The Recovery of Cytokinins during Extraction and Purification of Clubroot Tissue

Abstract

AbstractLosses of one naturally occurring cytokinin (zeatin) and one synthetic cytoknin (kinetin) were determined during purification of turnips (Brassica compestris) infected by Plasmodiophora brassicae (clubroot). A known amount of zeatin and 8‐14C‐kinetin was added after homogenization of plant material in ethanol or water. The commonly used practice to purify the aqueous residues of the homogenate by partitioning with petroleum ether was omitted because of emulsion formation. Losses due to emulsion formation and occlusion of 8‐14C‐kinetin into non‐water soluble plant material could be prevented by extractionof clubroot tissue with water instead of ethanol. To minimize enzyme activity the aqueous homogenate was kept at 100°C for 5 min. High molecular weight compounds were removed by dialysis against water and the diffusible fraction was partitioned with n‐butanol at pH 8.2.It was shown that a rapid evaporation of n‐butanol under reduced pressure at high temperature caused less breakdown of 8‐14C‐kinetin than prologned treatment at a low temperature.To minimize breakdown to zeatin riboside the butanol fraction was purified further on cation cellulose‐phosphate exchanger instead of on strong acid Dowex H+. 8‐14C‐kinetin was separated from zeatin by column chromatography on Sephadex LH20, and yielded 86% of the amount originally added to a plant homogenate.The zeatin containing fractions were further purified on thin layer chromatography (TLC) silicagel plates and injected into a high pressure liquid chromatograph. A yield of 60% could be estimated from the amount (15 μg) orignally added to 50 g clubroot tissue.