Topic 21.3

Various Methods Are Used to Detect and Identify Cytokinins

Naturally occurring molecules with cytokinin activity may be detected and identified by a combination of physical methods and bioassays. The introduction of immunoaffinity purification methods combined with liquid chromatography and mass spectrometry, along with the use of isotopically labeled cytokinins as internal standards, has resulted in major advances in the measurement of endogenous cytokinins (Redig et al. 1996).

Historically, bioassay was the only method available for the identification of molecules that act as cytokinins. Cell proliferation bioassays using tobacco pith tissue or carrot taproot continue to be important because the ability of these tissues to initiate and sustain cell proliferation is proportional to the concentration of active cytokinin in an extract, provided the extract does not contain growth inhibitors. In addition, some continuously cultured callus tissues of tobacco and soybean that cannot grow in culture without cytokinin have been used for cytokinin bioassays. All of these cytokinin-requiring tissues exhibit a linear increase in growth with increasing cytokinin concentration over a fairly broad range, although high concentrations of cytokinin usually inhibit growth (Web Figure 21.3.A).

Web Figure 21.3.A Tobacco tissues depend on cytokinin for their growth in culture. Cultured tobacco callus was transferred to fresh medium that contained an auxin and either zeatin or kinetin at the indicated concentrations. The tissues were weighed after growing for 1 month on the various media. The results show that zeatin is more effective than kinetin in supporting tobacco tissue growth. The maximum response was obtained with 5 × 10^–8 M zeatin, and higher zeatin concentrations either did not stimulate additional growth or were slightly inhibitory. The kinetin concentration had to be at least tenfold higher to achieve the same growth stimulation. (From Leonard et al. 1968.) (Click image to enlarge.)

Immunological methods are very useful for cytokinin identification and quantification. Researchers can produce antibodies against cytokinins by injecting rabbits or mice with cytokinin ribosides conjugated to a protein. Monoclonal antibodies also have been generated that are highly specific for individual cytokinins. These antibodies can be used to quantitate the amount of a cytokinin in a sample by means of a radioimmunoassay (Weiler 1980). For cytokinin isolation, plant extracts are first fractionated, usually by high-performance liquid chromatography (HPLC), and the cytokinins in the fractions are detected and measured by means of a cytokinin radioimmunoassay, similar to the auxin radioimmunoassay described in textbook Chapter 19.

The cytokinin antibodies can also be used to isolate the hormone from extracts by immunoaffinity chromatography (Akiyoshi et al. 1983). Immunological methods hold great promise for the identification and quantification of naturally occurring cytokinins because the antibodies are highly specific and more sensitive than most bioassays (Morris et al. 1991; Nicander et al. 1993). Furthermore, these immunological methods are very rapid.