Gamborg s vitamin solution

Seeds were washed with 100% ethanol for a second and sterilised with 2–10% (w/w) hypochlorite solution (Wako) containing 0.2% Tween-20 (Amersham Biosciences, Sweden) for 40 min. The sterilised seeds were washed with sterilised water and planted on Murashige and Skoog (MS) medium [MS salt (4.6 g l⁻¹), 0.2% (w/v) gellan gum (Wako), 0.1% (v/v) Gamborg’s vitamin solution (Sigma-Aldrich Co. LLC, MO), pH 5.8]. Sterilisation time, gellan gum concentration and sucrose concentration differed depending on the purpose of the experiment. Details of these differences are described in the result section separately. To determine the appropriate germination conditions, three replications were conducted for each treatment with ca. 30 seeds per plate. Plates were incubated at 24 °C under a 16L: 8D cycle.

Arabidopsis thaliana Columbia (Col-0) accession was used as a wild-type control. Transgenic plants possessing HTR10p::HTR10-mRFP or -Clover (Ingouff et al., 2007 (link); Kawashima et al., 2014 (link)), RPS5Ap::H2B-GFP or -tdTomato (Adachi et al., 2011 (link)), and RPS5Ap::tdTomato-LTI6b (Mizuta et al., 2015 (link)) genes were used to visualize the sperm cell nuclei, female gametophytic cell nuclei, and female gametophytic cell membrane, respectively. A tetraspore (tes) mutant, tes-4 (CS9353; Spielman et al., 1997 (link); Yang et al., 2003 (link)) was obtained from the Arabidopsis Biological Resource Center and crossed with the pollen from the HTR10p::HTR10-mRFP line.
A. thaliana seeds were sterilized in a solution containing 2% Plant Preservative Mixture^TM (Cosmo Bio, Tokyo, Japan), 50 μg/mL magnesium sulfide, and 0.1% Tween 20 for several days at 4°C. The seeds were sown on Murashige and Skoog (MS) medium [1 × MS salt (Duchefa Biochemie, Haarlem, Netherlands), 2% sucrose, 1 × Gamborg's vitamin solution (Sigma, St. Louis, MO, USA)] solidified with 0.3% Gelrite (Wako, Osaka, Japan) and adjusted to pH 5.7 with KOH. Plants were germinated and grown in a growth chamber at 22°C with continuous light. Two-week-old seedlings were transferred to soil and grown at 22°C in a plant growth room.

For all experiments, the A. thaliana accession Columbia (Col-0) was used as the wild type. All A. thaliana transgenic lines were in a Columbia (Col-0) background, and the myb98 mutant was previously described [10 (link)]. The following transgenic lines were also previously described: RPS5Apro::H2B–tdTomato [11 (link)], RPS5Apro::tdTomato–LTI6b [12 (link)], RPS5Apro::H2B–sGFP [13 (link)], FGR8.0 [14 (link)], MYB98pro::GFP [10 (link)], MYB98pro::GFP–MYB98 [15 (link)], EC1.2pro::mtKaede [16 (link)], FWApro::FWA–GFP [17 (link)], and ABI4pro::H2B–tdTomato [18 (link)]. The transgenic lines used are listed in S1 Table.
Arabidopsis seeds were sown on plates containing half-strength Murashige and Skoog salts (Duchefa Biochemie, Haarlem, the Netherlands), 0.05% MES-KOH (pH 5.8), 1× Gamborg’s vitamin solution (Sigma, St Louis, Missouri, United States of America), and 1% agar. The plates were incubated in a growth chamber at 22°C under continuous lighting after cold treatments at 4°C for 2 to 3 days in the dark. Two-week-old seedlings were transferred to soil and grown at 21 to 25°C under long-day conditions (16-hour light/8-hour dark).

Seventeen Rhizoctonia solani isolates were obtained from the Genebank of the National Agricultural Research Organization (NARO) in Japan. The sources of these isolates are summarized in Table 1.
R. solani isolates were maintained on potato dextrose agar (PDA) medium (24 g/L Difco™ potato dextrose broth and 2% agar). Arabidopsis seeds were surface-sterilized using sodium hypochlorite for five minutes, then suspended in sterile water and washed five times. The seeds were stored for three days at 4 °C in dark condition before sowing. Arabidopsis seeds were plated on half-strength Murashige and Skoog (1/2MS) medium plates (2.23 g/L MS salt (Nihon Pharmaceutical, Tokyo, Japan) enriched with 0.1% (v/v) Gamborg’s vitamin solution (Sigma-Aldrich, St. Louis, MO, USA), 1% (w/v) sucrose, 0.05% MES, and 0.8% agar, pH 5.7 with KOH). Two-week-old Arabidopsis seedlings were transplanted into the soil (Supermix-A; Sakata Seed, Kanagawa, Japan) and grown for 2–3 weeks in a long-day growth chamber with LED lights (Nippon Medical & Chemical Instruments, Osaka, Japan) under a 16 h light/8 h dark photoperiod at 23 °C.

Root explants (0 to 1 cm from the root tip) were excised from seedlings 7 days after germination and cultured on CIM containing Gamborg’s B-5 medium (Wako, Osaka, Japan) with 20 g L⁻¹ glucose (Wako), 0.5 g L⁻¹ MES (Wako), 1 × Gamborg’s vitamin solution (Sigma–Aldrich, St. Louis, MO, USA), 500 µg L⁻¹ 2,4-D (Sigma–Aldrich), 50 µg L⁻¹ kinetin (Sigma–Aldrich), and 0.8% gellan gum (Wako); the pH was adjusted to 5.7 using 1.0 M KOH. Continuous light was used for callus induction.
After culturing for 10 days on CIM, the explants were transferred to SIM containing Gamborg’s B-5 medium, 10 g L⁻¹ sucrose, 0.5 g L⁻¹ MES, 1 × Gamborg’s vitamin solution, 2 µg mL⁻¹ trans-zeatin, 0.4 µg mL⁻¹ indole-3-butyric acid, 1 µg mL⁻¹ d-biotin, and 0.8% gellan gum; the pH was adjusted to 5.7 using 1.0 M KOH. Continuous light was used for shoot induction. For the chemical treatment, Ky-2, 17β-estradiol, or DEX were added to CIM or SIM.
After incubation on SIM for 21 days, the number of explants that regenerated shoots on all explants was evaluated. All phenotypic assays and microscopic observations were performed at least twice.