Quantifying Phytohormones in Transformed Plants

Physicochemical techniques are very specific, sensitive, and accurate techniques widely used for phytohormone analysis (for a review see refs. 1 –3 ). Liquid chromatography mass spectrometry (LC-MS) or gas chromatography-coupled mass spectrometry (GC-MS) in particular, recently have become more important for both qualitative and quantitative analyzes of all phytohormones, except ethylene (for a recent review see ref. 3 ). Specific purification is, however, necessary prior to high-performance liquid chromatography (HPLC), gas chromatography (GC), LC-MS, or GC-MS. On the other hand, we have immunological techniques available through radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). The advantages of immunochemical techniques are their high sensitivity, high specificity, and an extremely short analysis time. There are a lot of reports on poly- and monoclonal antibodies for the analysis of plant hormones including cytokinins (4 –7 ). The mass spectrometric fragmentation pattern from electron impact (EI) GC-MS is a frequently used identification criterion. Using LC-MS for qualitative and quantitative cytokinin analysis, derivatization can be omitted (8 ). Moreover, cytokinins exhibit strong specific UV absorbance in the 220–300 nm range. Since UV spectroscopy is nondestructive, mass spectrometry and UV spectroscopy are complementary for the identification of cytokinins (9 ). Recently, qualitative LC-MS analysis has also been described for abscisic acid (ABA) (10 ), however, deuterated ABA obtained by active hydrogen exchange (11 ) is not suitable for thermospray conditions (our unpublished results).