L 1 agar

Seeds of all species were sterilized in 70% ethanol for 90 s and commercial bleach for 12 min and rinsed three times in sterile distilled water for 3, 7, and 10 min. Seeds were germinated on a solidified half-strength Murashige and Skoog (MS) medium [33 (link)] in sterile conditions for 2 weeks. Sterile leaves of the seedlings were placed on ½ MS media [33 (link)] or ½ MS with various combinations of auxins (2,4-dichlorophenoxyacetic acid; 2,4-D or α-naphthaleneacetic acid; NAA) and cytokinins (6-benzylaminopurine; BAP or kinetin; KIN) as follows: 2 mg L-1 2,4-D + 2 mg L⁻¹ BAP, 2 mg L⁻¹ 2,4-D + 2 mg L⁻¹ KIN, 2 mg L⁻¹ NAA + 2 mg L⁻¹ BAP, 2 mg L⁻¹ NAA + 2 mg L⁻¹ KIN, 0.5 mg L⁻¹ NAA + 5 mg L⁻¹ BAP.
All media were supplemented with 30 g L⁻¹ sucrose (Sigma-Aldrich, St Louis, MO, USA) and solidified with 8 g L⁻¹ agar (Duchefa Biochemie, Amsterdam, The Netherlands). The pH of the medium was adjusted to 5.7–5.8. The cultivation was carried out under stable artificial conditions, in a growth chamber at 25 ± 3 °C with a 16 h photoperiod under cool-white-fluorescent lamps (light intensity 70–100 μmol m⁻² s⁻¹). Culture media and instruments were sterilized in a steam autoclave (121 °C, 1.05 bar; Prestige Medical, Lancashire, UK). The tissue cultures were passaged onto fresh media every 2–3 weeks. The callus tissue was initiated to develop after 1–3 weeks, depending on the species.