An Enhanced Amaranthus Betacyanin Bioassay for Detection of Cytokinins

Abstract

An enhanced Amaranthus assay was developed. Cotyledons of A. caudatus viridis were obtained by ger- minating the seeds on agar medium (0.8 % w/v) at 22 to 24 °C in the dark for 72 h. Cytokinins, zeatin (Z), zeatin riboside ([9 R] Z), N6-(Δ2-Isopentenyl) adenine (iP), and N6-(Δ2-Isopentenyl) adenosine ([9 R] iP) were dissolved in 60 µL of 13.3 mmol · L-1 phosphate buffer (pH 6.3) containing 1 mg · mL-1 of L-tyrosine in test tubes, and 10 cotyledons were soaked in the sample solution. When the cotyledons were incubated at 30 °C in the dark for 24 h, 0.5 pmol of those cytokinins were successfully detected. The sensi- tivity was enhanced 20 to 40 times compared to those of conventional Amaranthus bioassay and soybean callus bioassay. Moreover, there was a nearly linear correlation between 0.5 and 15 pmol of each cytokinin and betacyanin production. Using the enhanced Amaranthus assay, cytokinin activity was observed in 5 HPLC fractions from each extract of Dendranthema vegetative shoots and Eustoma leaves. From the biologically active fractions in the Dendranthema shoots, presence of Z, [9 R] Z and [9 R] iP was confirmed by LC/MS and MS/MS.