Stability of adenine-based cytokinins in aqueous solution

Since the isolation of the first cytokinin almost 60 yr ago, cytokinins have become critically important for ornamental and agricultural crops in plant tissue culture. Despite the extensive research on this class of compounds, little information is available on the chemical stability of cytokinins in solution or following an autoclave cycle with Murashige and Skoog (MS) basal medium. This work describes the stability in aqueous solutions of five widely used adenine-based cytokinins: trans-zeatin (tZ), 6-(γ,γ-dimethylallylamino) purine (2iP), kinetin, benzyladenine (BA), and m-topolin. High pressure liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS) were used to quantify and identify their degradation. BA, kinetin, 2iP, and m-topolin were stable at 1.0 mg mL⁻¹ in 0.05 N KOH, with no statistically significant concentration changes (p > 0.05) after 90 d of storage at temperatures of −20°C, 2−6°C, or 25°C. The cytokinin tZ was used as a model compound to evaluate stability under alkaline and acid conditions as well as after repeated freeze-thaw cycles. Trans-zeatin retained >90% of the initial concentration of 1.0 mg mL⁻¹ when dissolved in 0.01 N KOH and stored at −20°C and 2–6°C for 90 d, with only the 2–6°C temperature treatment showing a statistical significant concentration change (p = 0.03). The 1.0 mg mL⁻¹ tZ solution in 0.01 N KOH was stable through six repeated freeze-thaw cycles over 90 d without any significant change in concentration compared to the initial freeze-thaw. Yet, tZ showed highly significant concentration changes when dissolved at 50 mg mL⁻¹ and 0.5 N KOH. All of these adenine-based cytokinins showed exceptional stability following an autoclave cycle at 121°C, 110 kPa for 30 min when in solutions of 1.0 mg mL⁻¹ in 0.05 N KOH, with no significant degradation detected. Trans-zeatin was also found to be stable after one autoclave cycle with 1× MS-basal salts.