Benzylaminopurine bap

The protocol was adapted from [70 (link),71 (link)] with minor modifications as described in [72 (link)]. The source material were cells that had grown for 1 d after sub-cultivation. We digested aliquots of 4 mL cell suspension (non-transformed WT, NtTPC1A-GFP, or PIN1-GFP, respectively) harvested under sterile conditions at day 1 after sub-cultivation. The cells remained for 1 h at 26 °C in 4 mL enzyme solution (1% (w/v) cellulase YC (Yakuruto, Tokyo, Japan), and 0.1% (w/v) pectolyase Y-23 (Yakuruto, Tokyo) in 0.4 mol/L mannitol at pH 5.5) under constant shaking on a KS260 basic orbital shaker at 100 rpm in petri dishes of 90 mm diameter. After digestion, we collected the protoplasts, using a mild centrifugation at 500 rpm for 5 min in fresh reaction tubes. The protoplast sediment was carefully re-suspended in 10 mL of FMS wash medium containing 4.3 g/L Murashige and Skoog salts, 100 mg/L (myo)-inositol, 0.5 mg/L nicotinic acid, 0.5 mg/L pyroxidine-HCl, 0.1 mg/L thiamin, and 10 g/L sucrose in 0.25 M mannitol [70 Kuss-Wymer]. After three washing steps, we transferred the protoplasts into 4 mL FMS-store medium, (FMS wash medium) complemented with 0.1 mg/L 1-naphthalene-acetic acid (NAA, Sigma Aldrich, St. Louis, MO, USA) and 1 mg/L benzylamino-purine (BAP, Sigma Aldrich, St. Louis, MO, USA). Then, the protoplasts were ready for microscopical observation.

For the initiation of aseptic culture of E. maritimum and E. alpinum, the shoot fragments with axillary buds isolated from plantlets were used as explants. The isolated primary explants were rinsed in distilled water for 5 min and dipped in 70% (v/v) ethanol-water solution for 30 s followed by rinsing in 1.33% (E. maritimum) or 2.5% (E. alpinum) sodium hypochloride solution, containing two drops of surfactant Tween 80 for 8 min. They were finally rinsed three times in sterilized double-distilled water.
The explants of both the species were placed in a Erlenmeyer flask with 50 mL of the solidified MS medium (Murashige and Skoog [39 (link)]) with plant growth regulators—benzylaminopurine (BAP; Sigma-Aldrich, Saint Louis, MO, USA), indole-3-acetic acid (IAA; Sigma-Aldrich, Saint Louis, MO, USA), and gibberellic acid (GA₃; Sigma-Aldrich, Saint Louis, MO, USA), each at the concentration of 1.0 mg/L [23 (link),40 (link)]. The culture vessels were placed in a growth chamber (21 ± 2 °C; with a 16 h light/8 h dark photoperiod; 55 µmol/m²∙s light) and subcultured every 5 weeks. Multiplication of shoots via the axillary branching method on MS medium was repeated many times, using at least 10 explants per repetition. Shoots were air dried.

Ceratopteris richardii strain Hn-n^{40 (link)} was used in this study to generate the transgenic plants. Gametophytes were grown on FM plates (pH 6.0) containing 0.5 × MS salts (PhytoTechnology Laboratories) and 0.7% (w/v) agar (Sigma-Aldrich). Sporophytes were formed on fertilized gametophytes and were transferred to soil, typically after 3–4 weeks of fertilization. Both gametophytes and young sporophytes were grown under continuous light at 28 °C. Adult sporophytes were grown in the LILY greenhouse facility at Purdue for harvesting spores.
Ceratopteris calli were induced from young sporophytes (shoot tips or fronds) on the callus induction medium (pH 5.8) that contains 1 × MS salts (PhytoTechnology Laboratories), 2% (w/v) sucrose, 1 mg/L benzylaminopurine (BAP), and 0.7% agar (Sigma–Aldrich). Calli were cultivated under continuous light at 28 °C in the growth chamber (Percival).