0.5% (w/v) sucrose, buffered to pH 5.8 with MES (2-Morpholinoethanesulfonic acid, Sigma), after 2 days of vernalization. Seeds were surface sterilized in 70% ethanol, 0.1% (v/v) Tween 20 for 5 min and rinsed in 99% ethanol. The Arabidopsis thaliana Columbia-0 (Col-0) accession was employed as the wild-type background and mutants were genotyped according to the description in the respective articles: yip4a-2 yip4b (Gendre et al., 2013 (link)) (called simply yip4a yip4b in this article), scn1-1 (Parker et al., 2000 (link)), 35S::CA-ROP2 (Li et al., 2001 (link)) and rop2 rop4i-4 rop6 (Ren et al., 2016 (link)). The rop4i-2, rop2 rop4i-8 and rop4i-5 rop6 mutants were generated by independent Agrobacterium tumefaciens-mediated transformation using the ROP4-RNAi construct under the native promoter of ROP4 (see Ren et al., 2016 (link)) introduced into Col-0, rop2 (SALK_055328C) and rop6 (SALK_091737C), respectively.
The following transgenic fluorescent-protein marker lines were used in the Col-0 background: EXP7::GFP (Cho and Cosgrove, 2002 (link)) and ROP2::EYFP-ROP2 (Xu and Scheres, 2005 (link)).

Unless otherwise indicated, seeds were stratified in sterile water for 4 days in the dark and 4°C, sown on soil and grown in environmentally controlled growth chambers under long day conditions (10,000 luxes, 16 hr light, 22°C/8 hr dark, 19°C).
For the hydroponic experiment, seeds were surface‐sterilized incubating them for 7 min in a 70% ethanol (Sigma‐Aldrich), 0.1% Triton X‐100 (Panreac Applichem) solution followed by five washes with sterile water. Seeds were then transferred to the Araponics seed holders (Araponics) filled with half‐strength pH5.7 Murashige and Skoog media (Duchefa Biochemie) supplemented with 0.65% Plant Agar (Duchefa Biochemie) and incubated for four days in the dark at 4°C before being transferred to the growth chamber (7,000 luxes, continuous day, 22°C). New Araponics nutrient solution (0.5 ml/L) was added every week supplemented with rac‐GR24 (Strigolab) or acetone as a control. After 6 weeks, plant height was recorded, pictures were taken, and stems were collected for toluidine‐O‐blue staining and cell wall analyses.

Seedlings of A. thaliana accession Col-0 and mutant opt3-2 (Stacey et al., 2008 (link)) were grown on a piece of nylon mesh (Nitex Cat 03-100/44, Sefar, Heiden, Switzerland) on standard Fe-sufficient growth medium consisting of modified Hoagland medium (Hoagland and Arnon, 1938 ) containing 5 mM KNO₃, 2 mM MgSO₄, 2 mM Ca(NO₃)₂, 2.5 mM KH₂PO₄, 70 μM H₃BO₃, 14 μM MnCl₂, 1 mM ZnSO₄, 0.5 mM CuSO₄, 10 μM NaCl, 0.2 μM Na₂MoO₄, 0.05% 2-ethanesulfonic acid (MES; Duchefa Biochemie, Haarlem, Netherlands), 50 μM FeNaEDTA, 1% sucrose, and 1% plant agar (Duchefa Biochemie). The pH of the medium was set to 5.7. Fe-deficient medium was prepared by omitting FeNaEDTA from the standard medium.
Typically, Arabidopsis seeds were sown at low density on standard Fe-sufficient growth medium (a single row of twenty seeds per square Petri dish of 120 × 120 mm). Seeds were sterilized by a 3-h exposure to the gas formed upon mixing 100 ml bleach with 3.2 ml hydrochloric acid fuming (37%) (Van Wees et al., 2013 (link)). After sowing, plates were sealed with a double layer of Parafilm and stratified in the dark at 4°C for 48 h. Plates were then placed vertically in a growth chamber under short-day conditions (14-h night, 10-h day; 21°C; 100 μmol m^–2 s^–1).