Influence of Sample Treatment on Apparent Hydrocyanic Acid Potential of Sorghum Leaf Tissue

When dhurrin [p-hydroxy-(S)-mandelonitrile-tJ-D-glucoside], the cyanogenic glucoside of sorghum [Sorghum bicolor (L.) Moench], is hydrolyzed by autoclaving, p-hydroxybenzaldehyde (P-HB) is released. The spectrophotometric determination of pHB concentration in autoclaved sorghum leaf extracts provides a measure of the hydrocyanic acid potential (HCN-p) of leaf tissue. Extracts of field-grown sorghum leaves contained substances that interfered with this procedure, but ether extraction effectively separated p-HB from these interfering materials. We observed that when flag leaf tissue from field-grown sorghum was dried at 75°C and then autoclaved, HCN-p values were about three times as high as those based on tissue that was autoclaved without drying. Investigations of this apparent enhancement supported the conclusion that when fresh field-grown sorghum leaf tissue was autoclaved, dhurrin was extensively altered or lost, but neither p-HB nor HCN was produced. Drying the tissue at 75°C prior to autoclaving effectively reduced this loss. Inclusion of tissue drying and ether extraction steps in the spectrophotometric assay made this procedure, which was designed for use with sorghum seedlings, satisfactory for use with field-grown sorghum leaves. Additional index words: Cyanogenesis, Dhurrin, p-Hydroxybenzaldehyde, Prussic acid, Sorghum bicolor (L.) Moench, Spectrophotometric assay. AN earlier paper from this laboratory described a procedure for the spectrophotometric assay of the hydrocyanic acid potential (HCN-p) of young chamber-grown sorghum [Sorghum bicolor (L.) Moench] seedlings (2). The procedure was based on determination of p-hydroxybenzaldehyde (P-HB) released when dhurrin [p-hydroxy-(S)-mandelonitrileI3-D-glucoside], the cyanogenic compound of sorghum, was autoclaved in water. The absorbance spectrum of p-HB in alkaline solution has a pronounced peak at 330 nm. Autoclaved extracts of leaves from young seedlings displayed absorbance spectra very similar to that of pure p-HB (2); for such leaves A3 30 values of extracts diluted in base, provided a reliable measure of HCN-p (2,6). This seedling assay procedure was used successfully in a program of divergent selection for HCN-p in sudangrass [So sudanense (Piper) Stapf] (3). Absorbance spectra of extracts from young fieldgrown sorghum tillers differed appreciably from that of pure p-HB (6). Spectra of some tiller extracts lacked a 330-nm peak; those of other extracts had peaks at 330 nm, but their shape suggested extensive non-pHB absorbance at 330 nm. Reliability of the spectrophotometric procedure for use with tillers was improved by fractionating the autoclaved tiller extracts with ether. The p-HB in these extracts was soluble in the ether phase whereas most of the interfering materials remained in the aqueous phase (5). We have observed that when extracts obtained by autoclaving fresh field-grown sorghum flag leaves were diluted in base and scanned, the resulting spectra lacked well defined peaks at 330 nm. However, spectra obtained from oven-dried samples had well 1158 defined 330-nm peaks. The objective of this study was to investigate the effects of leaf drying and other treatments on the spectra of leaf extracts and on the HCN-p values based on these spectra. In the course of these experiments, the spectrophotometric procedure for seedlings (2) was modified, making it satisfactory for use with field-grown sorghum leaves. MATERIALS AND METHODS