Assay of p-Hydroxybenzaldehyde as a Measure of Hydrocyanic Acid Potential

A method of assessing the hydrocyanic acid potential (HCN-p) of sudangrass [Sorghum sudanense (Piper) ~tapfl ~nd sorg~um [So bicolor (L.) Moench] seedlings IS described, ThIS procedure is based on the detennination of p-hydroxybenzaldehyde (p·HB), which is released upo,:, hydrolysis of dhurrin, the cyanogen normally prese;"t m plan~s of Sorghum species. Extraction and hydrolvs~s of d~urnn are accomplished by autoclaving young leaf trssue I~ water. The ~ontent of p.HB in the aqueous ~xtract .IS then .determmed by spectrophotometric assay m alkahne solution at 330 nm. Uniform samples for the comparison of widely divergent genotypes are obtained by using the first leaf of young, chamber-grown green seedlings. Relative ranks of a wide variety of s~rghum germplasm assayed for HCN content by this technique arc in good agreement with those obtained by other methods. The procedure shows promise of providing a rapid and precise tool for conducting genetic and breeding studies with sorghums. ---------Additional index words: Dhurrin, Prussic acid, Cyanide, Sudangr~ss, Forage sorghum, Sorghum sudanense, Sm'ghum bicolor. V ARI~US met~lOds .for the determination of cyanogemc glucosides In plants have been reported. Amo?g these are colorimetric (8, 11, 15, 18), Iluorometr~c (10, 28), potentiometric (4, 5, 12), and titrimetnc (1) procedures, all of which are based on the assay of hydrocyanic acid (HCN) released when the cyanogenic gluc.oside is chemically or enzymatically hydrolyzed to YIel~1 HCN, glucose, and aglycone. In most of tl?e published procedures, hydrolysis of the cyanogen IS accomplished enzymatically, using either endogenous or exogenous glucosidases. For colori~etri~ and fluorometric assays, the hydrolyzed sample IS subjected to diffusion, distillation, or aeration, and the volatilized HCN is trapped in an alkaline solution for subsequent assay. These time-consuming procedures help to reduce the e~fects of interfering compounds, but they may result in erroneous values due to incomplete hydrolysis of the cyanogen or incomplete recovery of the released HCN. Much of the early published work on the determination of the cyanogen content or HCN potential (HCNp) of sorghum [Sorghum bicolor (L.) Moench] and sudangrass [S. sudanense (Piper) Stapf] was based on variations of the sodium picrate procedure (9, 15, 24). Because these techniques usually depended upon 1 Contribu~ion from t~e ARS, USDA, and the Nebraska Agric. Exp. Stn., Lincoln. Published as Paper No. 5162, Journal Series, Nebraska Agric. Exp. Stn. The work reported was conducted under Nebraska Agric. Exp. Stn. Project No. 12-088. Received 23 Oct. 1976. 2 Supervisory r~search geneticists, ARS, USDA, and professor of agronomy, Um~. of Nebraska; former research associate, Dep. of Agron~)lny, Univ, of Nebraska, now assistant professor, Dep. of Chemistry, Appalachian State Univ.; assistant professor of agronomy; and foundation professor of agronomy, Univ. of Nebraska, Lincoln, NE 68583, respectively. 578 endogenous glucosidases for hydrolysis of the cyanogen, they s.ometimes permitted erroneously low readIngs resultmg from the incomplete release of HCN from the tissues. In more recent years, the cyanidespecific electrode has been utilized by many investigators to assess the HCN-p of various cultivars. Although the continuous potentiometric method reported by Easty et al. (5) involved volatilization of the HCN prior to measurement, other workers (1, 7, 12) have u~ed the electrod~ for direct measurement of the cyanide content of nssue extracts without volatilization. Blaedel et a1. (4) reported that most constituents of biological samples do not interfere significantly with the response of the electrode, but they noted that, because of unidentified interfering compounds, the electr.ode :vas not satisfactory for direct assay of low-evanide trssue extracts. In spite of this possible weakness, the cyanide-specific electrode has been used extensively in recent investigations (4, 6, 7). The direct potentiometric assay of HCN-p, however, has certain drawbacks for screening large numbers of tissue samples because each sample must be homogenized, and lo.w-HCN samples may require extensive equilibration WIth the electrode. In addition, "poisoning" of the ~lectrode membrane during extended use may further Increase the lag in electrode response, and erroneous readings may result (2). In assaying for plant cyanogenesis, the investigator also has the problem of obtaining uniform and comparable tissue samples from the lines or cultivars among which comparisons may be desired. In the case o.f dhurrin (P-hydroxy-L-mandelonitrile ,B-D-glucoSIde), the cyanogen of sorghum species, the choice of an optimum sampling time and tissue is complicated by the observations that: a) dhurrin content in plants d~cli?es .as the plant. n~atures (1, 16, 19, 22, 30); b) distribution of dhurnn IS not constant throuzhout the I b . '" pant ut IS generally highest in new growth, such as newly emerged seedlings, young leaves, and tillers (3, 19, 22); and c) environmental variables such as water stres~, (23), high N .fertility (13, 17,26), and frost (29) ca,? increase dhurrin content. It has been suggested (16) that tillers 13 to 18 em in length could be used t~ ~rovide uniform samples for assay. However, it is difficult to obtain tillers of uniform size and developn~e~t h:om all.genotypes, because many genotypes exhibit differential dates for the onset of tillerinz and rate of tiller growth (19). A sampling procedur~ that would minimize these difficulties has been greatly needed. Dlll~rrin is one of the few alkali-labile cyanogenic glucosides. Its aglycone, p-hydroxybenzaldehyde (pHil), exhibits strong absorption at 330 nm in alkaline solution (I, 20,21). Akazawa et a1. (1) demonstrated that the contents of HCN and p-HB in alkaline hvdrolysates of young sorghum seedlings were equivaPublished in Crop Science (July-August 1977) v. 17