Identification of soybean, Glycine max (L.) Merr., cytokinins

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Virginia Polytechnic Institute and State University


Soybean, Glycine max (L.) Merr., fruits 10-25 mm long contained nine fractions having cytokinin-like activity detectable by the Amaranthus bioassay. These fractions were derived from purification of extracts from 15 g of immature fruits. The fractions were purified by polyvinylpyrrolidone (PVP), paper, Sephadex LH-20 and high performance liquid chromatography. The fractions separated on the basis of polarity into polar, moderately polar and relatively non-polar groups, compared to zeatin. The polar fraction was the single most bioactive fraction and the active substance was tentatively identified as a zeatin glucoside or zeatin riboside glucoside. The moderately polar fraction contained bioactive substances that co-eluted with zeatin and zeatin riboside in four chromatographic procedures. The relatively non-polar fraction contained two bioactive components, one chromatographically resembling 2iPA, and the second less polar. A conjugated cytokinin was released from the original aqueous phase by alkaline phosphatase hydrolysis. This substance co-eluted with zeatin riboside on Sephadex LH-20. Further purification and analysis by multiple ion monitoring GC-MS did not yield conclusive identification of the cytokinins. Based on bioassay, the biological estimates of cytokinin-like substances present in the extracts were below the detection limit of the GC-MS. Obtaining sufficiently large samples of soybean fruits at the proper stage of physiological interest complicated definitive structural identification based on the purification and identification techniques used.