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

The level of cytokinin type hormones present in plant tissues, such as N6-(2-isopentenyl)-adenosine (IPA), N6-(2-isopentenyl)-adenosine (2-iP), trans-zeatin riboside (ZR) and trans-zeatin (Z) is a good indicator of the resistance of plants to abiotic environmental stresses and to necrotic pathogens. Hapten-homologous and hapten-heterologous competitive indirect enzyme-linked immunosorbent assays (ELISAs) were developed, allowing the use of minute amounts of plant extracts for cytokinin analysis. These assays were used for the detection of members of the cytokinin plant hormone family including IPA and ZR types. The assays, in optimized formats, readily detected these plant hormones at concentration levels of 2–5 ng ml−1, and showed high specificity for selected cytokinins. Certain assay parameters (e.g. the type of tracer enzyme, incubation and preincubation time, etc.) had a strong influence on detection sensitivity. Nonetheless, the assays appear robust showing tolerance to pH and to several water-miscible organic solvents. The described ELISA systems were sensitive enough to detect endogenous hormone levels in crude plant extracts without intense purification. In vitro selected transgenic tobacco and tomato lines showing tolerance to several stress factors proved to have higher levels of cytokinins than the corresponding control plants indicating that the developed immunoassay is suitable for the determination of stress resistance of plants by monitoring their cytokinin content.