Protein quality of corn hybrids differing for endosperm characteristics and the effect of nitrogen fertilization 1

Corn (Zea mays L.) is an important source of protein for humans and animals. Because dent corn is highly responsive to nitrogen (N) fertilization, substantial amounts of N are used for corn production. Application of N fertilizer may reduce protein quality of corn kernels through an increase in zein content. The objective of this study was to determine if corn endosperm characteristics influence the effect of N fertilization on protein quality. In 1988, six corn hybrids differing for endosperm characteristics were grown at two locations in Ohio and with two N rates, 34 and 200 kg/ha. The waxy hybrid had a greater concentration of fraction I protein than the non‐wary hybrid. These two hybrids did not differ for other fractions except fraction III at Columbus. The soft endosperm hybrid had a higher concentration of fraction I protein than the hard endosperms hybrids. Soft and hard endosperm hybrids differed for fraction II protein for the 34 kg N/ha fertilizer rate but not the 200 kg N/ha fertilizer rate. These two classes of hybrids did not differ for fraction III protein. Increasing N fertilizer increased fraction II concentration for all hybrids. Concentrations of the other two protein fractions did not respond to fertilizer rate. The increase in fraction II concentration with N fertilization may result in a decrease of protem quality and feed value. Although all hybrids responded to N fertilizer, some hybrids had bigger increases in fraction II proteins than other hybrids.     

Effect of Broken Corn Levels on Water Absorption and Steepwater Characteristics

Broken corn created by grounding sound corn kernels was added back at levels of 0, 4, 8, 12, or 16%, by weight, to whole kernels of three corresponding hybrids: FR27 × FRMo17 (a soft endosperm corn), FR618 × FR600 (amedium‐hard endosperm corn), and FR618 × LH123 (a hard endosperm corn). The samples had been dried from 28% moisture content to 15% moisture content either by using ambient air at ≈25°C or at 110°C. Samples were steeped for 36 hr at 52°C in 0.15% sulfur dioxide and 0.5% lactic acid steeping solution. The steepwater characteristics, such as water absorption, solids and protein content in the steepwater, and steepwater pH, were measured by periodic sampling and analyzed. Broken corn level has a significant effect on the amount of solids released during steeping and steepwater protein content for all samples. Both steepwater solids and protein content increased linearly as broken corn content increased. Corn drying temperature, kernel hardness, and interactions between drying temperature and kernel hardness has a significant effect on steepwater solids and protein content and steepwater pH in both broken and unbroken corn. Corn dried at low temperature released more soluble solids and protein into the steepwater than corn dried at high temperature. Soft endosperm and medium‐hard endosperm corn released more soluble solids and protein into the steepwater than hard endosperm corn. Soft endosperm corn resulted in a higher steepwater pH than medium‐hard and hard endosperm corn. No significant effect of broken corn content on final moisture content of steeped corn and steepwater pH was observed.     

Grain yield and mineral composition of corn as influenced by endosperm type and nitrogen

Abstract

Corn (Zea mays L.) is a major source of nutrition for humans and animals. Chemical and physical properties of corn endosperm vary among hybrids, are influenced by genotype and environment, and may affect the crop's response to nitrogen (N) fertilization. The objective of the study was to measure the responses of grain yield and grain N, phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), manganese (Mn), iron (Fe), boron (B), zinc (Zn,) and copper (Cu) concentration of different endosperm types to N fertilization. The study was conducted at two Ohio locations in 1988, where six endosperm types and two N rates (34 and 200 kg N/ha) were combined in a split plot arrangement. Nitrogen rate (main plots) had little effect on yield at either location, and the soft endosperm hybrid was the only hybrid to respond to N fertilizer. Within fertilizer level, hybrids differed in grain yield with the waxy hybrid out yielding the normal endosperm hybrid, and the hard endosperm hybrid out yielding the soft one at the 200 kg N/ha rate. Application of N fertilizer increased the grain N concentration of all hybrids. Grain of the waxy hybrid contained an equal or greater N concentration than the normal hybrid. In contrast, no difference in N level was found between hard and soft endosperm hybrids at either fertilizer level. Climatic conditions and soil fertility differences might have been partly responsible for location effects. Genetic make‐up could have been a factor in differing hybrid response since grain concentration of nutrients Varied by location, endosperm type, and N treatment.     

Compressive Strength of Wheat Endosperm: Comparison of Endosperm Bricks to the Single Kernel Characterization System

The three major classes of endosperm texture (grain hardness) of soft and hard common, and durum wheat represent and define one of the leading determinants of the milling and end‐use quality of wheat. Although these three genetic classes are directly related to the Hardness locus and puroindoline gene function, much less is known about the kernel‐to‐kernel variation within pure varietal grain lots. Measurement of this variation is of considerable interest. The objective of this research was to compare kernel texture as determined by compression failure testing using endosperm bricks with results of whole‐kernel hardness obtained with the Single Kernel Characterization System 4100 hardness index (SKCS HI). In general terms, the variation obtained with the SKCS HI was of similar magnitude to that obtained using failure strain and failure energy of endosperm brick compression. Objective comparisons included frequency distribution plots, normalized frequency distribution plots, ANOVA model R², and coefficients of variation. Results indicated that compression testing and SKCS HI similarly captured the main features of texture classes but also reflected notable differences in texture properties among and within soft, hard, and durum classes. Neither brick compression testing nor the SKCS HI may be reasonably expected to correctly classify all individual kernels as to genetic texture class. However, modest improvements in correct classification rate or, more importantly, better classification related to end‐use quality may still be achievable.     

Relationships Between the Microstructure,Physical Features,and Chemical Composition of Different Maize Accessions from Latin America

Chemical composition (moisture, total lipids, protein, and apparent amylose) and some physical features (1,000 kernel weight, hardness, and anatomical composition) were determined in 71 accessions representing races of maize from Latin America. Their microstructural characteristics (size and compaction of endosperm cell bodies, pericarp thickness, horny‐floury endosperm ratio, and morphology and size of starch granules) were also evaluated using environmental scanning electron microscopy (ESEM). Compaction was the most important microstructural feature of the maize kernels, representing kernel hardness. Highly compact kernels tended to be hard, with high protein, pericarp, and hard‐endosperm content and high pericarp thickness, but with low moisture, amylose content, and kernel weight and size. The opposite was observed in the least compact kernels. Highly compact kernels tended to have small, polygonal starch granules (<10 μm), while the least compact kernels contained large, spherical granules (>10 μm). These results suggest that microstructure is responsible for the physical features of maize kernels and that microstructure is related to chemical composition.     

Effects of nitrogen,phosphorus, and potassium nutrition on yield,rates of kernel growth and grain filling periods of two corn hybrids

Abstract

Depression of corn grain yields from nutrient stress has been studied extensively, but effects of nutrient stress on rates of corn development and yield determinants are less well understood. Nutritional effects on the number of kernels/unit area, growth rate/kernel, and duration of growth have implications concerning fertilization practices and yield potentials of crops. Two corn hybrids with equivalent silking dates but having different grain filling periods were grown in a field experiment. Fertility treatments consisted of a N series receiving 0, 112, or 336 kg of N/ha and a P‐K series receiving factorial combinations of 0, 22, or 112 kg of P/ha and 0, 56, or 224 kg of K/ha. Dates for grain initiation and maturity were determined for each plot along with tissue analyses of ear leaves, grain yields, and kernel weights. Concentrations of N and K in ear leaves generally corresponded to treatment levels of these nutrients, although Pioneer Hi‐bred 3390 appeared to be less efficient than Pioneer Hi‐bred 3334 in K uptake. Effects of nutrient stress on yield determinants depended on the determinant and nutrient under consideration. Severe N stress did not change length of grain filling periods, but decreased kernel numbers 30 to 70%. Stress for K, on the other hand, shortened grain filling periods about 13% and had only a slight effect on kernel number. Negligible P stress occurred in the experiment. The two hybrids produced equal quantities of grain/ha/day but the hybrid with a longer filling period (Pioneer 3334) filled many more kernels at a slightly slower rate and for a longer period of time to give a significantly greater grain yield compared to Pioneer 3390.     

Effects of Roll Gap,Kernel Shape,and Moisture on Wheat Breakage Modeled Using the Double Normalized Kumaraswamy Breakage Function

Flour milling separates endosperm from bran through repeated roller milling and sifting, in which the size distribution of particles produced by the initial breakage of the wheat kernels critically affects the process. The double normalized Kumaraswamy breakage function (DNKBF), previously developed to describe wheat breakage during roller milling, was extended to refine the modeling of the effect of roll gap on breakage. The DNKBF describes two populations of particles arising from roller milling of wheat, a narrow peak of mid‐sized particles and a wider distribution of both small and very large particles. A new dataset was obtained from milling a set of wheat samples bred to give a range of shapes by cross‐breeding a conventional wheat, Cappelle, with an almost spherical wheat, Triticum sphaerococcum. A residual analysis showed a statistically significant effect of kernel shape on breakage using this new dataset. This analysis supports earlier suggestions that more elongated kernels break to give slightly larger particles than more spherical kernels of equivalent hardness, because of the relatively greater bran content of elongated kernels. The extended DNKBF was also used to model effects of moisture content, showing a distinct disjunction at around 16% moisture that aligns with commercial practice for wheat milling.     

Factors Affecting the Alkaline Cooking Performance of Selected Corn and Sorghum Hybrids

Dent corn (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) sample sets representative of commonly grown hybrids and diverse physical attributes were analyzed for alkaline cooking performance. The influence of kernel characteristics including hardness, density, starch properties (thermal, pasting, and crystallinity), starch content, protein content, and prolamin content on alkaline cooking performance was also determined. Corn nixtamal moisture content was lower for hard, dense kernels with high protein contents; sorghum nixtamal moisture content was lower for kernels with low moisture contents and low starch relative crystallinities. Statistically significant (P < 0.05) regression equations showed that corn nixtamal moisture content was influenced by TADD (tangential abrasive dehulling device) index, kernel moisture content, starch content, and protein content; sorghum nixtamal moisture content was influenced by starch relative crystallinity, kernel moisture content, and abrasive hardness index. Pericarp removal was not strongly correlated with kernel characterization tests. Location (environmental) and hybrid (genetic) factors influenced most kernel characteristics and nixtamalization processing variables.     

Mechanical and Physicochemical Characterization of Vitreous and Mealy Durum Wheat Endosperm

The mechanical, physical, and biochemical characteristics of mealy and vitreous endosperm were investigated. Endosperm were obtained from four durum wheat cultivars grown under different nitrogen fertilization designs. The textural properties and the density of the endosperm were measured on hand‐shaped parallelepiped endosperm samples. Endosperm protein content and composition and also gliadin composition were investigated by HPLC. Mechanical tests showed that mealy and vitreous endosperm differed in hardness and vitreousness. Vitreousness increased with nitrogen fertilization supply whereas there was no variation among the different cultivars. Hardness seemed to be linked to genotype and insensitive to nitrogen supply. From this result, we concluded that hardness and vitreousness are not related. Endosperm protein content and gliadin‐to‐glutenin ratio were related to nitrogen supply and increased especially when nitrogen supply was applied at flowering. At the same time, endosperm vitreousness increased. Further biochemical analyses were performed on 270 kernels, mealy or vitreous, hand‐picked from 148 different crops. Results showed that protein content of vitreous endosperm exceeded 9.7% in >90% of the cases. The glia/glu ratio was a less accurate predictor of kernel vitreousness, indicating that, by itself, it cannot account for the change in kernel vitreousness. Endosperm vitreous texture would rise above a threshold content of 9.7% protein within the endosperm.     

Corn Kernel Structural Integrity: Analysis Using Solvent and Heat Treatments

Most corn (Zea mays, L.) processing is accomplished by causing a structural change to the kernel. Associations between corn endosperm structural components were characterized using textural analysis after solvent and heat treating kernels. Intact Asgrow 405W and B73xMo17 kernels were incubated and treated at 20, 40, 55, and 90°C for 1, 24, and 48 hr in static air, in acetone, and in aqueous solutions of water, calcium chloride, sodium chloride, sodium bisulfite, lactic acid, lime, lye, ethanol urea, and sodium dodecyl sulfate (SDS). After treatment, kernels were compressed between flat platens. Acetone did not significantly soften endosperm structure. Ethanol reduced kernel fracturability by weakening cell‐to‐cell (wall) bonds, but ethanol did not effectively reduce kernel hardness. Water and aqueous solvents swelled and softened kernels by plasticizing structural components. Bisulfite and SDS softened kernels more than water only soaks because they denatured matrix proteins. Alkaline soaks reduced fracturability and softened the kernel by dissociating both cell‐to‐cell and intracellular (starch‐protein) bonds. Soaking for longer periods and at higher temperatures increased aqueous‐based solvent softening effect. Urea imbibition into the kernel and its softening effects were highly dependent on time and temperature of soak. Endosperm structural integrity is the governed by a combination of cell‐to‐cell bonds and intra‐cellular (starch‐protein) bonds. Reagents that denatured the endosperm matrix proteins and disrupted hydrogen bonds resulted in the greatest alterations to kernel structural integrity. Ultimately a better understanding of kernel structural integrity will lead to the development of improved hybrids and process technologies designed to facilitate desirable structural changes.     

Description of a Micromill with Instrumentation for Measuring Grinding Characteristics of Wheat Grain

A new method for characterizing the grinding characteristics of wheat grain is described. A micromill was designed for this purpose and equipped with on‐line torque transducers to obtain accurate measurements of mechanical energy consumption during milling. This micromill can be used for testing the milling performance of small quantities of grain (100 g). It can distinguish between different types of wheat grain (soft wheat, hard wheat, durum wheat) on the basis of total specific energy during milling. Wheat characterization can be enhanced by taking particle sizes of the milled products into account. A milling index based on energy consumption and particle size reduction was developed to characterize wheat behavior during milling. This index had a high discriminatory potential, ranging from 100 kJ/kg for soft wheat flour to 600 kJ/kg for durum wheat flour. This micromill directly measures the grinding resistance of wheat kernels as a function of both the kernel hardness and vitreousness, contrary to standard kernel hardness measurements obtained by particle size index and near‐infrared reflectance analysis techniques that only reflect the fracture mode (fine particle reduction potential).     

Collaborative Analysis of Wheat Endosperm Compressive Material Properties

The objective measurement of cereal endosperm texture, for wheat (Triticum spp. L.) in particular, is relevant to the milling, processing, and utilization of grain. The objective of this study was to evaluate the interlaboratory results of compression failure testing of wheat endosperm specimens of defined geometry. Parallelepipeds (bricks) and cylinders were prepared from individual soft and hard near‐isogenic wheat kernels and compressed in two orientations (parallel and perpendicular to the long brush‐to‐germ axis). Compression curves were used to derive failure stress, failure strain, work density (area under the curve), and Young's modulus. In all five laboratories, the ability to delineate hard from soft wheat endosperm material properties was quite high. Four laboratories compressed endosperm bricks in the same orientation, on edge; texture class (soft vs. hard) was consistently the greatest source of variation in analysis of variance models (F‐values from 417 to 1401, Young's modulus and failure stress, respectively). Failure stress was found to be the best overall means of measuring the difference in what is known in the vernacular as wheat hardness. Across laboratories, the absolute measures of all four material properties ranged on the order of about two‐ to threefold from low to high, although within a laboratory, results were highly consistent. Laboratory by texture class interaction was deemed to be of minor importance. Brick size and moisture content within the ranges tested were not major sources of variation, and cylinders prepared from endosperm produced results similar to those obtained from bricks. The results suggested that wheat endosperm might express some level of anisotropic behavior, as specimens compressed in the kernel orientation parallel to the long axis failed at lower strain and stress values, with lower work density, when compared with kernel orientation perpendicular to the long axis. A key feature of interlaboratory variation was identified as being instrument rigidity, a subject of ongoing research. In conclusion, the preparation of endosperm specimens of defined size and shape, in combination with compression failure testing at low moisture content (<18%), is useful for objectively delineating the phenomenon known as hardness. The study presented here will advance our ability to objectively measure cereal grain texture and the material properties of endosperm.     

A Study of Maize Endosperm Hardness in Relation to Amylose Content and Susceptibility to Damage

The relative amounts of amylose and amylopectin in maize starch were determined in samples representing hard and soft endosperm. Although differences were small, amylose content differed significantly (P < 0.001 and P < 0.05) between the two types of endosperm, with hard endosperm containing a higher percentage of amylose. Scanning electron microscopy was used to determine that the surface appearance of starch granules from hard and soft endosperm differed. Starch granules from soft endosperm had randomly distributed pores on their surfaces, which had a rough appearance. Few pores were observed on granules from hard endosperm. A fairly common occurrence with starch granules from soft endosperm was the development of wrinkles or fissures upon prolonged exposure to the beam of the electron microscope. Thus, a correlation existed between endosperm hardness, amylose content, and susceptibility to wrinkling and fissures. The granules of the soft endosperm of maize, presumably less mature than the granules of the hard endosperm, have a lower amylose content (20.5 ± 1.9% vs. 23.0 ± 1.0%), exhibit more surface pores, and are more susceptible to wrinkling in an electron beam, compared with granules of the hard endosperm. Results suggested that the composition and internal architecture of the starch granule differ depending on the hardness of the endosperm from which it was obtained.     

Modeling First Break Milling of Debranned Wheat Using the Double Normalized Kumaraswamy Breakage Function

Debranning of wheat affects flour quality, initially by altering the breakage of wheat kernels during first break. The double normalized Kumaraswamy breakage function was applied to model the effect of debranning on wheat breakage during first break milling, in which type 1 breakage describes a relatively narrow distribution of midsized particles, whereas type 2 breakage describes a wide size range of predominantly small particles extending to very large particles. Mallacca (hard) and Consort (soft) wheats were debranned and milled at three roll gaps under sharp‐to‐sharp and dull‐to‐dull dispositions. Type 1 breakage increased at longer debranning times, whereas type 2 breakage decreased, for both wheat varieties under both dispositions. Sharp‐to‐sharp milling tended to produce more type 1 breakage than dull‐to‐dull. A mechanism of wheat breakage is proposed to explain the coproduction of very large and small particles via type 2 breakage and, hence, the effect of debranning. The proposed mechanism is that small particles of endosperm arise from scraping of large flat particles of wheat bran under the differential action of the rolls, such that removal of the bran reduces the production of the large bran particles and thus reduces the opportunity for the scraping mechanism that produces the very small particles.     

Relationship Between Single Wheat Kernel Particle-Size Distribution and Perten SKCS 4100 Hardness Index

The Perten Single Kernel Characterization system is the current reference method for determination of single wheat kernel texture. However, the SKCS 4100 calibration method is based on bulk samples. The objective of this research was to develop a single-kernel hardness reference based on single-kernel particle-size distributions (PSD). A total of 473 kernels, drawn from eight different classes, was studied. Material from single kernels that had been crushed on the SKCS 4100 system was collected, milled, then the PSD of each ground single kernel was measured. Wheat kernels from soft and hard classes with similar SKCS hardness indices (HI 40–60) typically had a PSD that was expected from their genetic class. That is, soft kernels tended to have more particles at <21 μm than hard kernels after milling. As such, a combination of HI and PSD gives better discrimination between genetically hard and soft classes than either parameter measured independently. Additionally, the use of SKCS-predicted PSD, combined with other low level SKCS parameters, appears to reduce classification errors into genetic hardness classes by ≈50% over what is currently accomplished with HI alone.     

Kernel Microstructure of Latin American Races of Maize and Their Thermal and Rheological Properties

Seventy‐one races of maize representing races from Latin America were analyzed for microstructural features such as the degree of compaction of the endosperm cell bodies, starch granule size and morphology, and hard‐soft endosperm relationship. Flours were analyzed using rapid visco analysis and differential scanning calorimetry. Compaction grade was the most important microstructural feature of the maize kernels that related to thermal and rheological properties. Highly compact kernels developed low peak and final viscosities; small, polygonal starch granules; and required more time and higher temperature to gelatinize. The opposite was the case for less compact kernels. This indicates that the characteristic protein matrix of highly compact kernels represents a physical barrier to water migration into the granules, retarding the gelatinization process.     

Effect of Soft Kernel Texture on the Milling Properties of Soft Durum Wheat

Worldwide, nearly 20 times more common wheat (Triticum aestivum) is produced than durum wheat (T. turgidum subsp. durum). Durum wheat is predominately milled into coarse semolina owing to the extreme hardness of the kernels. Semolina, lacking the versatility of traditional flour, is used primarily in the production of pasta. The puroindoline genes, responsible for kernel softness in wheat, have been introduced into durum via homoeologous recombination. The objective of this study was to determine what impact the introgression of the puroindoline genes, and subsequent expression of the soft kernel phenotype, had on the milling properties and flour characteristics of durum wheat. Three grain lots of Soft Svevo and one of Soft Alzada, two soft‐kernel back‐cross derived durum varieties, were milled into flour on the modified Quadrumat Senior laboratory mill at 13, 14, and 16% temper levels. Samples of Svevo (a durum wheat and recurrent parent of Soft Svevo), Xerpha (a soft white winter wheat), and Expresso (a hard red spring wheat) were included as comparisons. Soft Svevo and Soft Alzada exhibited dramatically lower single‐kernel characterization system kernel hardness than the other samples. Soft Svevo and Soft Alzada had high break flour yields, similar to the common wheat samples, especially the soft hexaploid wheat, and markedly greater than the durum samples. Overall, Soft Svevo and Soft Alzada exhibited milling properties and flour quality comparable, if not superior, to those of common wheat.     

A Novel Modified Endosperm Texture in a Mutant High‐Protein Digestibility/High‐Lysine Grain Sorghum (Sorghum bicolor (L.) Moench)

Development of high‐protein digestibility (HPD)/high‐lysine (hl) sorghum mutant germplasm with good grain quality (i.e., hard endosperm texture) has been a major research objective at Purdue University. Progress toward achieving this objective, however, has been slow due to challenges posed by a combination of genetic and environmental factors. In this article, we report on the identification of a sorghum grain phenotype with a unique modified endosperm texture that has near‐normal hardness and possesses superior nutritional quality traits of high digestibility and enhanced lysine content. These modified endosperm lines were identified among F₆ families developed from crosses between hard endosperm, normal nutritional quality sorghum lines, and improved HPD/hl sorghum mutant P721Q‐derived lines. A novel vitreous endosperm formation originated in the central portion of the kernel endosperm with opaque portions appearing both centrally and peripherally surrounding the vitreous portion. Kernels exhibiting modification showed a range of vitreous content from a slight interior section to one that filled out to the kernel periphery. Microstructure of the vitreous endosperm fraction was dramatically different from that of vitreous normal kernels in sorghum and in other cereals, in that polygonal starch granules were densely packed but without the typically associated continuous protein matrix. We speculate that, due to the lack of protein matrix, such vitreous endosperm may have more available starch for animal nutrition, and possibly have improved wet‐milling and dry‐grind ethanol processing properties. The new modified endosperm selections produce a range that approaches the density of the vitreous parent, and have lysine content and protein digestibility comparable to the HPD/hl opaque mutant parent.     

Hardness Measurement of Bulk Wheat by Single‐Kernel Visible and Near‐Infrared Reflectance Spectroscopy

Reflectance spectra (400 to 1700 nm) of single wheat kernels collected using the Single Kernel Characterization System (SKCS) 4170 were analyzed for wheat grain hardness using partial least squares (PLS) regression. The wavelengths (650 to 700, 1100, 1200, 1380, 1450, and 1670 nm) that contributed most to the ability of the model to predict hardness were related to protein, starch, and color differences. Slightly better prediction results were observed when the 550–1690 nm region was used compared with 950–1690 nm region across all sample sizes. For the 30‐kernel mass‐averaged model, the hardness prediction for 550–1690 nm spectra resulted in a coefficient of determination (R²) = 0.91, standard error of cross validation (SECV) = 7.70, and relative predictive determinant (RPD) = 3.3, while the 950–1690 nm had R² = 0.88, SECV = 8.67, and RPD = 2.9. Average hardness of hard and soft wheat validation samples based on mass‐averaged spectra of 30 kernels was predicted and compared with the SKCS 4100 reference method (R² = 0.88). Compared with the reference SKCS hardness classification, the 30‐kernel (550–1690 nm) prediction model correctly differentiated (97%) between hard and soft wheat. Monte Carlo simulation technique coupled with the SKCS 4100 hardness classification logic was used for classifying mixed wheat samples. Compared with the reference, the prediction model correctly classified mixed samples with 72–100% accuracy. Results confirmed the potential of using visible and near‐infrared reflectance spectroscopy of whole single kernels of wheat as a rapid and nondestructive measurement of bulk wheat grain hardness.     

Variation in Kernel Hardness and Associated Traits in U.S. Barley Breeding Lines

Kernel hardness is an important trait influencing postharvest handling, processing, and food product quality in cereal grains. Though well‐characterized in wheat, the basis of kernel hardness is still not completely understood in barley. Kernels of 959 barley breeding lines were evaluated for hardness using the Single Kernel Characterization System (SKCS). Barley lines exhibited a broad range of hardness index (HI) values at 30.1–91.9. Distribution of kernel diameter and weight were 1.7–2.9 mm and 24.9–53.7 mg, respectively. The proportion of hull was 10.2–20.7%. From the 959 breeding lines, 10 hulled spring barley lines differing in HI values (30.1–91.2) were selected to study the associations of HI with proportion of hull, kernel weight, diameter, vitreousness, protein, β‐glucan, and amylose content. Vitreousness, evaluated visually using a light box, showed a clear distinction between hard and soft kernels. Hard kernels appeared translucent, while soft kernels appeared opaque when illuminated from below on the light box. Kernel brightness (L*), determined as an indicator of kernel vitreousness, showed a significant negative correlation (r = –0.83, P < 0.01) with HI. Protein, β‐glucan, amylose content, proportion of hull, kernel weight, and diameter did not show any significant association with HI.