Immunoassays for plant cytokinins as tools for the assessment of environmental stress and disease resistance

The level of cytokinin type hormones present in plant tissues, such as N-6- (2-isopentenyl)-adenosine (IPA), N-6-(2-isopentenyl)-adenosine (2-iP), tran s-zeatin riboside (ZR) and trans-zeatin (Z) is a good indicator of the resi stance of plants to abiotic environmental stresses and to necrotic pathogen s. Hapten-homologous and hapten-heterologous competitive indirect enzyme-li nked immunosorbent assays (ELISAs) were developed, allowing the use of minu te amounts of plant extracts for cytokinin analysis. These assays were used for the detection of members of the cytokinin plant hormone family includi ng IPA and ZR types. The assays, in optimized formats, readily detected the se plant hormones at concentration levels of 2-5 ng ml(-1) and showed high specificity for selected cytokinins. Certain assay parameters (e.g. the typ e of tracer enzyme, incubation and preincubation time, etc.) had a strong i nfluence on detection sensitivity. Nonetheless, the assays appear robust sh owing tolerance to pH and to several water-miscible organic solvents. The d escribed ELISA systems were sensitive enough to detect endogenous hormone l evels in crude plant extracts without intense purification. In vitro select ed transgenic tobacco and tomato lines showing tolerance to several stress factors proved to have higher levels of cytokinins than the corresponding c ontrol plants indicating that the developed immunoassay is suitable for the determination of stress resistance of plants by monitoring their cytokinin content. (C) 2000 Elsevier Science B.V. All rights reserved.