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.
Internodal stem segments of 2–4‐month‐old Quercus rubra L. seedlings were cultured on Murashige and Skoog agar medium containing NAA and BA. Structures, termed organoids, were initiated from callus on medium containing 5 mg/1 NAA and 0.1 mg/1 BA. Organoids consisted of parenchymatous cells with a distinct epidermis and a continuous vascular system. Growth occurred principally by cell division throughout the parenchymatous tissue resulting in a variety of morphological shapes. Although no organized shoot meristems were observed, normal roots were produced; such roots were connected to the vascular system of the organoid.
The effects of repetitive subculturing, cytokinin species and concentration, basal medium, and gelatinizing constituent were studied to maximize shoot multiplication of Hydrangea macrophylla Thunb. ‘Rose Supreme’ shoot tip cultures. Cytokinin treatments of BA or 2iP at 2, 4, 8, 16, or 32 μm were incorporated into woody plant medium (WPM) solidified with 6 g·liter−1 Sigma agar. Cultures grown on 8 μm BA produced the greatest number of useable shoots per 4-week subculture. No treatment stimulated shoot multiplication until the 2nd subculture, after which shoot multiplication remained constant within treatments through the 5th subculture. Results indicate no difference in number of shoots produced per subculture on modified Gamborg's B5, modified Murashige and Skoog basal medium, or WPM when incorporating 8 μm BA and 6 g·liter−1 Sigma agar. No difference in shoot multiplication was observed between 6 g·liter−1 Sigma agar and 2 g·liter−1 Gel-Rite when incorporated into WPM containing 8 μm BA. However, culture fresh weight and shoot length was greater for cultures on Gel-Rite than for cultures on agar. Chemical names used: N-(phenylmethyl)-lH-purin-6-amine (BA) and N-(3-methyl-2-butenyl)-lH-purin-6-amine (2iP).
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