Gas Chromatography-Flame Thermionic Detector Analysis of Cytokinins in Etiolated Squash Seedlings using ^<14>C-BA as an Internal Standard

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Cytokinin contents in cotyledon, hypocotyl and root of etiolated squash (Cucurbita maxima Duch.) seedlings were determined by instrumental analysis using ^<14>C-benzyladenine (^<14>C-BA) as an internal standard. Crude extracts were purified using insoluble polyvinylpyrrolidone, cellulose-phosphate column and SEP-PAK C18 cartridge, then applied to a Sephadex LH-20 column to separate zeatin riboside (ZR), isopentenyl adenosine, isopentenyl adenine, ^<14>C-BA and a mixture of zeatin (Z) and dihydrozeatin (DHZ). The recovery rate for the cytokinin fractions after LH-20 was corrected by ^<14>C-BA. Each cytokinin fraction was further purified by HPLC which also separated Z and DHZ in the LH-20 fraction. Before permethylation, ^<14>C-BA was added to each of the cytokinin fractions to correct the methylation rates. Each methylated cytokinin fraction was again purified by HPLC, then subjected to gas chromatography with a capillary column and flame thermionic detector. The detection limit of cytokinins by this system was O.1 ng. cis-ZR was the most abundant cytokinin in all tissues of the etiolated squash seedlings. Active cytokinins such as trans-ZR and trans-Z were mostly found in cotyledons with lesser amounts in the roots. DHZ was most abundant in the cotyledon. All cytokinins isolated by this procedure were confirmed by gas chromatography-selected ion monitoring.
日本植物生理学会の論文