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Grain sorghum [Soirghum bocolor (L.) Moench] typically produces larger seeds than sudangrass [S. bicolor, formerly S. sudanense (Piper) Stapf]; and grain sorghum seedlings are higher in hydricyanic acid potential (HCN-p) than sudangrass seedlings. Previous studies have shown a seed-parent effect on seed weight and HCN-p in reciprocal F 1 hybrids of sorghum × sudangrass

Spectrophotometry is a useful assay for hydrocyanic acid potential (HCN-p) in sorghum and sudangrass [both Sorghum bicolor (L.) Moench] seedlings, but no systematic study of sources of variation in the procedure has been reported. Selfed seed was harvested from each of 12 ramets (two each from two sister plants from three low-HCN-p sudangrass parents), and seedlings for rumpling were grown in a growth chamber, in two rows from each ramet. Seven-day-old seedlings were harvested and divided into two samples per row for extraction; two aliquots per extract were assayed spectrophotometrically for HCN-p. The experiment was replicated three times. The three parents differed in HCN-p [...]

Sorghum [Sorghum bicolor (L.) Moench ‘Early Hegari’] seedlings were grown in nutrient solutions with various combinations of chlorides, nitrates, sulfates, and phosphates (dihydrogen) of ammonium, potassium, calcium, and magnesium to determine the effects of the mineral elements on hydrocyanic acid potential (HCN-p) in leaves. Growth was maximum or near maximum for the shoots of sorghum seedlings at 1 to 30 meq salt in solution and for the roots at 0.3 to 3 meq. The duration of exposure of seedlings to the treatment solution required for significant changes in HCN-p was at least 2 days. The effects of various concentrations of individual salts on HCN-p were: KCl, a slight decrease; K2SO4, NH4Cl, NH4NO3, (NH4)2SO4, NH4H2PO4, and KH2PO4)2, an increase; and KNO3, CaCl2, Ca(NO3)2, CaSO4, MgCl2, Mg(NO3)2, and MgSO4, no significant effect. When potassium and ammonium salts of a common anion were added in 3 × 3 factorial experiments, increasing levels of ammonium salts at the 1X potassium level generally increased HCN-p, but varied effects of higher levels of potassium salts were observed. In factorial experiments, different levels of pairs of potassium salts of the various anion had no significant effect on HCN-p. In experiments with pairs of ammonium salts, increases in salt concentration generally increased HCN-p. It was concluded that large changes in salt concentration affected alterations in HCN-p were associated with changes in ammonium salt levels

Two field studies involving different groups of germplasm were conducted to compare sorghum-sudangrass, Sorghum bicolor (L.) Moench X S. sudanense (Piper) Stapf, hybrid seed production of male-sterile FI's with their component A·lines. The FI's yielded 45 and 82% more grain than their A·line counterparts in Study I and 2, respectively, with the increase attributable to more seeds per panicle and a greater threshing percentage. Mean values of nine traits measured on nine groups of eight FI's in Study I and seven groups of six FI's in Study 2 were compared with their respective A·lines. All groups of FI's significantly outyielded their A·line counterparts in both studies except for A·line N35 in Study 1. Phenotypic correlations of yield and seeds per panicle were high in both A·lines and FI's in both studies, as were the correlations of yield and threshing percentage except for the FI's in Study 1. Application of these findings should facilitate the production of higher yields of sorghum-sudangrass hybrid seed at reduced cost per unit of seed. The best FI's to use and the magnitude of their superiority over A·lines will be influenced by the location in which seed is produced. Additional indexwords:Sorghum bicolor (L.) Moench, Sorghum sudanense (Piper) Stapf, Single-cross hybrids, Three-way hybrids. SORGHUM-SUDANGRASS, Sorghum bicolor (L.) Moench X S. sudanense (Piper) Stapf, hybrids are grown extensively to provide supplementary forage for animals as pasture, silage, or greenchop. These hybrids play an important role in the management plans of many livestock producers, particularly in drouth-prone regions such as the Great Plains. In a 1977 survey by Harvey (I), 81.5% of the commercially produced sorghum-sudangrass hybrids in the United States were F I 's, with 'Redlan' grain sorghum being the preferred male-sterile and 'Greenleaf sudangrass the most often used pollinator. Also, three-way cross hybrids made up I I% of the total seed production of sorghums and sudangrasses used for forage compared with less than 2% in grain sorghum (I). Despite this rather extensive use of three-way crosses in forage and sudangrass hybrids, only two preliminary reports were found in which the seed production of three-way and single cross hybrids was compared. In a I-year study of seed production of sorghum-sudangrass hybrids in Hungary (3), seed yields of F I male steriles were greater and production was more dependable than for Alines because the F1's matured earlier, but comparative seed yields of three-way and single cross hybrids were not presented. In Japan (6), four grain sorghum A-lines, six grain-sorghum male-sterile F I 's and six sorgo male-sterile F I 's were used in a comparative I Contribution of USDA-ARS and the NebraskaAgric. Exp. Stn. Published asPaper no. 7415, Journal Series, Nebraska Agric. Exp, Stn., Lincoln. Research was conducted under Nebraska Project no. 12-114. Received 29 Feb. 1984. 2 Supervisory research geneticist, USDA-ARS; research technologist inagronomy;George Holmes professorof agronomy; and research geneticist, USDA-ARS, Lincoln, NE68583, respectively. study of seed production of forage sorghum hybrids. Seed weight per head on the grain-sorghum malesterile F I 's was slightly lower than for the A-lines, while seed weight per head on the sorgo male-sterile F I 's was substantially greater than on either of the other two female types. Seed yield per land area was not given. The A-lines used differed in each of the three groups of material, and seed weights of F I 's could not be compared with those of their component A-lines. Although published information on comparisons of three-way with single cross forage-type hybrids is limited, more extensive literature is available for grain sorghums as indicated in a recent report (2). The objectives of this study were to I) compare seed yield and other agronomIC traits between malesterile F I seed parents and their component A-line seed parents in the production of sorghum-sudangrass hybrids in two different groups of germplasm, 2) determine phenotypic correlations among traits within parental groups, and 3) relate the findings to hybrid seed production. Evaluation of the hybrid seed produced in terms of forage production will be reported in a subsequent paper. MA TERIALS AND METHODS The seed parents used in Study 1 were nine combineheight cytoplasmic male-sterile A-lines and their 36 malesterile AX B-line crosses (FI'S) that were identical to those used in a similar study with grain sorghum hybrids (2). The A-lines included KS4, KS23, 'Martin', N30, N35, N36, N38, WD4, and 'Wheatland'. In Study 2, the seed parents were seven A-lines and their 21 male-sterile F t's. The A-lines were KS5 and KS9, described by Ross et al. (4); N38 and N48, described by Ross et al. (5); N4692, described by Webster et al. (7); N50I3, an experimental line; and the cultivar Redlan. The pollen source was produced from a composite of equal seed weights of the sudangrass cultivars Greenleaf and 'Piper', Nebraska 7035, and experimentallow-dhurrin strains of Greenleaf and Piper. This heterogeneous pollen source assured continuous and adequate pollen dispersal to the female parents over the range of their stigma receptivity. The sudangrass composite was seeded in double rows, with six rows of male-sterile sorghums being grouped between each pair of sudangrass rows. The outside rows of each group of six male-steriles were seeded to cytoplasmic male-sterile 'Combine Kafir-60', which served as unharvested border rows to minimize any effect of the taller sudangrass rows on the four randomly assigned male-sterile lines and F I hybrids that were harvested in each group. The experiments were planted 29 May 1979 and 27 May 1980 at the University of Nebraska Field Laboratory, Mead, in a medium textured Sharpsburg silty clay loam soil (fine, montmorillonitic, mesic Typic Argiudoll) to which 112 kg haI of N had been applied. Four replications of singlerow plots 7.6 m long and 0.76 m apart in a randomized complete block design were used each year. All plots were overseeded and thinned to 15 em between plants, giving a plant population of about 87 000 plants ha1• The exper1134 Published in Crop Science (November-December 1984) v.24, no. 6

A method of assessing the hydrocyanic acid potential (HCN-p) of sudangrass [Sorghum sudanense (Piper) Stapf] and sorghum [S. bicolor (L.) Moench] seedlings is described. This procedure is based on the determination of p-hydroxybenzaldehyde (p-HB), which is released upon hydrolysis of dhurrin, the cyanogen normally present in plants of Sorghum species. Extraction and hydrolysis of dhurrin are accomplished by autoclaving young leaf tissue in water. The content of p-HB in the aqueous extract is then determined by spectrophotometric assay in alkaline 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 sorghum germplasm assayed for HCN content by this technique are 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

The variation among 49 F1 forage sorghum [Sorghum bicolor (L.) Moench] hybrids from a 7 x 7 cross-classified design was explored in 1979 and 1980 for the following traits: dry matter (DM), crude protein (CP), in vitro dry matter disappearance (IVDMD), neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL). Differences were found among hybrids for all traits. Parental means for males generally had a wider range of variation than for females. The means of hybrids involving the male parent, 'White Collier', were highest in IVDMD, and lowest in NDF, ADF, and ADL. IVDMD was the only trait that showed significant differences among hybrids averaged over both males and females. General combining ability (GCA) effects were more important than specific combining ability effects. Our results suggest that the most rapid way to improve forage sorghum quality would be by improving IVDMD. Significant differences among hybrids averaged over both male and female parents were shown for this trait; it was affected relatively little by year effects and exhibited high GCA effects.

In a study of seedlings of 72 entries representing 39 species, 14 genera, and 2 tribes of grasses, only entries of the genus Sorghastrum were found to contain the cyanogenic glucoside, dhurrin [(S)-p-hydroxymandelonitrile β-D-glucopyranoside], Dhurrin was found in seedlings of each of the 10 entries of indiangrass [Sorghastrum nutans (L.) Nash] and the three entries of S. pellitum. Seedlings of five indiangrass cultivars were assayed for dhurrin concentration. Levels expressed as HCN potential (HCN-p) ranged from about 900 ppm for ‘Holt’ to about 1,200 ppm for ‘Llano’ and ‘Oto’.Further studies with Oto seedlings indicated that first leaves were higher and more uniform in HCN-p than were other portions of the shoot, but all shoot portions contained dhurrin. Seedling roots also contained dhurrin, but the HCN-p of roots was appreciably lower than that of shoots. Spectral evidence indicated that dhurrin was present in extracts of leaves of tillers harvested from field-grown plants. Dhurrin was not detected in all such tillers, however. Indiangrass tiller extracts contained more interfering substances than seedling extracts, in agreement with previous observations on Sorghum seedlings

Alteration in hydrocyanic acid potential (HCN-p) of the forage is one of the important objectives in sudangrass [Sorghum sudanense (Piper) Stapf] breeding programs. The effectiveness of recurrent phenotypic selection for increasing or decreasing HCN-p in sudangrass was evaluated in two cycles of individual plant selection in the cultivar 'Greenleaf'. In cycle 1, HCNP means of the high and low populations were higher and lower, respectively, than for Greenleaf, but only the low-HCN-p population was significantly different from Greenleaf. In cycle 2, mean HCN-p values of both populations differed significantly from Greenleaf. The average realized heritability for the two cycles was 0.40 while broad-sense heritability estimates averaged 0.86. After two cycles of selection, the low and highHCN-p populations differed from Greenleaf by about 17 and 30%, respectively. Additional index words: Prussic acid, Dhurrin, Forage quality, Heritability, Sorghum sudanense. SU D A N G R A S S [Sorghum sudanense (Piper) Stapf] and sorghum-sudangrass hybrids are used extensively to provide supplementary feed to animals as pasture or greenchop. Precautions in managing the crop are necessary to prevent animal losses due to prussic acid (hydrocyanic acid) poisoning. All known sudangrasses and sorghums [SO bicolor (L.) Moench] contain dhurrin [(S)-phydroxymandelonitrile (J-D-glucopyranoside] which yields hydrocyanic acid when hydrolyzed enzymatically in disrupted plant tissues or in the rumen of consuming anim';lls. Breed.ing sudangrasses with lowered hydrocyanic acid potential (HCN-p) would reduce the danger of hydrocyanic acid poisoning, and permit greater flexibility in the management of this crop. Also, sudangrasses with reduced HCN-p would be useful in the development of sorghum-sudangrass hybrids with lowered HCN-p. 'Contribution from USDA-ARS and the Nebraska Agric. Exp. Stn., ~incoln. Published as Paper No. 6173, Journal Series, Nebraska Agric. Exp. Stn. Received 18 Feb. 1981. The work reported was conducted under Nebraska Agric. Exp. Stn. Projects 12-088 and 12-114. 'Supervisory research geneticist, USDA-ARS; George Holmes professor of Agronomy, Univ. of Nebraska; and research agronomist, USDA-ARS, Lincoln, NE 68583, respectively. GORZ ET AL.: HYDROCYANIC ACID POTENTIAL IN SUDANGRASS 323 Breeding for lower HCN-p in sudangrass was facilitated by the recent development of a simple, rapid, nondestructive spectrophotometric procedure (6). This procedure involves the assay of individual first leaves from 7-day-old seedlings grown under controlled conditions. Following completion of the assay, selected seedlings can be transplanted to the field or greenhouse for the production of self or cross-pollinated seed for use in the next cycle of selection. In a review of studies of the inheritance of cyanogenesis in sudangrass and sorghum, Nass (9) reported that dominant or partially dominant factors were involved in the genetic control of both high and low HCN-p. Most studies suggested multigenic inheritance although one or two major genes also were hypothesized. Hogg and Ahlgren (8) evaluated 175 inbred lines ranging from low to high in HCN content during a 3-year period and reported that HCN content of the lines was stable over years. They also reported that low-HCN strains could be developed by crossing low-HCN inbred lines. Barnett and Caviness (1) reported broad-sense heritability estimates of 0.41 and 0.68 for HCN production in populations derived from two sorghum X sudangrass crosses. Sorghum is classified as a predominately self-pollinated crop with outcrossing averaging 6% (10). The outcrossing percentage of sudangrass is usually higher, but reported values vary quite widely. Garber and Atwood (5) observed 76.4, 18.2, and 34.4% cross-pollination in Pennsylvania for the years 1941, 1942, and 1943, respectively, while Hogg and Ahlgren (8) reported in 1943 that cross-pollination in Wisconsin ranged from 4.5 to 10%. Thus, extensive cross-pollination is possible, but the percentage that occurs in a specific seed field is apparently dependent upon the location, environmental factors, and the type of sudangrass being grown. Recent work by Foster et al. (4) demonstrated positive results for bi-directional mass selection in a grain sorghum population. Thus, selection based on population improvement techniques, developed for use with cross-pollinated crops, may be successfully used with crops such as sorghum that have a very low percentage of cross pollination. 'Greenleaf sudangrass is used extensively as the male parent in commercial production of sorghum-sudangrass hybrids (7). In 1977, a program of recurrent selection for low and high HCN-p in Greenleaf sudangrass was initiated at the Nebraska Station. The objectives of this research were to evaluate the effectiveness of recurrent selection for increasing or decreasing HCN-p in sudangrass populations, and to develop a reselected Greenleaf strain with lower HCN-p than the parent. MATERIALS AND METHODS All HCN-p values in this study were obtained by use of the spectrophotometric procedure (6). In this procedure, first-leaf samples from week-old seedlings were weighed, and dhurrin was extracted and hydrolyzed by autoclaving the samples in water. Aliquots of the extracts were then diluted in base, and absorbance was read at 330 nm, the absorption maximum of p-hydroxybenzaldehyde. HCN-p values were derived from the 330 nm absorbance (A330) readings by simple calculations (6). The initial population consisted of 184 seedlings of the cultivar Greenleaf grown from Kansas foundation seed. From this initial population, 19, 17, and 17 seedlings were selected to represent the lowest, intermediate, and highest HCN-p levels in further studies. The selected seedlings were transplanted to the field, heads were bagged prior to anthesis, and selfed seed was harvested. Five plants from each HCN-p group with adequate supplies of selfed seed were selected for further study. Seven replications of the seed from the 15 selected plants were planted for assay of HCN-po A total of 1405 S, seedlings were assayed with a range of 81 to 110 per line. This phase of the study will be referred to as cycle O. For the next step, cycle 1, only those seedlings with the highest and lowest levels of HCN-p were selected from the total of 1405 S, seedlings obtained from the 15 original plants. Selections were made across all replications and without regard to the original HCN-p group (high, medium, or low) from which the seedling originated. Thus, the 26 seedlings selected for lowHCN-p included 14, 9, and 3 seedlings from the low, medium, and high-HCN-p groups, respectively. Similarly, the 23 seedlings selected for high-HCN-p included 9, 5, and 9 seedlings from the low, medium, and high-HCN-p groups, respectively. The low and high-HCN-p populations were transplanted to separate field isolations in 1978, with plants on 61-cm centers. Open-pollinated seed was harvested from individual plants. Three replications of 10 seedlings each from each line (i.e., from each parent plant) were assayed for HCN-p. Greenleaf was included in each replication as a control. Plants for the next cycle of selection (cycle 2) were selected from individual seedlings of the 8 lines whose mean HCN-p was lowest among the 26 low-HCN-p lines, and from the highest 8 lines among the 23 high-HCN-p lines. The selected low and high-HCN-p populations included 54 and 56 plants, respectively. Each population was transplanted to a separate field isolation in 1979, with plants randomized on 107-cm centers. A severe infestation of chinch bugs [Blissus leucopterus leucopterus (Say)] destroyed some plants in the low-HCN-p isolation; seed was produced on only 33 of these plants. Open-pollinated seed was harvested from individual plants, and three replications of 10 seedlings from each plant that produced sufficient seed (31 plants from the low-HCN-p isolation and 55 from the highHCN-p isolation) were assayed for HCN-p. Greenleaf was included in each replication as a check. The breeding procedures used and the time period covered by each cycle are summarized in Table 1. A narrow-sense heritability estimate was obtained by the regression of offspring produced by selfing on their parents in cycle O. In this situation, bop, the regression of offspring on parents, was used as the heritability estimate (H,,). Realized heritability estimates (Hr) (3) were also determined for gcles 0, 1, ~d 2 using thefollowing equation: Hr = CX.,h Xo,)/(Xp" X p,) where the X's are means and the subscripts, 0, p, h, and 1 are offspring, parent, high and low, respectively. Variance components from the analyses of the replicated tests of the progeny of each cycle were used to calculate the ratio: Hv = a a'e)where a a the genetic and environmental components of variance, respectively. This ratio was difficult to interpret for this study because it was unknown whether the parents were homozygous or heterozygous individuals or whether the progenies were the products of crossing, selfing, or both. It is an estimate, however, of the variance among lines that is due to genotypic differences and as such can be used as a broad-sense heritability estimate. Gain from selection (Gs) was calculated both as a deviation from Greenleaf (G",) and as a deviation percentage from Greenleaf (G,p) for cycles 1 and 2. The equations used were as follows: (a) G'd for low HCN-p = (X-J X~. (b) G'd for high HCN-p = QCOh -_XgL (c) G,p for low HCN-p = Ph X~/~ X 100. (d) G,p for high HCN-p = (X"h Xg)/Xg X 100. 324 CROP SCIENCE, VOL. 22, MARCH-ApRIL 1982 Table 1. Means, standard deviations, and ranges for HCN·p of parents and their progenies for two cycles of divergent selection for HCN·p in Greenleaf sudangrass.

The hydrocyanic acid potential (HCN-p) of sorghum [Sorghum bicolor (L.) Moench] plants is recognized as a heritable trait, but previous tudies on the mode of inheritance of HCN-p have produced inconsistent results. The objective of this study was to investigate the inheritance patterns of seedling HCN-p and also of seed weight in reciprocal crosses of sorghum and sudangrass [formerly S. sudanense (Piper) Stapf]. Both traits were found to be inherited quantitatively. Generation means analysis indicated that additive genetic effects were most important for both seed weight (53% of variation) and seedling HCN-p (74% of variation). A maternal effect was found for both traits in the F~ and backcross generations. No evidence of this reciprocal effect was found in the F2, suggesting that cytoplasmic inheritance was not involved. A highly positive correlation between seed weight and HCN-p was found across all entries, across both parents, and in the F~ and backcross generations. However, correlation coefficients between seed weight and seedling HCN-p for individual entries, the pooled F2’S, or within types of seed parents in the F~ or backcross generations were generally nonsignificant. It was concluded that seed weight per se does not have a large effect on seedling HCN-p. Additional index words: Dhurrin, Prussic acid, Sorghum blcolor (L.) Moench, S. sudanense (Piper) Stapf. p REVIOUS studies of the inheritance of the cyanogenic glucoside, dhurrin [p-hydroxy-(S)-mandelonitrile-#-D-glucoside], in sorghum [Sorghum bicolor (L.) Moench] have not yielded consistent results. Nass (17) in 1972 reviewed published reports on the inhero itance of cyanogenesis in sorghum, various Lotus species, and white clover (Trifolium repens L.), and concluded that the situation in sorghum was more complex than that in other species. The studies agreed that the dhurrin content or hydrocyanic acid potential (HCNp) of sorghum leaves was a heritable trait, but disagreed on the presence or absence of dominance, dominance of high or low HCN-p, and the number of genes involved. Krauss (14), in a study not included in Nass’s review, concluded that HCN-p was conditioned by four genes with additive effects without dominance. Other studies conducted since Nass’s review reported different conclusions on the importance of additive and nonadditive effects (2, 4, 9). Recently, Gorz et al. (10) found that seedlings of sorghum lines KS8 and N32 were high in HCN-p, but the HCN-p of flag leaves from field-grown plants of KS8 was only about 1/10 as high as that of N32 flag leaves. A single gene pair was found to be primarily responsible for the large difference in mature leaves, and neither low nor high HCN-p was completely dominant. The lack of agree~ Contribution from the USDA-ARS and the Nebraska Agric. Res. Div., Lincoln, NE 68583. Published as Paper no. 8119, Journal Series, Nebraska Agaric. Res. Div. Research was conducted under Project 12-114. Recetved 7 Aug. 1986. -~ Former graduate research assistant, George Holmes professor of agronomy, and supervisory research geneticists, USDA-ARS, respectively. Published in Crop Sci. 27:522-525 (1987). ment among these studies was no doubt due in part to differences in lines or cultivars used, conditions of growth, types of tissue assayed, and the analytical procedures used. The primary objective of the present study was to investigate the inheritance of seedling HCN-p in crosses of grain sorghum and sudangrass [formerly S. sudanense (Piper) Stapf] lines. The sudangrass lines had been selected for low HCN-p; crosses of these lines to the high-HCN-p sorghum lines provided a wider range in HCN-p than was possible in most of the previous studies of HCN-p inheritance. Also, this study utilized the spectrophotometric assay described by Gorz et al. (8). This assay is independent of the activity of catabolic enzymes in the plant. MATERIALS AND METHODS Four inbred parental lines were used in this study. Two were lines from sudangrass populations, and two were grain sorghum cultivars. The two sudangrass lines, 81-1901-7 and 81-1904-74, hereafter referred to as 1901 and 1904, respectively, were selected primarily for low HCN-p. Both lines carried the ms3 gene for genetic male sterility (gms); this recessive gene caused 25 to 30% of the plants in each line to be sterile. Both sudangrass lines also restored fertility when crossed with cytoplasmic-male-sterile (cms) sorghum lines having A1 cytoplasm. The population from which line 1901 was selected has since been released and registered as NP25 low-dhurrin sudangrass germplasm (12). Line 1904 was selected from a population with most of its background from ’Piper’. Both 1901 and 1904 are very low in seedling HCNp compared to commercially available sudangrasses, and they are considered to be homozygous for low HCN-p. The two grain sorghums used were ’Redlan’ and ’Combine Kafir 60’ (CK60). A-lines (A 1 cytoplasm) and B-lines of these cultivars have been maintained for at least 10 yr by making paired crosses annually to produce A-line seed and also selfing the B-line plants used in the A-line crosses. The lines are considered to be homozygous for high seedling HCN-p. All possible crosses, including reciprocals, were made among the four parental lines. Crosses were made using gms plants from the sudangrass lines and cms plants from the sorghum cultivars as females. Use of these male-sterile plants allowed for production of a large number of FI seeds with minimal risk of contamination during crossing. The FI plants were selfed to produce F2 seed, and were also used as pollen sources in backcrosses to gms sudangrass and cms sorghum parental plants. To get backcross seed from the sorghum × sorghum F~’s, the cms hybrids were used as females, and pollen was taken from the parental sorghum B-lines. All crosses were made at the University of Nebraska Agronomy Farm, Lincoln, NE, in the summers of 1982 and 1983 or in the greenhouse in early 1984. All seedlings analyzed in the laboratory were grown from seed produced either in the field in 1983 or in the greenhouse in early 1984. In total, 52 entries were assayed in the laboratory, including 12 F~’s, 10 F2’s, 24 backcrosses, and 6 parental lines (1901, 1904, ARedlan, BRedlan, ACK60, and BCK60). Twenty-five seed packets were prepared for each of the 52

Vanillin-HCl procedures are widely used for the assay of tannins in plants. In attempts to adapt such procedures for use with sorghum [Sorghum bicolor (L.) Moench.] forage it was found that false positive reactions resulted, that is, red color developed in the presence of HCl with or without vanillin. Leucoanthocyanidins (monomeric proanthocyanidins) may be the constituents responsible for this red color. A "chloroform-HCl" procedure was developed for measuring leucoanthocyanidins in sorghum forage. The procedure avoids interference by chlorophyll or other chloroform-soluble constituents. With vanillin added to the solution, this procedure should also be useful for the assay of condensed proanthocyanidins (tannins). Additional index words: Sorghum bicolor L. Moench., Leucoanthocyanidin, Proanthocyanidin, Forage quality. A indicated by Sarkar et al. (14), tannins may be regarded as desirable (for their possible protection against bloat in grazing animals) or undesirable (for their adverse effect on digestibility) constituents of forages. Therefore, either increased or decreased tannin content might be a reasonable breeding objective, depending upon the nature and proposed use of the crop in question. Whether working toward increased or reduced levels of tannins, the breeder needs a simple, rapid, and reliable procedure for tannin assay. Vanillin-HCI procedures such as that described by Burns (2) have been developed for this purpose. In this procedure, dried and ground plant samples are extracted with methanol, and portions of the resulting extract are reacted with a solution containing vanillin and HCI in methanol. The development of a red color indicates the presence of tannins. Vanillin-HCI and vanillin-H2S04 reagents have been used extensively for the detection and assay of tannins and related substances in leguminous forage crops (2, 8, 13, 14) and in sorghum [Sorghum bieolor (L.) Moench] grain (1, 3,5,9, 11, 12). Reports of the use of vanillin-HCI proI Contribution from ARS, USDA, and the Dep. of Agronomy, Nebraska Agric. Exp. Stn., Lincoln. Published as Paper No. 6680, Journal Series, Nebraska Agric. Exp. Stn. Received 22 Apr. 1982. The work reported was done under Nebraska Agric. Exp. Stn. Project 12-114. 2 Former graduate research assistant; George Holmes professor of agronomy; and supervisory research geneticist, ARS-USDA, and professor of agronomy, respectively; University of Nebraska, Lincoln, NE 685830915. Current address of the senior author: FFR Cooperative, West Lafayette, IN 47906. 3 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA or the Univ. of Nebraska, and does not imply its approval to the exclusion of other products that may also be suitable. 197 cedures for the assay of tannins in sorghum forage are much less numerous. However, Cummins (4) and Paroda et al. (10) used the vanillin-HCI procedure of Burns (2), and Gourley and Lusk (6) used that of Maxson and Rooney (9) for estimates of forage sorghum tannins. In some of the cited studies precautions were taken to avoid false positive tests for tannins. For example, Price and Butler (11) and Sarkar and Howarth (13) used controls in which vanillin was omitted from the vanillin-HCI reagent, and EI Tuhami et al. (5) used extracts without either vanillin or HCI as controls. In the cited reports of tannins in sorghum forage, however, it appears that such controls were not used. In preliminary attempts to use a vanillin-HCI procedure to detect tannins in sorghum forage, we observed a false positive reaction for some cultivars, that is, leaf extracts from these cultivars produced a red color in the presence of HCI with or without vanillin. This paper presents the results of a series of experiments in which these false positive tests were investigated. MATERIALS AND METHODS Blades of upper leaves of field-grown forage sorghum plants were used in these experiments. Most of the plants were at or just beyond the stage of panicle emergence when sampled. Leaves were dried at about 75 C and were then ground in a Wiley3 mill to pass a I-mm screen. The ground tissue was stored in tightly covered jars in the dark at room temperature until used. The vanillin-HCl assays described by Burns (2) and Maxson and Rooney (9) served as the basis for the procedures used in this study. The Maxson and Rooney procedure differs from that of Burns in that 1 % concentrated HCl in methanol rather than pure methanol is used as the extractant. Unless otherwise indicated, leaf extracts were prepared by placing ground leaf tissue in test tubes or flasks, adding methanol, capping the vessels, and shaking the mixtures occasionally over a period of 16 to 24 hours at room temperature. Filtrates or supernatant solutions from these extractions were used in the assays. Further procedural details are given in connection with specific experiments. Reagent grade HCl and organic solvents were used in all experiments. Vanillin and D-catechin were obtained from the Sigma

Recent emphasis on energy problems has stimulated efforts to identify crops capable of producing high yields of biomass that can be converted into ethanol. The sorghum [ Sorghum bicolor (L.) Moench] crop includes cultivars and hybrids that vary widely in the relative amounts of grain and stalks produced, and also in the sugar content of the stalks. N39 × Wray, a relatively sweet hybrid with good grain potential, and Wray, a cultivar with sweet stalks, were compared for their grain and sugar yields, alcohol production potential, and other related characters. The hybrid produced heavier seeds, more seeds per head, and more heads per ha than the Wray. This resulted in a grain yield of 6630 kg/ha, about 3.4 times that of Wray. Wray produced about 10% more biomass with the stalk sugars accounting for about 80% of the total alcohol potential as compared to 45% for the hybrid. The total alcohol production potential (stalk plus grain) was 1.11 times as great for the hybrid as for Wray.

This study was conducted to evaluate a wider range of plant types and agronomic and quality traits than had been previously reported for blends of sorghum [Sorghum bicolor (L.) Moench]. Three groups of sorghum cultivars and hybrids, each including a i-dwarf (tall), a 2-dwarf (medium), and a 3-dwarf (short) sorghum, were used in a 2-year study. Within each group the following seven blends were compared: tall (T), medium (M), and short (S); T and M; T and S; M and S; T alone; M alone; and S alone. Principal emphasis was given to total dry matter (DM) production (sum of leaves, stems, and heads). It was concluded that blends consisting of entries differing in stature had no significant DM yield advantage over pure stands of T types. Yields of leaf and stem digestible dry matter and crude protein were closely correlated with leaf and stem DM yields. Yields of DM per stalk for S plants were usually highest when these plants were grown in pure stand, but DM yields/stalk for M and T entries were generally highest when these entries were grown in blends. Additional index words: Sorghum bicolor (L.) Moench, Forage yield, Crude protein, Digestibility, Intercropping. EX: ENSIVE .studies on. the theory and practice of many kmds of mtercroppmg have been published (4), but little published information is available on the intraspecific intercropping of sorghum [Sorghum hicolor (L.) Moench] cultivars and hybrids. In a 5-year study at Hays, Kansas, Ross (6) compared the yields of five grain sorghum hybrids with yields of all possible 1: 1 blends derived from them. No consistent advantage in grain yield was observed for any of the blends. Reich and Atkins (5) studied the performance of grain sorghum parental lines and hybrids, and two-component parental blends and hybrid blends in nine Iowa environments over 2 years. Averaged across all environments, most of the blends produced somewhat more grain than the mean of pure stand yields of their components. In each environment except one, however, the highest yield was produced by a hybrid grown in pure stand. More recently Skidmore and Hagen (7) studied the response of 2-dwarf and 3-dwarf isogenic hybrids of 'RS702' grain sorghum grown with the taller 2-dwarf hybrid as a shelter for the 3-dwarf hybrid. They observed that yields of grain and forage per culm of the 2-dwarf hybrid were higher in blends consisting of 13 or 25 % 2-dwarf seed than in a pure stand of the 2-dwarf hybrid. However, for the 3-dwarf hybrid yields of grain and forage per culm with this hybrid comprising 87 or 75% of the blend were not significantly different from yields per culm in a pure stand. The cited studies of Ross and of Reich and Atkins employed only grain sorghums; that of Skidmore and Hagen involved a single 2-dwarf, 3-dwarf pair of entries. In the study reported in this paper a wider range of plant types was utilized, and a more extensive list of traits was examined. MATERIALS AND METHODS Three groups, each consisting of three different entries, were included in this study. One of the entries in each group was a 1dwarf (tall) forage sorghum cultivar or hybrid; one was a 2-dwarf (medium) hybrid resulting from crosses involving germplasm from 'Early Hegari' and the forage sorghum cultivars 'Atlas', 'Rox', 'Sart', and 'White Collier'; and the third was a 3-dwarf grain sorghum hybrid (Table 1). Entries were chosen such that in each group the short (S) entry matured before the medium (M) entry which matured before the tall (T) entry. Within each group seven blends were prepared (Table 2). Blend trns, for example, consisted of a blend of the T, M, and Sentries, tm was a blend of T and M, etc. In some of the analyses the 12 individual components of the blends were considered. The relationship of these components to the blends also is shown in Table 2. The 'Contribution from USDA-ARS, and the Nebraska Agric. Exp. Stn., Lincoln. Published as Paper No. 6143, Journal Series, Nebraska Agric. Exp. Stn. Received 26 Jan. 1981. The work reported was conducted under Nebraska Agric. Exp. Stn. Projects 12-088 and 12-114. 'Supervisory research geneticist, USDA-ARS and professor of Agronomy; and George Holmes professor of Agronomy; Univ. of Nebraska, Lincoln, NE 68583, respectively. 224 CROP SCIENCE, VOL. 22, MARCH-ApRIL 1982 t Two-year means. entries grown in pure stands. Table 1. Groups of entries used in the sorghum blend experiment.

The concentrations of minerals in forages are important in satisfying animal requirements, but little attention has been given to determining these concentrations in forage sorghum [ (L.) Moench.] breeding programs. In this 2-yr study, the objectives were to determine the contents, genetic variability, combining ability effects, and correlations for 12 mineral elements in 49 experimental hybrids of forage sorghum. Mineral elements studied were N, Mg, Si, P, S, Cl, K, Ca, Mn, Fe, Cu, and Zn. General combining ability (GCA) effects exceeded specific combining ability (SCA) effects for all elements except P, Cl, and Fe in females, and S and Cl in males. The GCA and SCA effects in females were low for both Fe and Cu. Genetic ratios, resembling heritability, were higher for GCA than SCA except for Cl and Fe in females, and S and Cl in males. Thirty phenotypic correlations among the 12 elements were statistically significant. Some of the highest values were from the relationships of N with P, S, Cu, and Zn; Mg with Si and Cu; Si with Ca; P with Cu and Zn; S with K; and Cu with Zn. Some of the highest phenotypic correlations calculated among the mineral elements and 12 agronomic and quality traits were the positive association of protein with P, and the negative relationship of height with N and P, Brix of stem juice with K, and neutral detergent fiber with P. Data obtained in these studies show the feasibility of altering the mineral content of forage sorghum by breeding.

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

The KSS and N32 sorghum [Sorghum bieolor (L.) Moench) lines are low and high, respectively, in the hydrocyanic acid potential (HCN-p) of mature leaves. This difference is conditioned primarily by a single pair of alleles. The main objective of this study was to determine, at various stages of plant growth and various times during the growing season, the HCN-p of upper leaves and tillers of fieldgrown plants of these two parental lines and of two low-HCN-p F3 lines derived from crosses between KS8 and N32. The four entries were grown in a randomized complete block design with three replications in 1985. Samples of leaf tissue were dried, ground, and extracted, and cyanide in the extracts was assayed colorimetrically. Using a mean HCN-p level of SOO mg kgI dry wt to separate safe from unsafe sorghum forage, all samples of KS8 mature leaves and tillers would be considered safe, and all N32 samples would be considered potentially dangerous. Values for most of the samples of the FJ lines fell within the safe range, but some samples of young regrowth exceeded the 500 mg kg-I limit. Regressions of HCN-p on height for upper leaves of main stems and of tillers indicated a significant negative relationship for all entries except for leaves from the main stems of KS8. However, the relationship was not close enough to support the use of plant or tiller height as a reliable indicator of HCN-p. Levels of HCN-p also were determined for mature leaves and young regrowth of hybrids involving KS8, N32, and 'Redlan' sorghums as seed parents and NP25, 'Piper,' and 'Greenlear sudangrasses [So sudanense (Piper) Stapf) as pollinators. Results indicated that for minimizing the risk of cyanide poisoning, KS8 would be the seed parent of choice, and NP2S and Piper would be the preferred pollinators. Additional index words: Cyanogenesis, Prussic acid, Sorghum bicolor (L.) Moench, Sorghum sudanense (Piper) Stapf, Sorghum X sudangrass hybrids. A RECENT COMPARISON of the hydrocyanic acid potential (HCN-p) of the grain sorghum [Sorghum bicolor (L.) Moench] lines, KS8 and N32, indicated that seedling leaves of both lines were high in HCNp, but mature leaves from field-grown plants differed greatly, flag leaves of N32 being at least 10 times as high in HCN-p as KS8 flag leaves (3). The large difference in HCN-p between KS8 and N32 was detected in field-grown plants within about 5 weeks after planting (5), and was found to be conditioned primarily by a single pair of alleles (3). The objective of the present study was to determine, at various times during the growing season and at various stages of plant development, the HCN-p of upper leaves and tillers of KS8, N32, and two low-HCN-p F3lines derived from crosses 'between KS8 and N32. Determinations of this type are needed for appropriate management decisions concerning the safety of sorghum forage for grazing liveI Contribution from the USDA-ARS and the Nebraska Agric. Res. :Div., Lincoln, NE 68583. Published as Paper no. 8101, Journal Series, Nebraska A$ric. Res. Div. Research was conducted under Project 12-114. Received 31 July 1986. 2 George Holmes professor of agronomy; supervisory research ge~eticist, USDA-ARS; and research geneticist, USDA-ARS. respectively. Published in Crop Sci. 27:903-906 (1987). 903 stock. It would be unusual for farmers to use grain sorghums such as KS8 and N32 as pasture except during the period after grain harvest. However, sudangrass [So sudanense (Piper) Stapf] and sorghum X sudangrass hybrids are often used for pasture and greenchop, and the HCN-p of these forages at various growth stages is important to the livestock producer. Therefore, several other sorghum and sudangrass lines and their FI hybrids also were included in this study. MATERIALS AND METHODS

The variation among 49 Fl forage sorghum [Sorghum bicolor (L.) Moench.] hybrids from a 7 X 7 cross-classified design was explored in 1979 and 1980 for the following silage traits: dry matter (DM), crude protein, in vitro dry matter disappearance (IVDMD), neutral detergent fiber, acid detergent fiber, acid detergent lignin, ammonia, lactate, and Brix of the juice from fresh stalks. Wider ranges generally were found for male than for female parental means. Means for most traits were significantly different among entries. Significant differences among hybrid means over males and over females were found for only DM, IVDMD, and Brix. Interactions with years existed for most traits. Genetic ratios calculated from the mean squares indicated that general combining ability was important for DM, IVDMD, and Brix. Simple correlation coefficients between traits measured on silage and on fresh-dried samples from the same hybrids were all significant. In view of the effort required to make and evaluate silage samples, initial selection for traits used to estimate quality in fresh-dried samples appears to be the best approach for improving the quality of forage sorghum silage. Additional index words: Sorghum bicolor (L.) Moench., Protein, IVDMD, Fiber, Lignin, Ammonia, Lactate, Brix, Combining ability. T HE inheritance of several constituents used to estimate quality in dried samples from freshly harvested forage sorghum [Sorghum bicolor (L.) Moench.] hybrids was reported in an earlier paper (19). Forage sorghums often are preserved and utilized as silage (18), and some constituents used to estimate quality have been shown to be altered during the ensiling process (5). Information about the inheritance of silage constituents is needed in forage sorghum breeding programs. The large number of silage samples required in inheritance and breeding studies necessitates the use of simple, economical, miniature silos. Sealed glass canning jars meet this requirement and have been used experimentally for more than 50 years (1, 6, 11). It is recognized that the silage produced in miniature silos may differ from that produced in field scale silos, but small silos provide a feasible means of obtaining reasonable estimates of silage quality (16) from a large number of entries. Some factors that can affect silage quality include dry matter percentage at time of ensiling, lactic acid concentration, ammonia content, and availability of fermentable carbohydrates. Many traits such as in vitro dry matter disappearance (IVDMD), crude protein (CP), dry matter (DM) percentage, neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL), can be used to estimate quality in both freshly harvested sorghum forage and silage. Ward et al. (24) found that lower DM percentage of sorghum silage resulted in lower DM intake. However, this relationship was probably secondary since Thomas et al. (22) demonstrated that a change in the DM content of a silage at the time of feeding did not alter the DM intake. The intake depression associated with higher moisture silages may be due to their increased levels of organic acids, particularly lactic acid (9). Thomas et al. (22) reported a reduction in dry matter intake of silage when lactic acid was added to the silage or introduced directly into the rumen. McLeod et al. (15) found that increases in pH of the silage increased DM intake while decreases in pH reduced DM intake. The breakdown of proteins to ammonia is considered detrimental to silage quality (3) because palatability is reduced and some of the ammonia is lost through volatilization as the silage is fed. Some workers also have associated higher soluble N content with lowered intake of silage (17). McCullough and Cummins (14) reported that sorghum silages are deficient in fermentable carbohydrates, the energy source required for the desired lactate-type ensiling process. Similarly, Zimmer (25) stated that low sugar con1 Joint contribution of the USDA-ARS and the Nebraska Agric. Exp. Stn., Lincoln. Published as Paper No. 6783, Journal Series, Nebraska Agric. Exp. Stn. Received 12 Feb. 1982. The work reported was conducted under Nebraska Exp. Stn. Projects 12-088 and 12-114. 2 Formerly graduate research assistant (now assistant professor of agronomy, Auburn Univ., Auburn, AL 36849); George Holmes professor of agronomy; supervisory research geneticist, USDA-ARS; and associate professor of animal science, Univ. of Nebraska, Lincoln, NE 68583 respectively. PEDERSEN ET AL.: ESTIMATING SILAGE QUALITY OF SORGHUM HYBRIDS 377 centrations contributed to a less desirable heterofermentive ensiling process. However, Gourley and Lusk (8) proposed that if more soluble carbohydrates are available than the 6 to 8'70 required for fermentation, the excess may be metabolized to CO2 and water. They also stated that sweet sorghum silages generally are inferior to intermediateand graintype silages in terms of feeding value. The objectives of this study were to determine the extent of variation among 49 experimental forage sorghum hybrids for several constituents used to estimate silage quality and to estimate the proportion of the total variation that was caused by genetic variation. In addition, general combining ability (GCA) and specific combining ability (SCA) effects were estimated. MATERIALS AND METHODS The 49 Fl hybrids used, growing conditions, harvesting and sampling techniques, and statistical and genetic analyses were described in an earlier paper (19). Pertinent information relative to this study is repeated here. The 49 hybrids were produced by crossing each of seven male-sterile lines ('Redlan', 'N35', 'N38', 'N48', 'N4692', 'KS5', 'N5013'3) to each of seven pollinator lines ['Early Hegari-Sart' (EH -SAR T)3, 'Early Hegari-White Sourless' (EH-WS)3, 'Early Hegari-Rox' (EH-Rox)3, 'N6229', 'Rox', 'White Collier' (WC), and 'H60-29'3]. The 49 hybrids were grown in a randomized complete block design with three replications at the University of Nebraska Field Laboratory, Mead, in 1979 and 1980 using standard agricultural practices for that area. Plots were three 9.14-m rows spaced 0.76 m apart with plants hand-thinned to a spacing of approximately 15 cm. A 4.75-m section of the middle row of each plot was harvested after all entries had reached physiological maturity (24~ 27 Sept. 1979 and 6~ 7 Oct. 1980). Random subsamples were passed through a small chopper, mixed thoroughly, and packed as tightly as possible into the miniature silos which consisted of O.95-liter glass jars. The jars were immediately sealed with new canning lids. The lids and jar tops were dipped in melted paraffin at the end of each day to ensure an airtight seal. The miniature silos were then incubated at 28 C to promote a lactate-type fermentation (13). After 4 weeks the ensiling process was assumed to be complete, the jars were opened, and subsamples were withdrawn for drying. The jars were again closed, and jars and contents were held in a freezer until additional subsamples were withdrawn for the preparation of extracts. The subsamples withdrawn for drying were weighed, dried to a constant weight at 57 C in a forced-air oven, and reweighed. Data were collected for the following constituents used to estimate quality: Percent DM. Calculated from the difference in weights of wet and dried silage samples. Percent CP (Percent N X 6.25). Determined by the Kjeldahl procedure (10). IVDMD. Determined by the two-stage technique of Tilley and Terry (23) and expressed as a percentage. Percent NDF. Determined by the high concentrate procedure of Robertson and Van Soest (20). Percent ADF, ADL. Determined by the detergent fractionation procedures of Goering and Van Soest (7). Extracts were prepared from separate subsamples of the wet silage using a modification of Byer's (4) extraction procedure for organic acid analysis in fermented feeds. Twenty-five grams of silage, 70 ml of O.OlN H 2S04, and a thymol crystal to prevent bacterial growth, were placed in a 125-ml Erlenmeyer flask, mixed, and refrigerated for 24 hours to equilibrate. The liquid was filtered 3 Experimental forage sorghum lines. through Whatman #40 filter paper, and the filtrate was frozen and thawed once to aid in precipitation. Four milliliters of the thawed suspension was placed in a centrifuge tube with 1 ml of 10% (w Iv) trichloroacetic acid and centrifuged for 15 min at 12,000 X g. The supernatant was decanted and frozen for later analyses of lactate and ammonia. The determination of lactic acid content involved the use of Barker and Summerson's (2) co:orimetric technique, and ammonia content was determined by an adaptation of the indophenol method of McCullough (12). Measurements of Brix from three stalks/plot were made using a hand-held refractometer on 17 and 18 Sept. 1979 and on 9 Oct. 1980. Juice from the fifth internode, counting the peduncle as the first, was squeezed onto the refractometer stage for these readings. The genetic analyses of these data followed the method described by Ross et al. (21). Although described in an earlier paper (19), the procedure is repeated here for convenience. The entry source of variation was partitioned into females, males, and females X males, and the entry X year interactions were partitioned into females X years, males X years, and females X males X years. Mean squares (MS) were equated to their expected values and solved for components estimating the variance among the fixed effects in the corresponding sources of variation. F -tests were made for females as MSr/MSfy , for males as MSm/MSmy, for females X males as MSfm/MSfmy, and for females X males X years as MSrmy/MSe. Genetic ratios were estimated as follows: (lT~/RFY + lTfmy/FY + 8fm/F + IT;;'y/Y + 8;;')

Information about the extent of variation in quality traits among plants sampled at the same developmental stage but on different dates would be useful to forage researchers. The primary purpose of this study was to obtain such information for five forage sorghum [Sorghum bicolor (L.) Moench] cultivars. Percent dry matter, crude protein, and in vitro dry matter disappearance of leaf, stem, and whole plant samples were determined for field-grown samples harvested on three different days during early head emergence (EHE) and once during physiological maturity (PM) for each cultivar. Orthogonal comparisons between EHE and PM sampling stages and among the· EHE samples were made. Results of the 2-year study indicated that statistically significant differences existed among EHE samples and between EHE and PM samples. However, differences among EHE samples were generally much smaller than those between EHE and PM samples and may be of little importance for some applications. Additional index words: Protein, Digestibility, Dry matter, IVDMD, Sorghum bicolor. DIFFERENCES in quality traits of forage sorghum [Sorghum bicolor (L.) Moench 1 sampled at various stages of development or under different management regimes are well documented (1, 3, 5, 6, 9). Much less information is available, however, on possible differences among plants of the same genotype and within the same field plot that reach a given developmental stage at different ages in terms cf O:3.ys after planting. It would be useful for forage sorghum researcht, to know whether the quality traits of a plant that reaches a specific developmental stage on a certain day provide an accurate indication of the traits of other plants in the plot as they reach this stage several days later. An easily identified stage in the development of forage sorghum is the end of the boot stage which is signaled by the beginning of head emergence. This study was designed to determine the extent of variation in percentages of dry matter, crude protein, and in vitro dry matter disappearance occurring among forage sorghum plants that reached early head emergence at different times after planting. Plants also were sampled at physiological maturity for comparison with the samples taken at head emergence. Materials and Methods Five forage sorghum cultivars ('Atlas', 'Brawley', 'Coleman', 'Early Hegari-Rox', and 'White Collier') were grown in 1976 and 1977 in a randomized complete block design with three replications at the University of Nebraska Agronomy Farm, Lincoln, Nebr. 1 Contribution from the USDA-ARS, and the Dep. of Agronomy, Nebraska Agric. Exp. Stn., Lincoln. Published as Paper No. 6861, Journal Series, Nebraska Agric. Exp. Stn. Received 21 Apr. 1982. 2 Former graduate research assistant, Univ. of Nebraska, now assistant professor of agronomy, Auburn Univ.; George Holmes professor of agronomy; and supervisory research geneticist, USDA-ARS, and professor of agronomy, Univ. of Nebraska, Lincoln, NE 68583. 3 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA or the Univ. of Nebraska, and does not imply its approval to the exclusion of other products that may also be suitable. Each plot consisted of five 6.7-m rows spaced 0.76 m apart. Planting dates were 5 May 1976 and 16 May 1977. An application of 112 kg N/ha was disked into the soil [Kennebec silt loam (Cumulic Hapludoll) 1 prior to each planting, and plantings were followed immediately by applications of a mixture of propachlor (2-chloroN-isopropylacetanilide) and atrazine [2-chloro-4-(ethylamino)-6(isopropylamino)-s-triazinel at 3.75 and 1.25 kg a.i./ha. To supplement rainfall, the plots were flood irrigated once, at about midsummer, both in 1976 and 1977. The stage of early head emergence (EHE) was defined as occurring when the very tip of the head was first visible. When at least one culm in each row of a cultivar reached this stage, sampling of that cultivar was initiated. One culm at EHE was sampled from each row of the cultivar on sampling day one (EHE 1). Two and 4 days later, another single culm that had just reached the EHE stage was harvested from each row of the cultivar (EHE 2 and EHE 3, respectively). One set of samples also was taken for each cultivar at physiological maturity (PM) as indicated by black layer formation (4). The five culms harvested from a plot at each sampling day were cut off at the surface of the soil and were separated into leaf blade, stem plus leaf sheath, and head portions. Each culm was treated individually for assay. The immature head was considered to be part of the stem in all EHE samples. The separated samples were weighed and then dried at 75 C in a forced draft oven to constant weight. Dried samples were ground in a Wiley3 mill to pass a 1mm screen, and subsamples were stored for subsequent analysis. Percent crude protein (CP) was determined for each sample as Kjeldahl N X 6.25 (7), and in vitro dry matter disappearance (IVDMD) by a modification of the two-stage technique of Tilley and Terry (8). Percent dry matter (DM) was calculated for each sample, and values for CP, IVDMD, and DM were calculated for the total plant from the values for the leaf blade, stem, and, for PM samples, head portions. Analysis of variance with orthogonal comparisons (2) was completed for each of the traits under study. Results and Discussion Mean squares for some of the sources of variation involved in this study are shown in Table 1. For many of the measured traits, statistically significant variations were associated with year interactions. Therefore, the mean squares are presented on an individual year basis. Inspection of these mean squares shows that significant differences were detected between the EHE and PM stages for all traits except total plant IVDMD in 1977, and that significant (EHE vs. PM) X cultivar interactions were found in all cases except total plant CP in 1976. Significant differences among the three EHE sampling dates were shown for most traits. However, the mean squares for variation among EHE samples were generally much smaller than those for EHE vs PM. Means for each cultivar X sample time X year X trait combination are shown in Table 2. As expected, leaves and stems of all cultivars harvested at EHE were lower in DM than those harvested at PM. The difference between EHE and PM samples was especially pronounced for total plant values, reflecting the relatively high DM percentages of heads at the PM stage (separate data for heads not shown). Decreases in forage CP and IVDMD are generally expected as plants advance in maturity. Comparison of CP values of leaves, stems, and total plants indicates that such a decrease occurred for all cultivars between EHE and PM. Similarly, leaves of all cultivars decreased in IVDMD between these two stages. However, Early Hegari-Rox was

Seedlings of both KS8 and N32 sorghum [Sorghum bicolor (L.) Moench] were high in dhurrin [p-hydroxy-(S)-mandelonitrile-pD'glucoside] and thus in hydrocyanic acid potential (HCN-p), but the HCN-p of mature leaves from field-grown plants of KS8 was only about one-tenth as high as that of N32. A study of the inheritance of this large difference between KS8 and N32 revealed that a single major gene pair was responsible. There were no obvious maternal effects and F,'s were generally intermediate in HCN-p level between the two parents, indicating that neither high nor low HCN-p was completely dominant. HCN-p level was influenced by genetic background since mean values of the low and intermediate HCN-p classes increased slightly as the proportion of genetic background ascribable to N32 was increased. A survey of 102 additional sorghums revealed that the gene for low HCN-p carried by KS8 does not appear to occur widely because KS8 had the lowest HCN-p of all entries assayed. Additional index words: Cyanogenesis, Genetics, Hydrocyanic acid,p-Hydroxybenzaldehyde, Prussic acid, Sorghum bicolor (L.) Moench, Spectrophotometric assay. THE biosynthesis of the cyanogenic glucoside, dhurrin [p-hydroxy-(S)-mandelonitrile-l3-D-glucoside], in sorghum [Sorghum bicolor (L.) Moench] has been studied extensively, and much has been learned about the process (I). However, studies of the inheritance of dhurrin content in this species have been much less conclusive. Nass (10) reviewed published reports on the inheritance of cyanogenesis in sorghum, various Lotus species, and Trifolium repens 1., and concluded that the situation in sorghum was more complex than that in other species. There was general agreement that the hydrocyanic acid potential (HCN-p) of sorghum leaves was a heritable trait, but the reports failed to agree on such matters as dominance of low or high HCN-p and the number of genes involved. Lack of agreement may have been due in part to differences among the various studies with respect to lines and cultivars used, conditions of growth and sampling, and analytical procedures. Krauss (7) concluded that HCN-p in sorghum was governed by four gene pairs with additive effects and without dominance. His work was based on crosses among four sorghum cultivars differing in HCN-p. More recent reports have included information about the HCN-p of hybrids and their parents in forage sorghum (13, 15) and also in sudangrass and sudangrass-sorghum combinations (14), but these studies were not designed specifically to investigate the inheritance of HCN-p. Recently Haskins et al. (4) reported that the two sorghum lines, KS8 and N32, both had high HCNP levels as seedlings, but when upper leaves from , Contribution from the USDA-ARS and the Nebraska Agric. Res. Div., Lincoln, NE 68583. Published as Paper no. 7724, JournalSeries, Nebraska Agric. Res. Div. The work reported wasconducted under Nebraska Agric. Res. Div. Project 12-114. Received 27 Mar. 1985. 2 Supervisory research geneticist, USDA-ARS; George Holmes professor of agronomy; ana supervisoryresearch geneticist, USDAARS, respectively. 65 field-grown plants were compared in mid-August, the HCN-p of KS8 was only about one-tenth as high as that of N32. The existence of this large difference suggested that these lines might be useful in a study of the inheritance of HCN-p in mature sorghum leaves. MATERIALS AND METHODS

Spectral scanning was used to provide estimates of the leakage of the cyanogenic glucoside, dhurrin (p-hydroxy-[S]-mandelonitrile-beta-d-glucoside), and its metabolite, p-hydroxybenzaldehyde (p-HB), from young light-grown shoots of Atlas sorghum (Sorghum bicolor [L.] Moench) when these shoots were immersed in water, toluene, chloroform or mixtures of water and toluene or water and chloroform. Minimal leakage of dhurrin and virtually no leakage of p-HB occurred with water as the solvent. The 0.5% concentration (v/v) of both toluene and chloroform was more effective than either the 1.0 or 2.0% concentrations in effecting leakage of the two solutes. With either 0.5% toluene or 0.5% chloroform as the solvent, 80 to 90% of the total dhurrin was extracted from shoots in a 3-hour period. Breakdown of dhurrin during extraction was much more extensive with 0.5% chloroform than with 0.5% toluene. Some loss of p-HB occurred during 3- or 6-hour extractions in the water-organic solvent mixtures; spectral and chromatographic evidence suggested partial conversion of p-HB to p-hydroxybenzoic acid. With undiluted toluene or chloroform as solvents, extracts contained appreciable amounts of free p-HB but essentially no dhurrin. These solvents were less effective than the water-organic solvent mixtures in extracting the solutes from the shoot issue.

Flag leaves of 'Colman' forage sorghum (Sorghum bicolor) contain at least 25 times as much leucoanthocyanidin (LAC) and approximately half as much of the cyanogenic glucoside, dhurrin, as do flag leaves of 'White Collier' forage sorghum. Assays of flag leaves from 119 F2 plants and 11 F5 lines from crosses between these two cultivars revealed a statistically significant negative association between levels of LAC and dhurrin. Both LAC and dhurrin are aromatic compounds, and the negative association between the two may be the result of competition for intermediates or products of the aromatic biosynthetic pathway. This rationale appears to be quite different from that for the negative association reported for levels of tannin and cyanide in Lotus corniculatus. Although the negative relationship between LAC and dhurrin in sorghum was statistically significant, the association was not consistent enough to suggest that either trait could be used reliably in selecting or breeding to modify the other trait.

Week-old shoots of 50 Sorghum entries representing 22 species, plus four Sorghum entries of undesignated species, were dried at 75° and the dried tissue extracted with water at room temperature. The resulting extracts were diluted in 0.1 M sodium hydroxide and spectra were scanned immediately to provide a measure of free p -hydroxybenzaldehyde. Scans were repeated after the basic solutions had stood for 3 hr at room temperature to permit hydrolysis of dhurrin ( S - p -hydroxymandelonitrile β- D -glucopyranoside). Without exception, the quantity of free p -hydroxybenzaldehyde was very small in relation to the quantity released by dhurrin hydrolysis.

Flag leaves of KS8 sorghum (Sorghum bicolor (L.) Moench] are low in dhurrin (p-hydroxy-(S)-mandelonitrile-p-D-glucosidel content and thus in hydrocyanic acid potential (HCN-p), and they contain little (if any) leucoanthocyanidin (LAC). Comparable leaves of 'Colman' sorghum are intermediate in HCN-p and high in LAC. This study was conducted to investigate the inheritance of HCN-p and LAC in crosses of KS8 X Colman. Flag leaves from field-grown plants of both parents; the Flo KS8 X Colman; the backcross, KS8 X (KS8 X Colman); and the F 2, (KS8 X Colman) selfed, were assayed for both HCN-p and LAC. Assays for HCN-p indicated that the backcross values provided a good fit to 1 intermediate:l low (Xl = 0.05, P = 0.82) and F2 results to 3 intermediate:l low (Xl = 0.96, P =0.33). For LAC, backcross results were 1 LAC +:1 LAC(Xl = 0.20, P = 0.65) and F2 values were 3 LAC+:1 LAC(exact fit). Classification for both traits yielded good fits to 1:1:1:1 for the backcross (Xl = 0.30, P = 0.96) and 9:3:3:1 for the F 2 (Xl = 3.24, P = 0.36). These results indicated that the difference in HCN-p between Colman and KS8 depended primarily on a single gene, the difference in LAC depended largely on a separate single gene, and the HCN-p gene and the LAC gene were not linked. A 1986 REPORT (1) indicated that a single gene difference was primarily responsible for the difference in dhurrin content (expressed as hydrocyanic acid potential, HCN-p) of mature leaves between KS8 (lowHCN-p) and N32 (high-HCN-p) lines of sorghum. Similarly, the large difference in flag-leaf leucoanthocyanidin (LAC) content between 'White Collier' (LAC-) and Colman (LAC+) sorghums depended largely on a single pair of genes (2). Both KS8 and N32 were LAC-, and the HCN-p values for flag leaves of both Colman and White Collier were intermediate between the values for KS8 and N32 (EA. Haskins and H.J. Gorz, 1988, unpublished observations). Therefore, the crosses used in the previous studies were considered inappropriate for investigating the possible linkage of the HCN-p and LAC traits. The objective of the present study was to investigate the segregation of these two traits in a cross of KS8 (low-HCN-p, LAC-) X Colman (intermediate-HCN-p, LAC+). Materials and Methods Colman (male-fertile), AKS8 (cytoplasmic male-sterile), and BKS8(male-fertile, sterility maintainer) sorghum plants weregrown at the Agronomy Farm, Lincoln, NE, during the summer of 1985. The status of Colman with respect to fertilityrestoration was unknown; therefore, to ensure that malefertile F, plants would be produced, BKS8 plants were hand emasculatedand pollinated with Colman pollen. In NovemFA Haskins, Dep, of Agronomy, and H.J. Gorz, USDA-ARS and Dep. ofAgronomy, Nebraska Agric. Res. Div.,Lincoln, NE 68583. Published as Paper no. 8484, Journal Series, Nebraska Agric. Res. Div. Research was conducted under Project no. 12-114. Received 27 Nov. 1987. *Corresponding author. Published in Crop Sci. 28:864-865 (1988) ber, 1985, the F, seed was sent to Puerto Rico (Tropical Agricultural Research Station, USDA-ARS, Mayaguez)where F, plants were self-pollinated to produce F2 seed, and were also used as pollen parents in backcrosses to AKS8. AKS8 X Colman crosses also were made during the summer of 1985 to obtain larger quantities ofseed from which F , plants could be grown for sampling. Plants of BKS8, Colman, the F, (AKS8 X Colman), the backcross [AKS8 X (BKS8 X Colman)], and the F2 [(BKS8 X Colman) selfed] were started in the greenhouse during the spring of 1986 and were transplanted to the Agronomy Farm on 29 May. Plants were placed 0.61 m apart in rows with a 0.76-m spacing. The experiment was planted in four replications, with each replication including one 10-plant row of each parent and the F" two rows of the backcross, and five rows of the F2• Thus, the experiment was designed to include 40 plants of each parent and the F I , 80 backcross plants, and 200 F2 plants. Entries were assigned at random to the 10 rows in each replication. Insufficient plants of both parents and the F, were available for transplanting; therefore, seeded rows of BKS8, Colman and the F, were used in two, three, and one of the replications, respectively. On 30 July, when panicles were emerging from most plants, the blade of the flagleaf was harvested from each plant. One KS8 and 21 F2 plants were not sufficiently advanced to allow positive identification of the flag leaf; for these plants, the blade ofthe youngest leaf with a visible collar was harvested. Midribs were removed from the leaf blades, and the remaining tissue was dried at 70 to 75°C overnight. The dry tissue was ground through a I-mm screen and stored in plastic vials at -18°C prior to extraction for assay. Extraction and assay for HCN-p were conducted as described previously (I) except that dhurrin was hydrolyzed enzymatically rather than with NaOH. The enzyme preparation was an extract made by soaking defatted almond meal (Sigma Chemical Co., St. Louis, MO)' in distilled water (8 mg mL -I) and filtering the suspension through Whatman no. 1 filter paper. To hydrolyze dhurrin, I mL of this filtrate was added to I mL of leaf extract, and the mixture was incubated in a parafilm-capped tube at room temperature for 1.25 h. Following this incubation, 8 mL of 0.1 MNaOH was added, and a l-mL portion of the resulting solution was assayed colorimetrically as described previously (I). The procedure described by Haskins and Gorz (2) was used for extraction and assay of LAC. Absorbance at 540 nm was used as a measure of LAC content. Results and Discussion Segregation for HCN-p Mean HCN-p values for KS8 and Colman were 29 and 135 mg kg-I, respectively, and standard errors were such that these means appeared to be well separated (Table 1). However, ranges in HCN-p for these two parents overlapped slightly, which caused some uncertainty in the classification of backcross and F2 plants as either low (L) or intermediate (I) in HCNp. As shown in the table, with 50 mg kg-I as the dividing line between the classes, only one of 37 KS8 plants was classified as I-HCN-p and only two of 40 Colman plants were classified as L-HCN-p. With this dividing line, 41 of the backcross plants were I-HCNI Names ofproducts are included for the benefit of the reader and do not imply endorsement or preferential treatment by the USDA or the Univ. of Nebraska.

Free p -hydroxybenzaldehyde was not present in appreciable quantity on the surface or in the interior of week-old Sorghum bicolor shoots that had been heated to inactivate hydrolytic enzymes, nor was p -hydroxybenzaldehyde detected in epicuticular wax of greenhouse-grown sorghum ca 4.5 months old.

Hydrocyanic acid or prussic acid poisoning of livestock by sorghums [Sorghum bicolor (L.)Moenchl and sudangrasses [Sorghum sudanese (Piper) StapfJ is caused by the digestive liberation of hydrocyanic acid (HCN) from the cyanogenic compound, dhurrin [(S)-p-hydroxymandelonitrile ,8-D-glucopyranosideI found in tissue of these plants. Recent research documented that dhurrin is also present in indiangrass [Sorghastrum nutans (L.) Nash] seedlings. The purpose of this study was to determine the hydrocyanic acid potential (HCN-p) of forage from established stands of indiangrass. Five cultivars representative of indiangrass germplasm of the Great Plains were sampled during the growing season for 2 years from 2 sites in eastern Nebraska. The HCN-p of the indiangrass sampled in this study exceeded 750 mg-1 kg dry wt. (dangerous level) only in spring when new growth was 20 cm tall or less. Levels were less than 500 mg-1 kg (safe) when new growth was at least 40 cm tall and were very low (