Growth chamber

Seeds of Oryza sativa ssp. indica, cv. IR64 were surface sterilized with bavistin solution (0.1%), rinsed with distilled water and germinated hydroponically in half strength Yoshida medium as described previously (Mustafiz et al., 2011 (link)). Seedlings were grown under 16 h/8 h photoperiod at 28 ± 2°C with 70% humidity in the growth chamber (Panasonic, Japan). Ten day old seedlings were subjected to various stress treatments for 6 h (Tripathi et al., 2012 (link)). For salinity stress, seedlings were supplemented with half strength Yoshida medium containing 200 mM NaCl and for drought stress, seedlings were air-dried between folds of tissue paper as described (Singh V. K. et al., 2015 (link)). Untreated seedlings grown in half strength Yoshida medium were taken as control. The shoot tissues were harvested and immediately frozen in liquid nitrogen and stored at −80°C for RNA isolation.

Untransformed and transformed in-vitro banana plantlets were regenerated from embryogenic cell suspension cultures (ECS) of banana cultivar Rasthali [18 ].
Banana plantlets grown hydroponically on modified half strength Murashige and Skoog (MS) medium (pH 5.8) [19 ] were acclimatized for 3 weeks in a growth chamber (Panasonic Healthcare Co. Ltd, Japan) viz., under fluorescence white light (40W) for 16 h light/8h dark photoperiod, 70% RH, 25°C. These plantlets were transferred to modified half strength MS media with 250 μM and 350 μM concentrations of iron and allowed to grow for 10d. Similarly, a different set of plantlets were exposed to half strength MS devoid of iron and supplemented with 300 μM ferrozine for 10d. Plantlets exposed only to modified half MS were taken as experimental controls.
Regeneration of untransformed and transformed in-vitro plantlets was done as described later under the section “transformation”. The in-vitro plantlets were hardened in pre-autoclaved soil under controlled greenhouse conditions (60–65% RH and 25°C ± 2°C) for a period of three months. Plants were irrigated with tap water every two days.

Arabidopsis thaliana Columbia-0 (Col-0) was used as the wild type. The T-DNA insertion line, anac032 (SALK_012253) was obtained from the SALK collection and the ABRC. All seeds were sterilised by treating them with 1% bleach and 0.05% Triton X-100 for 5 min, and then washing 3 times with sterilised water. Seeds were germinated on MS (Murashige and Skoog, FUJIFILM Wako Pure Chemical, Osaka, Japan) medium supplemented with 1% sucrose and 1% agarose after two days at 4 °C. Plants were grown vertically in a growth chamber (Panasonic, Osaka, Japan) at 22 °C with a 16 h light/8 h dark cycle. For H₂O₂ and estradiol treatments, 5-day-old seedlings were transferred onto MS agarose plates containing 500 µM H₂O₂ and 5 µM estradiol (FUJIFILM Wako Pure Chemical).
SALK_012253 was genotyped using left-border primers on the T-DNA (LB) and right-side (RP) and left-side (LP) primers on the genome. The list of primers that were used in this study is provided in Supplementary Table S2.