Enzyme immunoassay (EIA) of endogenous cytokinins in Citrus

Two sensitive enzyme immunoassays (EIA) for the determination of zeatin riboside (ZR), isopentenyladenosine (IPA), and closely related cytokinins are described. ZR and IPA were conjugated to bovine serum albumin and used as immunogen for the production of two antisera. The assays utilized alkaline phosphatase-cytokinin tracers which were stable for at least 8 months. Only N super(6) substituted purines were detected; common naturally occurring purine bases and nucleosides do not interfere. As little as 50 pg of cytokinin could be detected by this method. For the analysis of cytokinins in plant tissues, extracts were fractionated by high-performance liquid chromatography (HPLC) and quantified by EIA. ZR, IPA, and isopentenyladenine (IP) were detected in Valencia orange and Ruby Red grapefruit flower buds. The levels of all three compounds were found to increase during maturation of Valencia orange blossoms.