Trans zeatin

LC-MS grade methanol (LC-MS LiChrosolv^®, Merck (≥99.97%), formic acid (98–100%), water (LC-MS Chromasolv), trans-zeatin (≥97%), solasodine (≥95%), tomatidine hydrochloride (≥85%), α-solanine (≥95%), solanidine (≥95%), phenylalanine (≥98%), tyrosine (≥98%), tryptophan (≥95%), methyl dihydrojasmonate (≥96%), and DMSO (≥99.9%) were purchased from Sigma-Aldrich (St. Louis, MO, United States) and α-chaconine (≥95%) (PhytoLab, Germany).

High-performance liquid chromatography (HPLC) grade acetonitrile, methanol and water were purchased from Burdick & Jackson (Morristown, NJ). Mass spectrometry grade formic acid and internal standards namely, Tryptophan-15N2, Glutamic acid-d5, Thymine-d4, Gibberellic acid, Trans-Zeatin, Jasmonic acid, Anthranilic acid, and Testosterone-d3 were purchased from Sigma- Aldrich (St.Louis, MO). The calibration solution containing multiple calibrants in acetonitrile/trifluroacetic acid /water was purchased from Agilent Technologies (Santa Clara, CA).

The concentration of endogenous cytokinins was determined in the abscission zones in X50, X33, the GhCKX3-RNAi transgenic lines (CR-3, CR-13), and the WT control line. The experiments were performed as described previously (Zeng et al., 2012 ) with modifications. Samples were extracted in 80% cold methanol overnight at 4 °C, which contained [²H₅] trans-Zeatin (Olchemim, Olomouc, Czech Republic) and [²H₆] N⁶-isopentenyladenine (Olchemim) as internal standards. After centrifugation at about 13 200 g for 15 min at 4 °C), the supernatant was passed through Oasis HLB columns (Waters). The filtered liquid was then evaporated using N₂, dissolved in 2 ml 1% formic acid, and passed through Oasis MCX columns (Waters). The columns were first washed with 2 ml methanol, and then washed with 2 ml 0.35 M NH₄OH. Finally, the columns were washed with 0.35 M NH₄OH in 60% methanol and the elution was collected and evaporated using N₂. The residue was dissolved in 100 μl 50% acetonitrile and stored at –80 °C until measurement. Cytokinins were quantified using a HPLC-MS/MS system with trans-Zeatin (Sigma) as the external standard.

Both the 1× MS agar medium (0.44% MS Basal medium, 0.5% sucrose, 0.05% MES hydrate, pH adjusted to 5.7 with Tris–HCl, 0.8% agar) contained in the PCR tubes, and the 1/500× MS liquid medium (0.00088% MS Basal medium, 0.5% sucrose, 0.05% MES hydrate, pH adjusted to 5.7 with Tris–HCl) in the nurseries, were supplemented with 10 nM trans-zeatin (Sigma–Aldrich Z0876) as previously suggested (Miyawaki et al., 2006 (link)). Still in their PCR tubes, seedlings were transferred into V-boxes, filled with 1/500× MS liquid medium supplied with 10 nM trans-zeatin to conduct the electrotropism experiments.
Sample sizes N and number of replicates R were the following: WT + tZ, N = 14, R = 3; cyp735a1 +tZ at 1.5 V/cm, N = 13, R = 3; cyp735a1 +tZ at 0 V/cm, N = 14, R = 3.

Seeds (n = 500) of Olea europaea subsp. europaea var. sylvestris were collected from sylvestris plants growing in open field at the locality “Pietra del Demanio” in Cosenza, Italy, and vernalized for 3 weeks at 4°C [26 ]. After mechanical removal of endocarp, they were soaked for two days, sterilized with 70% alcohol for 1 min and then treated with sodium hypochlorite 5% (v/v) plus tween 20 0.1% (v/v) for 12 min. Seeds were rinsed five times with sterile distilled water, treated with the “Plant Preservative Mixture” (PPM) 1.5% (v/v) (Micropoli, Milan, Italy), supplemented with 50 mg/L of magnesium salts (chloride magnesium, sulphate magnesium, and nitrate magnesium). The PPM treatment was performed for 7 hours under continuous stirring. Finally, seeds were placed in culture on a substrate consisting of sterile water and bacto-agar 0.7% (w/v), pH 5.8. Seeds were kept at 24 ± 1°C under 16 h light per daily photoperiod. Irradiance intensity during the light period was 55 μmol m⁻² s⁻¹ PAR obtained by a cool-light fluorescent lamps. To induce a rapid vegetative growth, after 30 days of culture, root-excised seedlings were transferred to olive medium (OR) [27 ], enriched with 30 g L⁻¹ mannitol, 5 mg/L (23 μM) trans-zeatin (Sigma, Milan, Italy), 0.8 g L⁻¹ bacto-agar, and 0.1%. PPM [27 , 28 ]. This medium was named ORZ₅.