An enhanced Amaranthus betacyanin bioassay for detection of cytokinins

An enhanced Amaranthus assay was developed. Cotyledons of A. caudatus virid is were obtained by germinating the seeds on agar medium (0.8 % w/v) at 22 to 24 degrees C in the dark for 72 h. Cytokinins, zeatin (Z), zeatin ribosi de ([9R]Z), N-6-(Delta(2)-Isopentenyl) adenine (iP), and N-6-(Delta(2)-Isop entenyl) adenosine ([9R] iP) were dissolved in 60 mu L of 13.3 mmol . L-1 p hosphate buffer (pH 6.3) containing 1 mg . mL(-1) of L-tyrosine in rest tub es, and 10 cotyledons were soaked in the sample solution. When the cotyledo ns were incubated ar 30 degrees C in the dark for 24 h, 0.5 pmol of those c ytokinins were successfully detected. The sensitivity was enhanced 20 to 40 times compared to those of conventional Amaranthus bioassay and soybean ca llus 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 H PLC fractions from each extract of Dendranthema vegetative shoots and Eusto ma leaves. From the biologically active fractions in the Dendranthema shoot s, presence of Z, [9R] Z and [9R] iP was confirmed by LC/MS and MS/MS.