HIGH-THROUGHPUT SCREENING TECHNOLOGY FOR MONITORING PHYTOHORMONE PRODUCTION IN MICROALGAE(1)

New miniaturized techniques for multiplying microalgae and estimating their phytohormone production were developed; in these methods, the strains to be tested are cultivated in microtitre plates, and the phytohormones in suspensions of the cultures are measured by direct ELISAs. Specific and sensitive ELISAs for determining abscisic acid (ABA), indole-3-acetic acid (IAA), cis- and trans-zeatin riboside, isopentenyladenosine (iPR), and other less common cytokinins were developed for this purpose. Polyclonal antibodies used in the ABA and IAA assays were raised against C1- and C1'- conjugates of the compounds with BSA, respectively, and thus were specific for the free acids and their respective C1-derivatives. The use of cytokinin ribosides coupled via their sugar residues to BSA as haptens generally led to antibodies that bound free bases, 9-glycosides and nucleotides, but with high specificity for the corresponding N(6) -side chains. Using internal standards, dilution assays, and authentic [(2) H] and [(3) H] recovery markers, it was shown that the ELISAs could be used to estimate contents of the selected phytohormones in the cultures. The ELISAs provided reliable and very fast estimates of the selected phytohormones, at concentrations ranging from 0.01 to 10 pmol · mL(-1) in various microalgal strains. In addition, a recently developed HPLC selected ion monitoring mass spectrometry (HPLC-SIM-MS) method was used to calibrate and validate the ELISA results and confirm the presence of the detected phytohormones in immunoaffinity-purified extracts. Where independent validation of results is deemed necessary, the use of quantitative HPLC-MS is recommended for each new microalgal strain to be tested.