A PROCEDURE FOR QUANTIFICATION OF CYTOKININS AS FREE BASES INVOLVING SCINTILLATION PROXIMITY IMMUNOASSAY

Cytokinins occur in a diversity of forms and determination of their in dividual levels requires extensive purification. However, determinatio n of the total level of each major base in free, riboside and nucleoti de forms would often be adequate. Hence, a methanolysis procedure whic h releases cytokinin bases from 9-ribosyl derivatives was developed an d applied to plant extracts. A simple procedure, involving low pressur e column chromatography, for purification of the cytokinin bases in tr eated extracts, and a scintillation proximity immunoassay for their qu antification, were developed. The total level of each cytokinin base [ N-6-(2-isopentenyl)adenine, zeatin and dihydrozeatin] in free and ribo sylated forms determined by these methods is reported for several plan t tissues and the results are compared with those obtained after addit ional purification by HPLC. Values for zeatin were not changed by HPLC but isopentenyladenine and dihydrozeatin levels were usually reduced indicating the presence of unknown compounds which cross-react in the immunoassay. Modifications to the above purification method to quantif y O-glucosyl cytokinins are also described. The methods described faci litate the quantification of the total amount of each cytokinin base i n forms closely associated with cytokinin action, and the detection of cytokinin biosynthesis by labelled precursor incorporation.