Murashige and skoog ms medium

Arabidopsis thaliana qrt1-2 (CS8846; Col-3) was used as the wild type. The gex1-1 (CS817261; Col-3) mutant allele has been described [13 (link)]. Seeds of rapid-cycling Brassica rapa (Fast plants) [24 (link)] were purchased from In The Woods Group (Aomori, Japan). The seeds were surface-sterilized and sown on soil or Murashige and Skoog (MS) medium (Fuji Film Wako, Osaka, Japan) containing 0.7% agar and 1% sucrose. The plants were grown at 22 °C under continuous light.

Seeds of Arabidopsis thaliana cv. Columbia (2n = 10) from Kobe University were sterilized in 1 mL 10% (v/v) kitchen bleaching solution (KAO, Tokyo, Japan) for 30 min, rinsed several times, vernalized at 4 °C for 2–3 days and germinated on 0.5 × Murashige and Skoog (MS) medium (Wako, Tokyo, Japan) supplemented with 0.5% agar for 10–15 days at 25 °C. DNA was extracted from seedlings in DNA Suisui buffer (RIZO Inc., Tsukuba, Japan) and ethanol precipitated.

The WT used in this study was A. thaliana Colombia-0 (Col-0), and mutants and all transgenic plants were on the Col-0 background. Also, fugu5-1 was isolated and characterized as a loss-of-function mutant of the vacuolar type H⁺-PPase (Horiguchi et al., 2006b (link); Ferjani et al., 2007 (link), 2011 (link)). In addition, the two previously described independent transgenic lines expressing yeast sPPase IPP1 on the fugu5-1 mutant background (AVP1_pro::IPP1#8-3 and #17-3) were used (Ferjani et al., 2011 (link)). The seeds were sown on rockwool (Nippon Rockwool), watered daily with 0.5 g L^–1 Hyponex solution, and grown under a 16-h light and 8-h dark cycle with white light from fluorescent lamps at approximately 50 μmol m^–2 s^–1 and 22°C. The sterilized seeds were sown on Suc-free Murashige and Skoog (MS) medium (Wako Pure Chemical) or MS medium with 2% (w/v) Suc, where indicated. 0.1% (w/v) 2-(N-morpholino) ethanesulfonic acid (MES) was added, then pH was adjusted to 5.8 with KOH, and solidified with 0.2–0.5% (w/v) gellan gum (Murashige and Skoog, 1962 ) to determine the effects of medium composition on phenotype. The seeds were sown on MS plates and stored at 4°C in the dark for 3 days as cold treatment. After the cold treatment, the seedlings were grown in the light (as above) or in the dark for the indicated periods of time.