Simultaneous analysis of cytokinins, auxins and abscisic acid by combined immunoaffinity chromatography, high performance liquid chromatography and immunoassay

A method has been developed for the rapid and simultaneous extraction and analysis from plant material of 3‐indolylacetic acid (IAA), naphthalene acetic acid (NAA), abscisic acid (ABA) and the cytokinins benzyladenine (BA), zeatin, zeatin riboside, dihydrozeatin, dihydrozeatin riboside, isopentenyl adenine and isopentenyl adenosine. The method involves extraction with 80% (v/v) methanol, pre‐purification of the extracts through reversed phase C18 Sep‐Pak cartridges and immunopurification. The separation of the different compounds was accomplished by reverse‐phase high performance liquid chromatography for cytokinins, and by partition with diethyl ether for IAA, NAA and ABA. After methylation of the IAA fraction with diazomethane, quantification of all plant growth regulators (PGRs) was made by immunoassay. The percentage recovery at each step was monitored following the addition of radioactive compounds at the beginning of the process. The final recovery was 68% for IAA, 92% for NAA, 76% for ABA and 75% for BA. The method was applied to the analysis of PGRs in tissues and callus of kiwifruit (Actinidia deliciosa Liang and Ferguson).