6 benzylaminopurine ba

The 7-day-old rice seedlings in 50 mL polypropylene conical tubes containing 30 mL water were first treated with varying levels of epibrassinolide (BR; Sigma-Aldrich, St. Louis, MO, USA), ethephon (Sunmoon Green Science, Seoul, Korea), 6-benzylaminopurine (BA; Sigma-Aldrich), and indole-3-acetic acid (IAA; Sigma-Aldrich). The BR biosynthesis inhibitor brassinazole (100 µM; Sigma-Aldrich) and GA biosynthesis inhibitor paclobutrazol (10 µM; Sigma-Aldrich) were used to inhibit BR and GA biosynthesis, respectively. GA₃ was purchased from Duchefa Biochemie (Harrlem, The Netherlands). BR, BA, and IAA were dissolved in 0.1% ethanol. The control (C) contained 0.1% ethanol in water, except for ethephon, where the control was water. Treatments were applied for 1 day under cool daylight fluorescent lamps (60 μmol m^–2 s^–1) (Philips) with a 14 h light/10 h dark photoperiod at 28 °C/24 °C (day/night), followed by treatment with 0.5 mM CdCl₂ for 3 days under continuous light conditions (60 μmol photons m^–2 s^–1) for melatonin detection by high-performance liquid chromatography (HPLC). The upper parts of leaves and stems were harvested and stored in liquid nitrogen for further analyses.

Sinningia hybrida ‘Isa’s Murmur’ was purchased from Horticulture Co., Ltd. in Sichuan. The plant was certified by Professor Minghua Luo of Mianyang Normal University. Leaf was excised from mature greenhouse-grown plantlets. Explants were initially rinsed for 30 min employing water, and then surface-sterilized, adopting 75% ethanol, for 15 s before 0.1% (w/v) HgCl₂-mediated disinfection for 5 min, then rinsed five times, applying distilled water.
They were placed on Murashige and Skoog (MS) basal medium [46 (link)] with different plant growth regulators (PGRs) for shoot induction. All culture medium contained 3% (w/v) sucrose and 0.7% agar. The pH of media was adjusted to 5.6 with 0.1 mol L⁻¹ NaOH before autoclaving for 15 min at 121 °C. All PGRs (thidiazuron (TDZ), zeatin (ZT), kinetin (Kin), 6-benzylaminopurine (BA), 3-Indole-butyric acid (IBA), naphthalene acetic acid (NAA), and Gibberellin A₃ (GA₃) were purchased from Sigma-Aldrich (St. Louis, MI, USA). The cylindrical culture bottle had a diameter of 7.0 cm and a height of 8.0 cm. All explants and plantlets were cultured at 25 ± 1 °C under a 12 h light cycle with a light intensity of 30 µmol m⁻² s⁻¹. At the same time, we selected the different length of experiments according to the development time of adventitious shoots, which is different to that of adventitious roots.

The third and fourth leaves of 3-week-old plants were harvested and floated on 3 mM MES (pH 5.7). The samples were incubated at 22 °C in the dark for up to 11 d. The photochemical efficiency of PSII (Lee et al., 2016 (link)) and chlorophyll content (Vernon, 1960 ) of leaves were measured as described previously. For chemical- or plant hormone-mediated senescence assay, detached leaves were floated abaxial side up in 3 mM MES (pH 5.7) with or without 50 μM 1-aminocyclopropane-1-carboxylic acid (ACC; Sigma, USA), 0.2 μM 6-benzylaminopurine (BA; Sigma, USA), 100 mM sodium chloride (Samchun, Korea), or 10 mM hydrogen peroxide (Junsei, Japan). All treatments were performed at 22 °C in the dark. Photochemical efficiency and chlorophyll content were measured as described above. For developmental leaf senescence assays, the third and fourth rosette leaves at the indicated leaf age were harvested from individual plants and used for measurements of chlorophyll content, photochemical efficiency, and gene expression. Leaves used for gene expression and microarray experiments were harvested 4 h into the subjective day.

Seeds collected at 105 DAP were used to investigate the influence of cytokinins on asymbiotic germination. In this experiment, modified Norstog medium was supplemented with either 6-(γ,γ-dimethylallylamino)purine (2iP, Sigma-Aldrich Co.), 6-benzylaminopurine (BA, Sigma-Aldrich Co.) or kinetin (KN, Sigma-Aldrich Co.) at concentrations of 1, 2, 4 and 8 μM. Modified Norstog medium without cytokinin supplementation was used as a control.