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Plant and Cell Physiology, 1987, Vol. 28, No. 6 1033-1041
© 1987

Article

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

Susumu Kuraishi, Takashi Ohara and Naoki Sakurai

Department of Environmental Studies, Faculty of Integrated Arts and Sciences, Hiroshima University Higashisendamachi, Hiroshima 730, Japan

Cytokinin contents in cotyledon, hypocotyl and root of etiolated squash (Cucurbita maxima Duch.) seedlings were determined by instrumental analysis using 14C-benzyladenine (14C-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, 14C-BA and a mixture of zeatin (Z) and dihydrozeatin (DHZ). The recovery rate for the cytokinin fractions after LH-20 was corrected by 14C-BA. Each cytokinin fraction was further purified by HPLC which also separated Z and DHZ in the LH-20 fraction. Before permethylation, 14C-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 0.1 ng.

cis-ZK 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 chromatographyselected ion monitoring.

(Received December 26, 1986; Accepted June 1, 1987)
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R. Joseph Neil Emery, L. Leport, J. Edith Barton, N. Clifford Turner, and C. Anthony Atkins
cis-Isomers of Cytokinins Predominate in Chickpea Seeds throughout Their Development
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