High Performance Liquid Chromatographic Analysis of Cytokinins in Sorghum bicolor Leaves

High performance liquid chromatography with octadecylsilica (Bondapak C₁₈/Poracil B) column packing was used to purify and separate cytokinins in Sorghum leaf extracts. The column size was 56 × 0.21 cm i.d. By gradient elution, using acidified water with increasing amounts of methanol, the major peaks of cytokinin activity, as determined by the callus tissue bioassay. were effectively separated from large amounts of extraneous impurities. These cytokinins were further separated on a microoctadecylsilica column (μBondapak C₁₈, 30 × 0.4 cm i.d.) with a gradient of acidified water-acetonitrile. Zeatin and zeatin riboside gave distinct ultra violet absorption peaks which could be used for quantitative estimation. Biological activity corresponded to the elution of these peaks. These two cytokinins are the major cytokinins in Sorghum leaves.     

Diffusible Cytokinins in shoot apices of Dahlia variabilis

Cytokinin activity (soyabean callus assay) has been determinedin excised apical buds of Dahlia variabilis before and aftera period of 3 h with cut surfaces in contact with agar gel,in the agar gel and in xylem exudates from cut shoot stumps. Buds before diffusion gave three peaks of activity in the butanolfraction, one in the aqueous fraction, following paper chromatography.Two of the former diffused into agar gel, the third (in whichmost activity was recorded) decreased in level during the 3-hperiod but was absent from the agar diffusate. The water-solublecytokinin remained at its original level and was absent fromthe agar diffusate. The three peaks of activity in the butanolfraction were also present in xylem exudates. Ether and ethylacetate fractions contained callus-growth inhibitors which diffusedinto agar gel.