6 benzylaminopurine bap

All used chemicals were of analytical grade: MgSO₄ was purchased from POCh (Gliwice, Poland); zinc hydroaspartate was from Farmapol (Poland); conc. HNO₃ and H₂O₂ suprapure were from Merck (Darmstad, Germany); and anthranilic acid, l-tryptophan, and serine (purity ≥98 % by HPLC) were from Sigma-Aldrich (St. Louis, USA). The growth regulators naphthalene-1-acetic acid (NAA) and 6-benzylaminopurine (BAP) were also from Sigma-Aldrich. Water was filtered through Millipore Millex-GP, 0.22 μm, and was purified by quadruple distillation.

Commercial S. tuberosum L. cultivars, Agria, Désirée and Kennebec, were used in experiments. The three unrelated potato varieties were selected to validate the presence of investigated genes and compare StSODs expression in tetraploid genotypes that differ in heat tolerance. Based on our unpublished data, Désirée was considered relatively heat tolerant, Kennebec as moderately sensitive, and Agria as heat-sensitive genotype.
Virus-free tubers of three potato cultivars were obtained from Solanum Komerc, Guča, Serbia. In vitro cultures were established from surface-sterilized sprouts, which were transferred on the basal medium (BM) consisting of Murashige and Skoog macro and micro-mineral salts [51 (link)], Linsmaier and Skoog vitamins [52 (link)], 0.7% agar, 3% sucrose, 100 mgL⁻¹ myo-inositol and supplemented with 0.5 mgL⁻¹ 6-benzylaminopurine (BAP; Sigma Aldrich, St. Louis, MO). Shoots obtained on this medium gave rise to plantlets when transferred to BM without BAP. Microplants were grown in a controlled environment (21 °C, 16 h light period, light flux 90 μmol m⁻² s⁻¹) and were routinely subcultured every four weeks on BM using single-node stem cuttings (SNC).

Dexamethasone (Dex; Sigma-Aldrich Company Ltd, Darmstadt, Germany), indole-3-acetic acid (IAA; Sigma-Aldrich Company Ltd, Darmstadt, Germany) and luteolin were dissolved in 70% ethanol. AVG and 6-Benzylaminopurine (BAP; Sigma-Aldrich Company Ltd, Darmstadt, Germany) were dissolved in water. Mock treatments were equal volumes of each solvent in the agar media. For BAP plate treatments (100 nM) and IAA plate treatments (100 nM) 2-day old seedlings were grown on BNM plates for 24 h with either BAP or IAA and supplemented with 1 μM AVG to replicate spot inoculation conditions. For the lateral root primordium assay 2-day old seedlings were transferred to modified Fahraeus medium supplemented with IAA (100 nm) or mock for 24 h. For Dex treatments, 3-week old plants with transformed roots were transferred to BNM agar plates supplemented with KNO₃ (potassium nitrate, 2.5 mM) and either Dex (1 μM) or mock treatment, with a piece of filter paper placed over the root systems to ensure full contact with the additives.

Seeds of Arabidopsis thaliana (L.) transgenic lines ARR5: GUS and DR5: GUS were obtained from ABRC (ABRC OH, USA). Seeds of wild type (Col-0; Lehle Seeds, Round Rock, TX, USA) and transgenic lines were surface sterilized using 2% (v/v) NaOCl by soaking for 1 min followed by four rinses with sterile distilled water and stratified at 4 °C for 2 days. Seedlings were produced as described by Rathor et al. (2020) (link). In brief, sterilized seeds were placed on plates containing half-strength Murashige and Skoog (MS) medium (Fisher Scientific, ON, Canada) supplemented with 1% (w/v) sucrose and solidified with 0.4% (w/v) Gellan Gum (Fisher Scientific, ON, Canada). Plates were maintained at 22 °C with 16 h light/8 h dark cycle, with a light intensity of 200 µmol m⁻²s⁻¹. Four days old seedlings were transferred into 24 well plates (Fisher Scientific, ON, Canada) containing half-strength MS medium supplemented with either/or HA (0.1%), IAA (25 μM) (Sigma, ON, Canada) and 6-benzyl aminopurine (BAP) at 10 μM (Sigma, ON, Canada). Each treatment had three biological replicates. After 24 h the seedlings were fixed in 90% cold acetone and GUS staining was performed as described by Weigel and Glazebrook (2002) .

The shoots were trimmed to about 1.0 cm and cultured in baby food jars each containing 30 ml shoot initiation medium. The shoot initiation medium was MS medium containing 6-Benzylaminopurine (BAP) (0.5, 0.75, 1.0, 1.25, 1.5 mg/l) (Sigma-Aldrich, St. Louis, Missouri, USA) in combination with 0.5 mg/l thidiazuron (TDZ) or TDZ alone (0.5, 0.75, 1.0, 1.25, 1.5 mg/l) (Sigma-Aldrich, St. Louis, Missouri, USA), 30 g/l sucrose, 0.7% (w/v) agar (HiMedia, Mumbai, Maharashtra, India). The pH of the medium was adjusted to 5.8 before addition of agar. Basal MS medium was used as control. The cultures were maintained at a temperature of 25 ± 2 °C under light intensity of 20 μmol m⁻² s⁻¹ and 16 h photoperiod provided by cool-white fluorescent lamp (OSRAM GmbH, Munich, Germany). For each treatment, a total of 30 explants were used. There were six explants per jar with five replications. The experiment was repeated once. Number of dead and initiated shoots was recorded.