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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.Electronic supplementary materialThe online version of this article (doi:10.1007/s11627-015-9734-5) contains supplementary material, which is available to authorized users.

Callus induction, from mature caryopses, and callus maintenance and plantlet regeneration, using 14‐month‐old stock callus (SC), were investigated in Penncross creeping bentgrass (Agrostis palustris Huds.). Greatest callus induction occurred on Murashige and Skoog (MS) medium with 1.0 mg 2,4‐dichlorophenoxyacetic acid (2,4‐D) under light incubation and 1.0 or 10.0 mg 2,4‐D/1 under dark incubation. Kinetin supplements were not required for callus induction, however, caryopses incubated in the dark with 0.01 mg kinetin had greater callus formation than caryopses incubated without kinetin when combined with 1.0 mg 2,4‐D/1. Under dark incubation, caryopses produced the greatest callus yields (47 and 57 mg) and callus removal from caryopses required less time and effort. Callus induced from caryopses originated from the scutellum, root cortex and along the coleoptile up to and adjacent the first node. Callus growth using SC was greatest at 1.0 mg 2,4‐D/1. Callus previously cultured on 1.0 mg 2,4‐D maintained good plantlet regeneration capacity; whereas, callus previously cultured at 10 and 100.0 mg 2,4‐D/1 had poor and no plantlet formation, respectively. The best combination for shoot and root development in SC occurred at 0.10 mg and 1.0 mg kinetin/1 without 2,4‐D. All plantlets were potted in a sand:peat (1:1) mix and maintained in a glasshouse. Callus induced plants were similar to seeded plants. Less than 1% of the plantlets formed were albino.

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