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Journal of Cereal Science 54 (2011) 354e362

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Journal of Cereal Science

journal homepage: www.elsevier .com/locate/ jcs

Microstructure of hard and soft kernels of barley

Sindhu Nair a, Michael Knoblauch b, Steven Ullrich a, Byung-Kee Baik a,*

aDepartment of Crop and Soil Sciences, Washington State University, Pullman, WA 99164-6420, USAb School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA

a r t i c l e i n f o

Article history:Received 31 August 2010Received in revised form2 June 2011Accepted 27 June 2011

Keywords:BarleyHardnessStructureMicroscopy

Abbreviations: HI, Hardness index; LM, Light mmission; SEM, Scanning electron microscopy; SKCS, Ssystem; TADD, Tangential abrasive dehulling device.* Corresponding author. Tel.: þ1 509 335 8230; fax

E-mail address: bbaik@wsu.edu (B.-K. Baik).

0733-5210/$ e see front matter Published by Elseviedoi:10.1016/j.jcs.2011.06.014

a b s t r a c t

Kernel hardness, an important quality trait of cereal grains, is known to influence pearling properties andmalting quality of barley. To understand the endosperm micro-structural features of kernels and theirrelationship to kernel hardness, endosperms of three hard and three soft hulled spring barley lines basedon single kernel characterization system hardness index were observed under light (LM) and scanningelectron (SEM) microscopy. Under LM, endosperm cell wall of the three hard kernel lines was signifi-cantly thicker than that of the three soft kernel lines. Hard and soft lines showed differences in thedegree of starch-protein association and continuity of protein matrix under the SEM. Hard kernel lineswith a continuous protein matrix exhibited greater starch-protein adhesion than the soft kernel lines,suggesting that starch-protein binding may be one of the factors influencing barley kernel hardness. SEMof flour particles of soft kernel lines showed numerous well defined individual A and B-type starchgranules, while, flour of hard kernel lines mostly showed small flour aggregates with few individualstarch granules.

Published by Elsevier Ltd.

1. Introduction

Barley, Hordeum vulgare subsp. vulgare, is one of the mostversatile cereal crops suitable for cultivation in extreme environ-ments. As one of the founder crops of Old World Agriculture, barleywas domesticated from its wild relative H. vulgare subsp. sponta-neum about 10,000 years ago in the Fertile Crescent of the MiddleEast (Badr et al., 2000). Despite its historical significance as animportant food source in parts of Asia, Africa and Europe, onlyabout 2% of the total barley produced is currently used as food (Baikand Ullrich, 2008). Proven health benefits of barley along with theU.S Food and Drug Administration approval of health food claimsand the easy availability of this crop is gradually reviving humaninterest in barley based food products. Though extensive informa-tion about the nutritional benefits of barley is available, not much isknown about the functionality of barley grain components in termsof processing and food product development (Baik and Ullrich,2008).

Endosperm texture is one of the most important functionalparameters known to significantly influence processing and

icroscopy; LTm, Light trans-ingle kernel characterization

: þ1 509 335 8674.

r Ltd.

product quality in wheat, where kernel hardness is the primarymarket class determinant. Kernel hardness is defined as the resis-tance of the kernel to deformation with hard kernels being moreresistant to destruction and soft kernels being easily damaged.Unlike kernel hardness, which describes a mechanical property ofkernels, vitreousness is a visual description of the kernel based onthe endosperm packing with the terms mealiness and vitreousnessor steeliness used to describe the endosperm structure (Turnbulland Rahman, 2002). Malting, a process of controlled germinationwhich involves a series of enzyme degradations of cell walls andstarchy endosperm, is strongly influenced by the structure andchemical composition of the endosperm. Palmer (1991), Chandraet al. (1999), and Holopainen et al. (2005) reported relationshipsbetween endosperm structure and several barley kernel and maltcharacteristics. Though such studies indicate a significant role ofendosperm structure onmalting quality, it also suggests that barleyendosperm structure may have some influence on other functionalfood traits and food processing parameters.

Kernel hardness, an important quality parameter in wheat, hasbeen shown to significantly influence the malting quality in barley(Henry and Cowe, 1990; Psota et al., 2007; Vejra�zka et al., 2007).While the significance of kernel hardness and endosperm structureon malting has been relatively well established, not much is knownabout the structural basis for variation in barley kernel hardness.The objective of the research reported here in was to understandthe structural features of barley kernels differing in single kernelcharacterization hardness index.

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2. Materials and methods

2.1. Materials

Kernels of 25 genetically diverse hulled spring barley linesincluding eight lines from the USDA-CSREES sponsored barleyCoordinated Agricultural Project grown in Pullman, WA in 2007were tested for their single kernel characterization system (SKCS)hardness index (HI). From these 25 barley lines, six lines of variableSKCS HI were selected for the final study.

To prevent clogging of the SKCS, barely kernels were abraded for80 s to remove the tightly attached hull using the TangentialAbrasive Dehulling Device (TADD) (Venables Machine Works,Saskatoon, Canada) before measuring their SKCS hardness. The lossof kernel mass after 80 s abrasion ranged from 14.8 to 15.6% for thesoft lines and 14.2e14.6% for the hard lines. Barley kernel hardnesswas determined on whole, dehulled, non-shriveled bright kernelsof each line using the SKCS 4100 (Perten Instruments, Springfield,IL). In addition to average hardness of 100 kernels expressed as HI,the SKCS also provides average kernel diameter (mm), weight (mg),and moisture (%) measurements.

A subsample of dehulled barley kernels of the six selected lineswere ground using a Cyclone sample mill (Udy, Fort Collins, CO)fitted with 0.5 mm screen to produce flour. The flour was mixedwell and stored in tightly sealed glass jars.

2.2. Vitreousness

Visual evaluation of vitreousness of barley kernels was per-formed using a light box. Fifty dehulled kernels of each of the sixselected barley lines were placed with their crease side down ona bottom lit light box and observed for the illumination trend (Nairet al., 2010a). Only the illumination pattern of the six barley lineswas noted without any numerical interpretation of the visualobservation. Brightness (L*), as an indicator of vitreousness, wasmeasured using a chromameter (CR-310, Minolta, Osaka, Japan).Dehulled barley kernels were packed into a container with blackbackground provided in the kit and the light reflected by thekernels was measured (Nair et al., 2010a).

2.3. Light microscopy (LM)

2.3.1. Specimen fixation and embeddingTen dry mature dehulled kernels of each barley line were cut

into quarters at room temperature with stainless steel uncoatedsingle-edge industrial blades. The cut kernels were fixed overnightin 2e2.5% glutaraldehyde/2% paraformaldehyde in 0.05 M caco-dylate buffer (pH 7.2) at 4 �C. Following aldehyde fixation, speci-mens were rinsed thrice (10 min each) with 0.05 M cacodylatebuffer to completely remove the primary fixative and thenimmersed overnight in 1% osmium tetroxide in 0.05 M cacodylatebuffer at 4 �C for post fixation (Karnovsky, 1965; Parlade, 1952). Thespecimens were rinsed twice (10 min each) with 0.05 M cacodylatebuffer and then dehydrated in ethanol series (30%, 50%, 70%, 80%,95%, 100%) followed by ethanol and acetone in 1:1 ratio and 100%acetone. Infiltration was done with SPURRs resin and acetone ina ratio of 1:3, 1:2, 1:1 and 3:1, and each ratio involved overnightinfiltration in a hood. This was followed by overnight infiltration in100% SPURRs in a hood (three times). Embedding was done inSPURRs, overnight at 70 �C (Spurr, 1969).

2.3.2. Sectioning and stainingResin embedded blocks were sectioned with glass knives using

a Reichert-Jung Ultracut R ultramicrotome (Leica Wetzlar,Germany) set at speed 2.0 mm/s with section thickness of 0.5 mm.

Sections were cut from the same central part of each kernel for eachline and transferred to moist gel-coated glass slides. Dual staincombination of 2% Safranin O and 1% potassium iodide (KI) wasused for LM. Sections were first stained with 2% Safranin O withheating at 61 �C for 20 s. They were then washed with distilledwater, heat-dried and stained with 1% KI with heating for 13 s.Sections weremounted in immersion oil tomake permanent slides.

2.3.3. Image analysis and measurementsSpecimens were observed at 200 and 400�magnifications with

a bright field microscope (LEICA DM-LFSA, Leica Microsystems,Heidelberg, Germany). Five images of 200� magnification fromdifferent kernels of each line were selected for cell wall measure-ments. Twenty random cell wall measurements per image wererecorded using the publicly available Java-based image processingImage J software. Two replicates of 100 measurements were takenfor each line.

2.4. Scanning electron microscopy (SEM)

Dry mature dehulled barley kernels were cut into halves withstainless steel uncoated single-edge industrial blades at roomtemperature and placed on aluminum specimen mounts coveredwith double coated carbon conductive tabs (Ted Pella Inc., Redding,California, USA). For the SEM of barley flour, a thin single layer offlour of each line was spread on the double coated carbonconductive tab. Both the kernel sections and the flour particleswere gold coated at 10 mA for 6 min to get an approximately15e20 nm thick coating (Technics Hummer V Sputter Coater,Technics, San Jose, California, USA). Barley kernel sections and flourparticles were examined using a scanning electron microscope at12 kVwith aworking distance of 15 and 10mm, respectively (S-570Hitachi Ltd., Tokyo, Japan). Images were captured and stored digi-tally with Quartz PCI v. 4.2- Scientific Image Management System(Quartz Imaging Corporation, Vancouver, British Columbia,Canada).

2.5. Statistical analysis

Statistical significance of the differences between the hard andsoft kernel barley lines was calculated using paired Student t-test.All the tests were performed at least in duplicate.

3. Results and discussion

3.1. Relationship between SKCS kernel hardness and vitreousness

Visual observation using a diffusion light box showed consistentdifferences in light transmission patterns between hard and softkernel lines. On the diffusion light box, hard lines appeared mostlytranslucent while soft lines appeared dark and opaque; suggestingthat vitreousness or endosperm packing affects SKCS HI with hardkernels being vitreous and soft kernels being mealy (Fig. 1). Whenkernels are illuminated from below, endospermic pores and theloosely packed starch granules present in the soft mealy kernelsmay scatter a large proportion of the incident light, giving anopaque appearance. On the other hand, hard vitreous kernelsowing to their compact endosperm composed of tightly packedstarch and protein, may transmit most of the incident lightresulting in a translucent appearance. The L* values of soft kernellines ranged from 64.2 to 65.9 with an average of 64.8 while that ofhard kernel lines ranged from 62.4 to 63.9 with an average of 63.1(Table 1) and was significantly different (P¼ 0.01) between the twogroups. These results concur with the visual evaluation of vitre-ousness using the light box. Unlike the light box which provides

Fig. 1. Dehulled soft (S1eS3) and hard (H1eH3) kernel barley lines on a light box illuminated from below. Soft kernel lines appear opaque, while hard kernel lines appear mostlytranslucent. Dark patches seen on both soft and hard kernel lines are the remnants of the hull still attached to the kernels after 80 s abrasion.

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illumination from below, the Minolta chromameter illuminateskernels from above. Consequently, when kernels are illuminatedfrom above, vitreous kernels owing to their compact endospermpacking, allow most of the incident light to pass through thekernels. This gets absorbed by the black background of thecontainer, giving a reduced reflection of light resulting in lower L*values. Mealy endosperm allows little transmission of light throughthe kernel and reflects more incident light than vitreous endo-sperm, resulting in higher L* values. These results implicateendosperm packing in barley kernel hardness and agree with theprevious report by Nair et al. (2010a), suggesting that degree ofendosperm packing may be one of the probable factors responsiblefor variation in barley endosperm texture.

Nair et al. (2010a) reported significant associations betweenbarley SKCS HI and vitreousness measured by both the Minoltachromameter and the light box. They observed that hard barleylines were more vitreous than the soft barley lines. Using a rapidlight transmission meter (LTm meter) that measures the lightpassing through individual barley kernels, Chandra et al. (2001)

Table 1Physical characterization of softa and hardb kernel hulled spring barley lines basedon single kernel characterization system hardness index (SKCS HI).

Line SKCS Cell wall L*

HI Thickness (mm)

S1 31.8 2.7 64.2S2 41.8 2.7 64.3S3 45.9 3.5 65.9Mean 39.8** 2.9** 64.8**

H1 72.3 4.6 63.9H2 85.8 5.3 62.9H3 94.1 4.4 62.4Mean 84.1** 4.8** 63.1**

**Soft vs. hard mean differences significance at P < 0.01 based on paired Studentt-test.L* or brightness was measured as an indicator of kernel vitreousness. Small L*indicates a high degree of kernel vitreousness.

a Soft kernel barley lines-S.b Hard kernel barley lines-H.

reported that mealy kernels hinder light transflectance due toincreased scatter, giving low LTm values, while, vitreous kernelsfacilitate light transflectance resulting in higher LTm values.

3.2. Micro-structure of barley kernel and its relationship withkernel hardness

For light microscopy (LM), various stains such as Periodic AcidSchiff’s, Fast Green, Toluidine Blue, Stevenel Blue, Analine BlueBlack as well as different combinations involving these stains weretried, but none of these stains prominently highlighted the cell wallalong with other kernel features. Calcofluor, a fluorochrome withan affinity for b-glucans, was also tried but it failed to properly stainthe SPURRs embedded specimens. Safranin O clearly demarcatedprotein and non-starch areas including the cell walls of the barleyendosperm. Therefore, a two-stain combination of Safranin O andpotassium iodide (KI) was used. Safranin O stained protein dark,opaque orange and the cell walls, a translucent pink-orange shade.Starch granules were stained bluish-purple by KI.

Both soft and hard kernel lines showed cellular organizationtypical of a barley caryopsis. Overall, cross sections of barley kernelsshowed from outside-in several layers of outer pericarp, cuticularand nucellar cell walls, aleurone layer, and inner endosperm(Fig. 2). The sub-aleurone layer, comprised primarily of protein, ispresent at the junction of the aleurone and the inner endosperm(Fulcher et al., 1977). The aleurone region, which ranged from oneto three cell layers, showed somewhat cubical cells which wereabundant in spherical aleurone grains. The sub-aleurone layershowed small starchy endosperm cells which were separated bythe endosperm cell wall material and contained small starchgranules that appeared to be embedded in a relatively thick proteinmatrix. The sub-aleurone region has a higher proportion of proteinthan the inner endosperm region. The major portion of the kernelwas composed of starchy endosperm cells which contained big (A-type) and small (B-type) starch granules surrounded by the proteinmatrix. Under LM, no systematic differences were observed in theorganization of the aleurone and sub-aleurone regions or thepacking of the endosperm cells between the hard and soft kernellines.

Fig. 2. Light micrographs of the outer endosperm region of three soft (S1eS3) and three hard (H1eH3) kernel spring barley lines at 400� magnification. Sections show outerpericarp (P), cuticular (C) and nucellar (N) cell walls. Aleurone (A) region ranges from one to three cell layers thick with the sub-aleurone (SA) layer interior to the aleurone. Starchyendosperm (E) forms the major part of the kernel.

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Though sections of all six lines were taken from the same kernelregion and stained for the same time period, intensity of staining orthe stain uptake by the sections varied between lines and alsowithin lines. Both within a line and between lines, some sectionswere more brightly and noticeably stained than the others. Varia-tion within sections of one kernel of a line may probably be due toany errors in infiltration and sectioning, while variation withinsections of different kernels of the same line may also be due to theheterogeneity of the kernels from the same cultivar. Variation instain intensity between sections of different linesmay be due to anyvarietal differences between the lines that affects its stain uptake.Within a section, cells in some areas appeared to be shrunken andwithdrawn from the cell walls leaving gaps between cells and thesurrounding cell walls. These gaps are artifacts resulting from thefixation and alcohol dehydration procedures. In certain places, the

cells were intact but starch granules were missing, resulting in gapswithin the cells. This probably was due to the loss of starch granuleseither while cutting the kernels into quarters or during sectioning.While cytoplasmic components are strongly affected by fixation,the alteration of cell wall dimensions is minimal (Mullendore et al.,2010).

Cell wall thickness was measured only in the central endospermregion inside of the sub-aleurone layers. Safranin O stained the cellwalls a distinct translucent orange and this greatly facilitated thecell wall thickness measurement under the light microscope. Sincemicrographs showed cell walls sectioned in different angles (crosssections to tangential sections), measurements were taken fromcross sections only in order to receive accurate statistics on cell wallthickness. Cell wall thickness of hard kernel lines ranged from 4.4 to5.3 mm with an average of 4.8 mm and was significantly thicker

Fig. 3. Scanning electron micrographs of the central endosperm region of three soft (S1eS3) and three hard (H1eH3) kernel spring barley lines at 1000� magnification. (S - starchgranule; P - protein matrix).

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(P ¼ 0.01) than that of soft kernel lines whose cell wall thicknessranged from 2.7 to 3.5 mmwith an average of 2.9 mm (Table 1). Thisresult suggests that cell wall thickness may be one of the probablefactors responsible for the observed textural variation in barley.Though barley cell walls have been extensively investigated formalting purposes, very limited information is available on differ-ences in cell wall thickness of barley lines of variable hardness. Ina study on the characterization of induced low b-glucan barleymutants, Aastrup (1983) found that mutants with low b-glucancontent had thinner cell walls compared to Minerva, the progenitorcultivar. In SEM studies on the barley microstructure, both Fornalet al. (2000) and Holopainen et al. (2005) reported thicker cellwalls in vitreous barley kernels compared to the mealy kernels. Inan SEM study on barley near-isogenic lines (NILs) containing bglgene, the recessive mutant gene for (1/3) (1/4)-b-D-glucan lessbarley grain, Tonooka et al. (2009) observed that the NILs had thinendosperm cell walls and softer grain texture.

3.3. Scanning electron microscopy (SEM) analysis of barleyendosperm structure

Both SKCS hard and soft kernel lines showed similar patterns ofcell size and distribution under the SEM. The bulk of the kernel wascomposed of the starchy endosperm which showed both A-typeand B-type starch granules. The amount of protein increasedgradually from the central endosperm to the sub-aleurone region.

Fig. 4. Scanning electron micrographs of the central endosperm region of three soft (S1eS3granule; P - protein matrix).

Cells in the central region of the endospermwere irregular in shapeand highly disorganized in arrangement, whereas more organizedcells were observed in the peripheral region of the endosperm.

Hard and soft kernel lines showed differences in the degree ofstarch-protein association and continuity of protein matrix (Figs. 3and 4). Soft kernel lines showed both A-type and numerous B-typestarch granules within the endosperm cells. A-type starch granuleswere smooth, well defined and showed little amounts of theassociated protein matrix. B-type starch granules were also prom-inently visible and showed some residual protein still attached tothem. Interface between the A-type and B-type starch granulesshowed small amounts of the matrix protein. Hard kernel linesshowed a continuous protein matrix and exhibited greater starch-protein adhesion than the soft kernel lines. A-type starch gran-ules appeared to be embedded in a dense network of the associatedprotein. This continuous protein sheet appeared to cover thepresence of B-type starch granules such that only the A-type starchgranules were mostly visible within the starchy endosperm (Figs. 3and 4). The differences were most visually prominent between thesoftest (S1) and the hardest (H3) lines. This is expected as there isa difference of almost 60 hardness units between the two. Thoughthe other two soft lines, which had hardness values in the forties,also showed smooth, well defined starch granules with discontin-uous protein matrix, S3 showed features slightly closer to the hardlines, possibly due to its hardness value being the highest amongthe three soft lines.

and three hard (H1eH3) kernel spring barley lines at 2000� magnification. (S - starch

Fig. 5. Scanning electron micrographs of flour particles of three soft (S1eS3) and three hard (H1eH3) kernel spring barley lines at 1300� magnification.

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These results suggest that continuity of protein matrix and thedegree of starch-protein adhesion may also contribute to the vari-ation in barley kernel hardness. Continuous protein matrix whichfirmly embeds the starch granules may provide resistance to frac-ture and the subsequent disintegration of the kernel; therebyimparting a harder texture. This protein network may also preventthe easy release of starch granules, subsequently producing coarseparticles in hard kernels duringmilling. Additional force required togrind the coarse particles during milling may further result inadded starch damage in hard kernels. Accordingly, continuity of theprotein matrix and its association with starch granules may be themajor factors responsible for the differences in particle size distri-bution and starch damage observed between hard and soft barleylines (Nair et al., 2010b). Similar structural observations have beenreported in wheat (Turnbull and Rahman, 2002).

Brennan et al. (1996) reported similar observations in SEM ofstructural features of good and poor malting barley cultivars. In theendosperm of good malting barley, the A-type granules weresmooth and lacked the associated protein matrix. In poor maltingbarley, the A-type starch granules were embedded in the networkof the protein matrix which appeared to completely embed andmask the presence of B-type starch granules within the endosperm.Palmer (1991) and Chandra et al. (1999) observed that vitreouskernels had dense endosperm with tightly packed protein andstarch granules, while in mealy kernels, the endospermwas looselypacked with frequent air spaces between starch granules. Fornalet al. (2000) reported that vitreous Vs mealy kernels had thickercell walls and more densely packed endosperm with starch gran-ules embedded in the protein matrix.

3.4. Scanning electron microscopy (SEM) analysis of barley flour

In the SEM analysis of soft and hard kernel barley flours, particlesize appeared to increase with kernel hardness. Among the sixbarley lines, difference in flour particle size was most conspicuousin the softest (Fig. 5[S1]) and the hardest (Fig. 5[H3]) lines.Numerous independent A-type and B-type starch granules spreadall throughout were observed for the soft kernel lines. Hard kernellines appeared to show a greater proportion of flour clustersinstead of independent starch granules (Fig. 5). In soft kernel lines,the A-type starch granules appeared smooth and well defined withlittle residual matrix protein attached to them. Numerous B-typegranules were also clearly seen (Fig. 5). Surface of A-type starchgranules appeared to be rough probably due to the presence ofremnant protein matrix in hard kernel lines. B-type granulesappeared to be clustered within the protein matrix in hard kernellines (Fig. 5).

The observed differences in flour particle size may be due to thedegree of starch-protein association and continuity of the endo-sperm protein matrix. It appears that while milling barley using anUdy grinder, it is relatively easier to release starch granules fromthe loosely attached discontinuous protein matrix surroundingthem, resulting in smaller flour particles in soft kernels. By contrast,during grinding of hard kernels, it may be difficult to release starchgranules from the strongly attached protein matrix, making endo-sperm disintegration difficult, resulting in bigger particle size.These observations appear to agree with the results of our previousstudy. In a study on the influence of barley kernel hardness on foodprocessing parameters, Nair et al. (2010b) reported positive corre-lation between SKCS kernel hardness and proportion of barley flourparticles >106 mm. SEM of both barley flour particles and kernelsections, and flour particle size distribution determined in ourprevious study (Nair et al., 2010b) suggest that the degree of starch-protein adhesion and the continuity of protein matrix may be oneof the major influences for the observed textural variation in barley.

In wheat, kernel hardness is a simply inherited trait controlledby a single gene with other minor genes being responsible forvariation in hardness within classes of hard and soft wheat cultivars(Symes, 1965). The explanations for this variation in hardnessinclude the degree of adhesion between starch and the proteinmatrix or the continuity of the protein matrix and the strength withwhich it physically entraps starch granules (Barlow et al., 1973;Stenvert and Kingswood, 1977). It appears that similar mecha-nisms may also be involved in barley hardness variation. Pur-oindoline proteins, coded for by the Pin genes, are the primarycomponents of friabilins and have been implicated in wheathardness. Friabilins bind to the starch granular lipids and preventtight adhesion between starch granules and the surroundingprotein matrix. Starch granules can be easily separated from theprotein matrix in soft wheat, whereas, in hard wheat, the tightadhesion between starch and protein matrix makes separationdifficult, resulting in an increased energy requirement and starchdamage during milling (Turnbull and Rahman, 2002). Friabilinproteins have been investigated for their involvement in barleykernel hardness variation.While Morrison et al. (1992) observed nofriabilins in barley, Jagtap et al. (1993) found small amounts offriabilins in barley. Darlington et al. (2000) found equal amounts offriabilins in starches obtained from both hard and soft barleykernels. Darlington et al. (2001) reported the presence of hor-doindolines (puroindoline homologs of wheat) in barley endo-sperm but found no association between kernel texture andhordoindolines. Other workers observedminor allelic variations forthe hordoindolines but found no clear relationship between thesequence variation and grain hardness (Beecher et al., 2001; Foxet al., 2007).

Since the role of friabilins in barley is not very conclusive, theexact mechanism controlling the observed starch-protein adhesionneeds to be investigated. It may be possible that some otherprotein, either alone or in combination with other proteinsincluding the friabilins, may be responsible for this observedassociation. Since only 22% of the observed textural variation hasbeen attributed to the hardness locus in barley (Beecher et al.,2002), it appears that there may be other genomic loci involvedin controlling barley kernel hardness. Also, the environmentaleffect on starch-protein association and the continuity of proteinmatrix needs to be investigated.

4. Conclusion

Cell wall thickness detected under light microscopy appears tobe associated with barley kernel hardness. SKCS hard kernel lineshave thicker cell walls than soft kernel lines. Under the scanningelectron microscopy (SEM), hard and soft lines showed differencesin the degree of starch-protein association and the continuity ofprotein matrix. Hard kernel lines with a continuous protein matrixexhibited greater starch-protein adhesion than the soft kernel linessuggesting that starch-protein bindingmay be another major factordetermining barley kernel hardness. As observed from SEM offlours, soft kernel lines appeared to produce smaller sized flourparticles than the hard kernel lines.

Acknowledgments

This research was carried out as a part of the USDA-CSREESsponsored Barley Coordinated Agricultural Project. We would liketo thank the staff of the Franceschi Microscopy and Imaging Centerat the Washington State University, Pullman, for providing tech-nical assistance in equipment training, sample preparation andimage analysis.

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