IMMUNODETECTION AND IDENTIFICATION OF N(6)-(O-HYDROXYBENZYLAMINO)PURINE AS A NATURALLY-OCCURRING CYTOKININ IN POPULUS X CANADENSIS MOENCH CV ROBUSTA LEAVES

A highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for 9-beta-d-ribofuranosyl-N6-(o-hydroxybenzylamino) purine [(oOH)[9R]BAP] and structurally related cytokinins. As little as 3 femtomoles of the compound could be detected by this method. Cross-reactivity studies demonstrated the specificity of four polyclonal antibodies for (oOH)[9R] BAP and its free base in preference to a range of natural cytokinins and other purines. After evaluating the method by internal standardization employing [2-H-3](oOH)[9R]BAP of high specific radioactivity as recovery marker by dilution analyses and by immunohistograms, it was possible to apply ELISA to quantify (oOH)[9R]BAP in plant extracts. In addition to (oOH)[9R]BAP, an unknown cytokinin reacting with the same antibody was detected in high performance liquid chromatography-fractionated extracts of mature Populus x canadensis Moench cv Robusta. The structure of the new compound was determined by gas chromatography-mass spectrometry and finally confirmed by synthesis as N6-(o-hydroxybenzylamino)purine.