Precautions in the Use of Lanolin as an Assay Diluent for Plant Growth Substances.

Lanolin is frequently used as a diluent for applying growth substances 'to plants. Its earliest use was described by LAIBACH (1). He applied unilaterally, to the first internodes of Phaseolus multiflorus seedlings, a lanolin paste containing the test substance. The presence of growth substances induced negative stem curvatures. Subsequently, Laibach and co-workers (2, 3) applied lanolin pastes containing dissolved ether-extractible materials of urine and orchid pollen to the cut stems of Coleus and Tradescantia. Callus formation and rooting were induced. Numerous other workers have since adopted lanolin as a diluent. The use of lanolin has persisted largely because of its desirable physical characteristics, and its apparent inertness toward plant tissues and growth substances. An adaptation of the bio-assay method described by LAIBACH (1) was adopted in studies designed for the eventual isolation of the growth regulating substances in corn pollen. The degree of enrichment obtained from any particular fractionation was measured by the curvature produced on bean seedlings. By successive dilutions, determinations were made of the lowest concentrations dissolved in lanolin, which would produce curvatures of the same magnitude as would the non-diluted starting material. In earlier isolation attempts, even under the best processes, a considerable loss of activity sometimes occurred. It was found that upon standing for 24 hours at room temperature in contact with a 1%o -aqueous solution of hydrogen peroxide much of the growth regulator present in the crude pollen extracts was destroyed. Presumably, other oxidizing agents might also react with the growth regulator(s). Removal of peroxides from all solvents employed in extraction and enrichment processes did not eliminate the loss of activity indicated by the lanolin dilution assays. The probable source of this loss was detected when a qualitative test with acidified potassium iodide showed that the sample of lanolin employed as a diluent contained unidentified oxidizing agents. Subsequently, it was found that treatment of the lanolin with sodium hydrosulphite removed -the oxidizing agents. A product satisfactory for bioassay of natural growth substances may be obtained by shaking out a solution of 25 gm. of lanolin in 250 ml. of ether-with 100 ml. of water in which are dissolved 15 gm. of sodium hydrosulphite and 5 ml. of glacial acetic acid. After separating the two phases, the less dense phase (ether) lJournal Article No. 1105 (n.s.) of the Michigan Agricultural Experiment Station. 356