Items from the United States - Kansas.




Room 200, 632 S.W. Van Buren, Topeka, KS 66603, USA.

E.J. Thiessen, Sherri Hand, and Ron Sitzman.

Kansas wheat varieties.

Jagger remains number one. Jagger was the leading variety of wheat seeded in Kansas for the 2000 crop, according to Kansas Agricultural Statistics (Table 1). Jagger gained popularity in all districts except the north central and southeast, accounting for 34 % of the wheat in the state. Jagger made the biggest gains in the west-central and southwest districts. The KSU-maintained variety 2137 ranked second over all, with 23.1 % of the acreage, and ranked first or second in all districts except for the southwest, where it was third. TAM 107 remained in third position, but dropped to 6.3 %of the acreage statewide. Ike moved up to fourth place with 4.1 %of the acreage, but dropped 1.4 % from last year. The fifth most popular varieties were Karl and improved Karl with 3.5 % of the acreage. The KSU-maintained variety 2163 ranked in the top five varieties in all but the western districts of the state and accounted for 2.3 %. Seventh was AGSECO 7853, with 1.5 %. New to the top 10 are Dominator, with 1.4 %, and TAM 110, with 1.3 %. Larned remained in the top 10, with 1.2 percent. Blends were used more extensively in the central one-third of the state, accounting for 7.5 % of the acres planted statewide. Out of the total state acres planted with blends, 87 % had Jagger in the blend and 76 % had 2137 in the blend. All HWWW varieties accounted for 0.2 % of the acreage.

Table 1. Top 10 wheat varieties grown in the state of Kansas for the 1999 crop and percent of seeded acreage.

 Variety % of acreage  Variety  % of acreage
 1. Jagger  34.0  6. 2163  2.3
 2. 2137  23.1  7. AGSECO 7853  1.5
 3. TAM 107  6.3  8. Dominator  1.4
 4. Ike  4.1  9. TAM 110  1.3
 5. Karl/Karl 92  3.5  10. Larned  1.2

Table 2. Top wheat varieties planted in Kansas by district and percent of seeded acreage.

 Variety  % of acreage  Variety % of acreage   Variety % of acreage 
 District 10 (Northwest)  District 40 (North central)  District 70 (Northeast)
 Jagger  21.0  2137  26.9  2137  31.7
 2137  17.5  Jagger  19.1  Karl/Karl 92  28.3
 TAM 107  14.1  Karl/Karl 92  12.5  Jagger  12.2
 Vista  8.7  Dominator  6.5  2163  6.6
 Niobara  3.8  2163  2.7  Dominator  4.8
 District 20 (West central)  District 50 (Central)  District 80 (East central)
 Jagger  22.2  2137  35.0  2137  36.4
 2137  21.5  Jagger  29.5  Karl/Karl 92  21.7
 TAM 107  20.8  Karl/Karl 92  4.8  Jagger  17.2
 Ike  9.5  2163  4.3  2163  6.2
 TAM 110  5.6  Dominator  3.2  Dominator  2.9
 District 30 (Southwest)  District 60 (South central)  District 90 (Southeast)
 Jagger  23.9  Jagger  49.2  Jagger  36.7
 TAM 107  16.9  2137  20.2  2137  33.5
 2137  16.1  2163  2.5  Karl/Karl 92  6.9
 Ike  16.1  AGSECO 7853  2.5  2163  2.6
 TAM 110  4.3  Karl/Karl 92  2.4  AgriPro Big Dawg  2.3


Table 3. Distribution of Kansas winter wheat cultivars, 2000 crop.

Table 4. Distribution of Kansas winter wheat cultivars, specified years.


Monthly Crop. Wheat cultivars, percent of acreage devoted to each cultivar. Wheat quality, test weight, moisture, and protein content of current harvest. $10.00

Crop-Weather. Issued each Monday, March 1 through November 30 and monthly, December through February. Provides crop and weather information for previous week. $12.00

County Estimates. County data on wheat acreage seeded and harvested, yield, and production on summer fallow, irrigated, and continuous cropped land. December.

Wheat Quality. County data on protein, test weight, moisture, grade, and dockage. Includes milling and baking tests, by cultivar, from a probability sample of Kansas wheat. September.

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Throckmorton and King Halls, Manhattan, KS 66506-5501, USA.

Wheat transformation using the hva1 gene for drought and salt tolerance.

C. Detvisitsakun 1, G.H. Liang 1, and S. Muthukrishnan 2.
Department of Agronomy 1 and Department of Biochemistry 2.

Environmental stresses, such as water deficit, increased salinity of soil, and extreme temperatures are major factors limiting plant growth and productivity. For this reason, we are attempting to introduce the hva1 gene, a late embryogenesis-abundant (LEA) protein gene from barley that confers drought and salt tolerance, by wheat transformation.

Two wheat cultivars, Jagger (HRWW) and KS115 (HWWW) were used in this study. The transformation process was accomplished using microprojectile-bombardment. The genes were delivered to wheat embryogenic calli using a Bio-Rad Biolistic® PDS-1000/HE. Cobombardment using two gene constructs was applied in the experiment. One bombardment provides the hva1 gene, which is regulated by the rice actin promotor, and the other provides the bar gene, which is regulated by a ubiquitin promotor. The bar gene confers resistance to L-phosphinothricin (PPT) and bialaphos (a derivative of PPT), which serves as a selectable marker. The bombarded calli were cultured on a modified MS media containing 5 mg/l bialaphos for more than 1 month. So far, 19 Jagger and 11 KS115 plants have been obtained from the transformation and selection processes. All regenerated lines are being tested by molecular analytical techniques including PCR and western and Southern blotting. Bioassays will be made on the T1 generation.



Department of Agronomy, Waters Hall, Kansas State University, Manhattan, KS 66506-5501, USA.

Phytoremediation of closed animal-waste lagoons.

Liansheng Zhu and M.B. Kirkham.

Animal waste storage lagoons are used on farms and at concentrated animal operations. However, due to environmental regulations in some states and falling meat prices (especially pork) nationwide, many lagoons in the U.S. are being closed. The soils at the bottom of lagoons are laden with ammonium nitrogen and salts and need to be remediated. The overall objective of our work is to determine if phytoremediation can be used to restore contaminated soil at abandoned animal waste lagoons after closure. Phytoremediation is the use of green plants to remediate soils and is advantageous, because it is cheap (the soil does not need to be carted away) and prevents erosion, and the plants can be harvested and used. The specific objective of the research reported here was to determine if wheat could be grown in animal waste-lagoon soil. We studied two HRWWs: Turkey, an old cultivar, and 2137, a modern cultivar developed by Rollin Sears at Kansas State University. Vernon Schaffer, in charge of Foundation Seed Production at Kansas State, supplied the seeds.

Soil at the bottom of the animal-waste lagoon serving the Department of Animal Sciences and Industry at Kansas State University in Manhattan, KS, was sampled at three different locations on 21 October, 1999, when the lagoon was being cleaned out in accordance with environmental regulations. The soil was spread out on a greenhouse bench and allowed to air-dry for 1 month. The soil was extremely hard upon drying and felt like rocks. The soil was ground and sieved through a 2-mm sieve. The two wheats, 10 seeds/pot, were planted on 20 December, 1999. There were three pots/soil for a total of 18 pots (three soils x two cultivars x three replications). Germination began on 28 December, 1999. Germination and height were measured until 56 days after planting. No differences in germination occurred among the treatments. On average, nine seeds/pot germinated. In soil from two of the locations in the lagoon, the height of the two wheats did not vary, but in one of the soils where the height was lowest, 2137 grew taller than Turkey. The plants are now being analyzed for elemental composition. The results to date showed that both wheats germinated and grew in the polluted soil, but 2137 grew taller (Fig. 1). The work is being carried out by graduate student Liansheng Zhu, whose Graduate Research Assistantship is funded by KCARE, the Kansas Center for Ag Resources and the Environment. We acknowledge with thanks this financial aid. Dr. Jay M. Ham and Dr. Loyd R. Stone also are part of the project.



Liangsheng Zhu from China, a MS degree student and Stanley Liphadzi from South Africa, a Ph.D. student and Fulbright Fellow, have joined the group. Both are studying phytoremediation of animal-waste lagoon soil.


Department of Plant Pathology, Throckmorton Hall, Kansas State University, Manhattan, KS 66506-5502, USA.

W.J. Raupp, B.S. Gill, B. Friebe, J.H. Hatchett (USDA-ARS), R.G. Sears, D.L. Wilson, J.D. Faris, M. Dhar, P. Zhang, L.L. Qi, K.M. Haen, and R.G. Kynast (University of Minnesota, St. Paul).


Transfer of wheat-rye translocation chromosomes conferring resistance to Hessian fly from bread wheat into durum wheat.

B. Friebe, R.G. Kynast, J.H. Hatchett, R.G. Sears, D.L. Wilson, and B.S. Gill.

Breeding for host-plant resistance is the most agronomically desirable way to control the Hessian fly in wheat. Twenty-seven major genes conferring resistance to Hessian fly have been identified and used in wheat improvement. These genes confer resistance to specific biotypes of the Hessian fly. Recently, new sources of Hessian fly resistance derived from cultivated rye were reported that confer resistance to all known biotypes of the Hessian fly. The resistance gene H21 is present on the wheat-rye whole-arm translocation T2BS·2R#2L. H25 is present on an interstitial rye segment in the 4AL arm of the wheat-rye translocation chromosome Ti4AS·4AL-6R#1L-4AL. The objective of the present study was to transfer H21 and H25 to tetraploid durum wheat, thereby making these genes available for the improvement of durum wheat. Homozygous T2BS·2R#2L and Ti4AS·4AL-6R#1L-4AL translocation durum lines were recovered that expressed the H21 and H25 resistance. The H25 durum translocation lines was vigorous and set seed similar to the durum wheat parent cultivar. Thus, the H25 transfer can be used directly for the improvement of durum wheat. Plant vigor and seed set of the H21 durum translocation line were reduced drastically, indicating that the missing 2BL arm in this translocation has genes that are essential for normal plant vigor and fertility. Further chromosome engineering is required to shorten the rye segment in this translocation before H21 can be used in durum breeding.


Cytogenetic and molecular mapping of the leaf rust resistance gene Lr39 in wheat.

W.J. Raupp, S. Singh, G.L. Brown-Guedira, and B.S. Gill.

Leaf rust is one of the most serious diseases of wheat worldwide. Growing resistant cultivars is an efficient and economical method of reducing losses to leaf rust. A new leaf rust resistance gene, Lr39, was transferred from Ae. tauschii into common wheat and conditions both seedling and adult plant resistance to the pathogen. The inter- and intra-chromosomal mapping of the Lr39 gene showed that it is different from all described Lr genes. We used monosomic analysis for the interchromosomal mapping and wheat microsatellite markers for the intrachromosomal mapping. The monosomic and ditelosomic analysis indicated that Lr39 is independent of the centromere on the short arm of chromosome 2D. Eight microsatellite markers for 2DS were used for linkage analysis on a population of 57 F2 plants derived from a cross of an Ae. tauschii-derived, wheat cultivar Wichita line (TA4186, with Lr39) with the Wichita monosomics for the D-genome chromosomes. Microsatellite marker analysis confirmed the location of the gene on 2DS. Three markers were polymorphic and linked to the gene. The closest marker Xgwm210 mapped 10.7 cM from Lr39. The location of Lr39 near the telomere of 2DS distinguishes it from the Lr2 and Lr22 loci, which are located on 2DS proximal to Xgwm210.


The targeting of markers to a specific genomic region of wheat using chromosome deletion lines.

J.D. Faris and B.S. Gill.

The chromosome deletion lines of wheat are valuable genetic stocks that have been used extensively for the construction of physical maps of the wheat genome. We used mRNA differential display and AFLP analysis on deletion lines 5AL-7 and 5AL-23 to target markers to the submicroscopic segment of chromosome 5A that differs between the two lines. The segment accounts for less than 1 % of the chromosome arm and is estimated to contain about 5 Mb of DNA. We used 90 and 64 primer combinations for differential display and AFLP analysis, respectively. Both techniques yielded approximately 80 bands per primer combination. Sixteen differential-display bands and 10 AFLP bands were present in 5AL-23 but absent in 5AL-7. These 26 bands were excised from the polyacrylamide gels, cloned, and physically mapped on the deletion lines. Only three of the differential-display fragments detected loci within the targeted interval, but 11 of the 16 bands resulted in 48 loci on group-5 chromosomes, suggesting the presence of a regulatory gene within the targeted interval. Three of the 10 AFLP bands were highly repetitive and could not be used as RFLP probes, but the remaining seven bands resulted in single or low-copy clones that detected loci within the targeted interval on chromosome 5A. These results show that specific regions of the wheat genome can be saturated with molecular markers using the chromosome deletion lines.


Chromosome healing by telomere addition is a gradual process.

B. Friebe, R.G. Kynast, M. Dhar, P. Zhang, and B.S. Gill.

The gametocidal factor on chromosome 4Mg of Ae. geniculata was used to induce the formation of dicentrics involving the satellite chromosomes 1B and 6B of common wheat. C-banding analysis identified the dicentric chromosomes as T1BS·1BL-2AL·2AS and T6BS·6BL-4BL·4BS, respectively. The dicentrics initiated breakage-fusion-bridge cycles that ceased 2 and 4 weeks after germination and resulted in deficient chromosomes 1B and 6B with break points in proximal regions of the 1BL and 6BL arms. The process of chromosome healing was analyzed in root-tip meristems, at meiotic metaphase I, and in root-tip meristems of the derived progenies by fluorescence in situ hybridization analysis using the telomeric probe pAtT4 from Arabidopsis thaliana. The results showed that chromosome healing by de novo addition of telomeric repeats to the break points in wheat is a gradual process and that broken chromosome ends have to pass through several cell divisions in the sporophyte to acquire the full telomeric repeat length.


High-density physical map of the group-5 short arm chromosomes of bread wheat.

L.L. Qi and B.S. Gill.

A high-density physical map of the short arm of the group-5 chromosomes was constructed by mapping 41 DNA markers on a set of 17 homozygous deletion lines. Ninety-six RFLP loci were mapped: 35 on 5AS, 34 on 5BS, and 27 on 5DS. The RFLP loci were distributed nonrandomly in the three chromosome arms. In the 5AS arm, 74 % of markers mapped between deletion break points 0.75-0.97. The proximal 43 % of the 5BS arm had poor marker coverage. The deletion-interval between FL 0.63 and FL 0.67 in 5DS represents only 4 % of the chromosome arm and contains 10 of the 25 (40 %) probes mapped in the arm; a 10-fold enrichment in marker density. A consensus, physical map was aligned colinearly with a consensus genetic map of the group-5 short arms. Sixteen of the 17 markers in the consensus genetic map encompass a genetic distance of 25 cM and correspond to the distal region (0.56-0.97) of the consensus physical map. Two rice probes, Xrg463 and Xrg901, previously linked to the marker Xcdo344 (group-5S of wheat) in chromosome 12 genetic map of rice, map between FL 0.56 and 0.63 in the consensus map, confirming that at least a part of the short arm of the group-5 chromosomes is homoeologous to a region of chromosome 12 of rice.


Saturation mapping of a gene-rich recombination hot-spot region in wheat.

J.D. Faris, K.M. Haen, and B.S. Gill.

Physical mapping of wheat chromosomes has revealed small chromosome segments of high gene density and frequent recombination interspersed with relatively large regions of low gene density and infrequent recombination. We constructed a detailed genetic and physical map of one highly recombinant region on the long arm of chromosome 5B. This distally located region accounts for 4 % of the physical size of the long arm and at least 30 % of the recombination along the entire chromosome. Multiple crossovers occurred within this region, and the degree of recombination is at least 11-fold greater than the genomic average. Characteristics of the region, such as gene order and frequency of recombination, appear to be conserved throughout the evolution of the Triticeae. The region is more prone to chromosome breakage by gametocidal gene action that gene-poor regions, and evidence for genomic instability was implied by loss of gene collinearity for six loci among the homoeologous regions. These data suggest that a unique level of chromatin organization exists within gene-rich recombination hot spots. The many agronomically important genes in this region should be accessible by positional cloning.


Wheat-alien translocation lines.

B. Friebe, W.J. Raupp, and B.S. Gill.

Wild relatives of common wheat are important sources for disease and pest resistance. Recently, we reviewed the status of wheat-alien translocations conferring resistance to diseases and pests (Friebe et al. 1996. Euphytica 91:59-87). Since then, several new transfers were reported and markers linked to resistance genes identified. The tables update the available information on wheat-alien translocations.



USDA-ARS Plant Science and Entomology Research Unit

Throckmorton Hall, Manhattan, KS 66506-5502, USA.


Changes in the Plant Science and Entomology Research Unit at Manhattan.

D.Z. Skinner and M.E. Eversmeyer.

Dan Skinner was appointed acting research leader of the unit effective 5 April, 2000, replacing Merle Eversmeyer who requested to return to full-time research on wheat rust after having served as research leader for the past 28 years.

Cathy Sue Katsar was hired to replace Jimmy Hatchet as leader of the Hessian fly project at Manhattan. She assumed her duties on 1 August, 1999. She earned her Ph.D. at Texas A&M University working on the greenbug and was in an ARS postdoctoral position at Cornell University.


QTL mapping of Karnal bunt resistance genes in hexaploid wheat.

S. Singh, G.L. Brown-Guedira, T.S. Grewal*, H.S. Singh*, B.S. Gill, and H.S. Dhaliwal*.
* Punjab Agricultural University, Ludhiana, India.

Identification of markers linked to genes conferring resistance to Karnal bunt is paramount to enhancing resistance of U.S. wheats to the pathogen. One hundred fifty RILs developed from a cross of a Karnal bunt-(KB) susceptible (WL711) and a resistant line (HD29) were evaluated for resistance to three isolates of N. indica prevalent in northern India. The data on incidence of KB indicated that at least three genes for resistance were segregating in the population. Molecular analysis of the RILs was made using mapped wheat mirosatellites and eight AFLP primer combinations. One hundred ten polymorphic loci between WL711 and HD29 were scored on the RILs, and an analysis of variance indicated significant effects of the markers mapped on chromosomes 7B, 6B, 3A, and 4B for KB reaction. Additional markers are being added to the map to establish linkage with genes for resistance.


Mapping a leaf rust-resistance gene transferred to common wheat from Triticum timopheevii subsp. armeniacum.

G.L. Brown-Guedira, R. Malik, and S. Singh.

Identification, characterization, and tagging of new leaf rust resistance genes is important to develop resistance gene pyramids. Wheat germ plasm lines KS96WGRC35, KS96WGRC36, and KS98U662, having genes transferred from accessions TA 28, TA 870 and TA 874, respectively, of T. timopheevii subsp. armeniacum, each have a single dominant gene for resistance. Our data showed that TA 870 and TA 874 donated the same gene for resistance. Segregation ratios of crosses between the germ plasm lines having resistance genes from TA 870 and TA 874 with lines having resistance from TA 28 did not differ from a 15 resistant : 1 susceptible ratio, indicating that the gene derived from TA 28 is independent of the gene transferred from TA 870 and TA 874.

Leaf rust-resistant BC3 lines were compared with their recurrent wheat parent at 112 SSR loci, and populations segregating for leaf rust resistance were evaluated with polymorphic markers to identify markers linked to the resistance genes. The wheat microsatellite primer pair GWM382 amplifies orthologous loci that mapped distally on the long arm of wheat homoeologous group 2 chromosomes (Roder et al. 1998). This SSR primer pair amplified a polymorphic band that was closely linked to the resistance gene derived from TA 870 and TA 874 when tested on BC4F2 populations. No other named genes for resistance to leaf rust have been mapped to the long arm of chromosomes 2A or 2B. The T. timopheevii subsp. armeniacum-derived gene linked to Xgwm382 has been assigned the temporary designation LrArm.

Virulence to LrArm exists in races of P. triticina in North America. However, low to intermediate infections types were observed in adult plants in the field at locations in Kanasa, Texas, Lousiana, and Georgia. This gene should be useful in the Great Plains if used in gene combinations. Identification of a microsatellite marker closely linked to LrArm provides a tool to incorporate this gene into resistance gene pyramids.


Response of wild wheat relatives to boron toxicity.

A. Ryser and G.L. Brown-Guedira

Soil with a high concentration of boron has become a micronutrient problem that limits yield of wheat in the Central Anatolian Plateau of Turkey and southern and western Australia. Many experiments have looked at the tolerance of wheat cultivars to boron toxicity but few have examined the response in progenitor species. Boron toxicity leads to root growth reduction and decreases in height and shoot growth. To evaluate the response of wild wheat relatives to high levels of boron, experiments were conducted using a hydroponic system in a 20°C growth chamber with a 12-hour day length. Twenty-six accessions of Ae. tauschii, 22 accessions of T. monococcum subsp. aegilopoides, and 30 accessions of T. timopheevii subsp. armeniacum of diverse geographic origins were tested along with wheat controls Chinese Spring, Kenya Farmer, and Langdon durum. Eight seed of each accession were germinated on filter paper for 2 days and then placed in containers holding aerated Hoagland's solution with boron concentrations of either 0 µM or 10 µM. Root length was measured before placing seedlings in the containers using the Marsh line intersect method (Tennant 1975). After 10 days of growth, the roots were measured again. The percent reduction in root growth was calculated as (G0-G10)/G0, where G0 is the change in length after 10 days at 0 µM boron and G10 is change in length after 10 days at 10 µM boron. Seven accessions of Ae. tauschii were tolerant to moderately tolerant to high concentrations of boron (TA 1602, TA 1620, TA 1681, TA 2119, TA 2374, TA 2572, and TA 2581) (see Table 1). Four accession of T. timopheevii subsp. armeniacum were moderately tolerant (TA 956, TA 967, TA 1518, and TA 2894) and one accession of T. monococcum subsp. aegilopoides was moderately tolerant (TA 332). Aegilops tauschii has a higher frequency of tolerance to high concentrations of boron than either Triticum species and represents a new source of tolerance that has not been utilized by breeding programs aimed at improving tolerance to boron toxicity of wheat.


Table 1. Reduction in root growth of accessions after 10 days of growth in boron solutions. Percent reduction in root growth was calculated as (G0-G10)/G0, where G0 is the change in length after 10 days at 0 µM boron and G10 is change in length after 10 days at 10 µM boron. All TA numbers are accession numbers of the Wheat Genetics Resource Center, Kansas State University, Manhattan.
 Ae. tauschii    T. timopheevii subsp. armeniacum    T. monococcum subsp. aegilopoides
 Accession  % reduction  Accession  % reduction  Accession  % reduction
 TA 1582  99  TA 2  94  TA 175  91
 TA 1588  100  TA 11  96  TA 179  97
 TA 1590  71  TA 25  78  TA 184  94
 TA 1599  97  TA 28  88  TA 200  81
 TA 1602  45  TA 30  93  TA 204  82
 TA 1603  87  TA 34  88  TA 214  92
 TA 1620  39  TA 40  72  TA 228  95
 TA 1623  75  TA 44  73  TA 249  86
 TA 1626  79  TA 104  93  TA 252  88
 TA 1643  91  TA 145  91  TA 258  90
 TA 1674  86  TA 161  72  TA 264  90
 TA 1678  82  TA 870  100  TA 295  86
 TA 1681  50  TA 895  83  TA 306  91
 TA 1715  93  TA 896  91  TA 332  61
 TA 2119  64  TA 909  83  TA 357  90
 TA 2371  75  TA 921  85  TA 519  93
 TA 2374  36  TA 935  75  TA 522  86
 TA 2386  0  TA 940  87  TA 591  92
 TA 2391  76  TA 956  68  TA 618  77
 TA 2409  45  TA 960  77  TA 762  95
 TA 2479  73  TA 967  62  TA 2007  86
 TA 2486  90  TA 976  86  TA 2012  88
 TA 2500  99  TA 1475  89    
 TA 2536  0  TA 1485  85    
 TA 2572  50  TA 1497  84    
 TA 2586  63  TA 1518  28    
 TA 1536  76        
 TA 1540  88        
 TA 1549  67        
 TA 2894  51        
 Wheat controls
 Chinese Spring  91  Chinese Spring  85  Chinese Spring  83
 Kenya Farmer  94  Kenya Farmer  85  Kenya Farmer  71




USDA, Agricultural Research Service, Manhattan, KS 66502, USA.

O.K. Chung, F.E. Dowell, G.L. Lookhart, J.B. Ohm, S.R. Bean, J.L. Steele, L.M. Seitz, D.B. Bechtel, M.S. Ram, A. Sayaslan, D. Wang, C.R. Martin, I.Y. Zayas, J.D. Hubbard, M. Tilley, B.W. Seabourn, M.S. Caley, R. Rengarajan, J.M. Downing, S.H. Park, J.D. Wilson, J.E. Baker, J.E. Throne, P.W. Flinn, D.B. Sauer, C.S. Chang, and D.E. Koeltzow.


A comparison of polymers for SDS-CE separations of wheat proteins.

S.R. Bean and G.L. Lookhart.

Non-cross linked polyacrylamide (PAA), polydimethyl acrylamide (PDMA), dextran, polyethylene oxide (PEO), polyvinyl alcohol (PVA), and a commercial polymer from BioRad were evaluated for use in SDS-capillary electrophoresis (CE) separations of wheat proteins. Each polymer was optimized (where possible) by manipulating polymer concentration and buffer (pH, ion type) concentration and through the use of organic modifiers (methanol and ethylene glycol (EG)). The addition of 15 % EG to the separation buffer was found to generally improve the resolution of the separations, except for some polymers such as PEO. The addition of EG was necessary to resolve several pairs of HMW-GS, such as 1 and 5. Several HMW-GS showed migration patterns different from those typically seen in SDS-PAGE (e.g., 1 and 5). Once optimized, all polymers provided good separations of wheat proteins, though each had particular advantages and disadvantages. In addition, the molecular weights of the wheat proteins predicted by the various buffer systems differed from each other and the predicted cDNA mass. Separations done with a commercial polymer from BioRad modified by the addition of EG showed high resolution separations of both high molecular weight and low molecular weight glutenins. Good repeatability was found with this method for both migration time (<1 % RSD) and corrected peak area (about 1 %).


Size based separations of wheat proteins by capillary electrophoresis. I. Uncoated capillaries.

S.R. Bean and G.L. Lookhart.

Four different polymer/buffer systems: a commercial polymer from BioRad, dextran, poly(ethylene oxide), and non-crosslinked polyacrylamide were evaluated for their ability to separate wheat proteins by size. These polymers were chosen based on published reports of these polymers being used in uncoated or dynamically coated capillaries. Each polymer was optimized (where possible) by manipulating the polymer concentration, buffer concentration, and through the use of organic modifiers such as methanol and ethylene glycol. The addition of ethylene glycol to the separation buffer was found to improve the resolution of the separations, except when PEO was used as the sieving polymer. Once optimized, all polymers provided good separations of wheat proteins, though each had particular advantages and disadvantages. Each polymer also differed in the predicted molecular mass of the various wheat-protein fractions separated. Further work was done with the BioRad buffer modified by the addition of ethylene glycol. Several different wheat protein fractions as well as proteins extracted from several different cultivars were separated with this buffer and compared.


Preliminary characterization of wheat proteins by HPLC-multi-angle laser light scattering (MALLS).

G.L. Lookhart and S.R. Bean.

Multi-angle laser light scattering is capable of providing absolute molecular masses (without the need for generating calibration curves to standards), as well as molecular geometry of biomolecules. MALLS was used in conjunction with HPLC to characterize gliadin and glutenin proteins of wheat. This methodology has been applied previously to the study of wheat starch by several research groups. Here we describe preliminary studies on the use of this technique for the study of gluten proteins. Reversed-phase (RP)-HPLC-MALLS is being applied to study individual glutenin and gliadin proteins. Preliminary data gathered on five HMW-GS show that the masses determined by RP-HPLC-MALLS closely matched those of their respective cDNA masses (< 5 %). Unreduced polymeric proteins are being characterized with size exclusion chromatography (SEC)-MALLS. Development of extraction conditions (solvents, stirring/vortexing, temperature, or sonication) for analysis of unreduced proteins by SEC-MALLS is in progress. The use of HPLC-MALLS provides an easy, automated, high resolution method for further characterizing both reduced and unreduced glutenin proteins.


Ultrafast capillary electrophoretic analysis of cereal storage proteins and its applications to protein characterization and cultivar differentiation.

S.R. Bean and G.L. Lookhart.

The isoelectric-buffer compound iminodiacetic acid (IDA) was optimized for separation of cereal storage proteins. Several organic solvents, including methanol, ethanol, 1-propanol, t-butanol, acetonitrile, and ethylene glycol were tested as buffer additives at several concentrations (5-20 %). Buffer concentration (25-150 mM), separation voltage (20 to 30 kV), and separation temperature (25-45°C) also were optimized. Optimum separation conditions for wheat, oat, and barley storage proteins were 50 mM IDA containing 20 % acetonitrile and 0.05 % hydroxypropylmethyl cellulose. Separations generally were complete in 3-4 min for all classes of proteins, except for wheat gliadins, where ~ 8 min were required for w-gliadin separation. Cultivars of all cereals could be differentiated in 3 min, including wheat using either prolamin or glutelin protein patterns. Resolution was similar to, or higher, than that of similar separations in other acidic buffers. Migration time repeatability was excellent with run-to-run variability < 1 % RSD, day-to-day < 1.4 % RSD, and capillary-to-capillary < 3.5 % RSD. Because larger inner-diameter capillaries (50 µm) could be used with this buffer, sensitivity was improved and capillary rinse times could be reduced in comparison to smaller capillaries (25 µm i.d.). Total separation time also was reduced so that the majority of cereal storage protein from several types of cereals could be analyzed with total analysis times of 3-5 min with extremely high resolution and repeatability. This method would allow unattended, high throughput (~ 180-400 samples/24 hours) analysis of cereal proteins without generating much organic solvent waste and also automated data analysis and storage.


Introduction and overexpression of HMW-glutenin gene 1Ax1 in field grown wheats: protein characterization and baking quality data.

J. Zhao, S. Bean, P. McCluskey, G. Lookhart, H.-P.Zhou1, W. Goure1, F. Altpeter, V. Vasil, and I.K. Vasil.

Bobwhite wheat contains six HMW-glutenin genes (1Ay, Ax2, Dx5, Bx7, By9, and Dy10), but only five are expressed, as 1Ay is always silent. We have introduced a seventh HMW-glutenin gene, 1Ax1, into Bobwhite and demonstrated its expression and accumulation in the endosperm. The transgenic lines expressing 1Ax1 were analyzed for stability of gene expression and their effect on food functionality. The expression of the sixth HMW-glutenin (1Ax1) in the R4-generation plants under field growing conditions did not disrupt the expression of the five native HMW-glutenin and was similar to that found in R2-generation plants from a growth chamber, indicating that the high level expression of 1Ax1 under its own promoter is stable. Agronomic yields of the transgenic wheats and the control were similar, as were flour yields. Two transgenic lines showed slightly higher protein levels than the control cultivar. Analysis of the gross protein composition showed that two lines had slightly increased levels of 50 % propanol insoluble polymeric proteins (IPP) and decreased levels of gliadins. Capillary electrophoretic (CE) analyses demonstrated that introduction of the 1Ax1 gene did not alter gliadin or LMW-glutenin proteins. However, SDS-PAGE, CE, and HPLC all revealed additional proteins present in several of the transgenic lines. Line ax31 exhibited substantially different HMW-glutenin patterns when analyzed by CE and HPLC. Quantification of the expression levels of the 1Ax1 transgene by CE showed the same levels as those reported with greenhouse-grown samples. The transgenic lines showed similar loaf volumes, crumb grain, and bake absorption as the control. Six of the lines showed increased mixing times compared to the control. Some 1Ax1 transgenic lines had increased levels of insoluble polymeric protein (IPP), indicating that the overexpressed 1Ax1 was incorporated into the large polymeric protein network. Correlations were found between IPP and dough properties; these results are consistent with the hypothesis that increasing HMW-glutenin level will increase dough strength and bake mix times of wheat flour.


Quality effects of rye chromosome arm 1RL transferred to wheat.

R.A. Graybosch, C.J. Peterson, and O.K. Chung.

Quality effects of rye chromosome arm 1RL transferred to wheat were characterized by comparison of a group DS1R(1B) substitution lines and T1BL·1RS translocation lines. The experimental materials were sister lines derived from the cross 'Mironovskaya 10/NE7060//NE80413'. DS 1R(1B) lines were identified by the presence of rye w- and g-secalins among 70 % ethanol-soluble proteins, combined with the presence of HMW-secalin proteins in total grain protein extracts. Genes on 1RL reduced grain weight, grain hardness, Mixograph time, Mixograph tolerance, and SDS sedimentation volumes. Chromosome arm 1RL had no effect on flour yield or grain and flour protein concentrations. The HMW-secalin proteins encoded by genes on 1RL most likely caused the decline in dough strength seen in DS 1R(1B) lines relative to that of T1BL·1RS sister lines. Reduced grain hardness also might be related to the presence of HMW secalins, although a role for additional, unidentified genes on 1RL could not be discounted.


Genetic, agronomic, and quality comparisons of two T1AL·1RS wheat-rye chromosomal translocations.

R.A. Graybosch, J.-H. Lee, C.J. Peterson, D.R. Porter, and O.K. Chung.

The T1AL·1RS wheat-rye chromosomal translocation originally found in Amigo wheat possesses resistance genes for stem rust, powdery mildew, and greenbug biotypes B and C, but also has a negative effect on wheat processing quality. Recently, a second T1AL·1RS translocation carrying Gb6, a gene conferring resistance to greenbug biotypes B, C, E, G, and I, was identified in the wheat germ plasm line GRS1201. Protein analytical methods and PCR were used to identify markers capable of differentiating the 1RS chromosome arms derived from Amigo and GRS1201. The secalin proteins encoded by genes on 1RS differed in Amigo and GRS1201. A secalin of Mr 70 kDa was found in the Amigo T1AL·1RS but did not occur in the GRS1201 T1AL·1RS. Polymorphisms detected by PCR primers derived from a family of moderately repetitive, rye DNA sequences also differentiated the two translocations. When GRS1201 was mated with a non-1RS wheat, no recombinants between 1RS markers were observed. In crosses between 1RS and non-1RS parents, both DNA markers and secalins would be useful as selectable markers for 1RS-derived greenbug resistance. Recombination between 1RS markers did occur when 1RS from Amigo and 1RS from GRS1201 were combined, but in such intermatings, the molecular markers described herein still could be used to develop a population enriched in lines carrying Gb6. No differences in grain yield or grain- and flour-quality characteristics were observed when lines with 1RS from Amigo were compared to lines with 1RS from GRS1201. Hence, differences in secalin composition did not result in differential quality effects. When compared to sister lines with T1AL·1RS derived from the wheat cultivar Redland, lines with the GRS1201 had equal grain yield, but produced flours with significantly shorter mix times, weaker doughs, and lower SDS-sedimentation volumes.


Characterization of gluten proteins from Aegilops tauschii lines that conferred improved quality to bread wheat crosses.

M. Tilley, S.R. Bean, P.A. Seib, R.G. Sears, and G.L. Lookhart.

Aegilops tauschii has been used in crosses with established cultivars to develop plants bearing disease and insect resistance and new combinations of gluten proteins. Crosses of the cultivar Century with the Ae. tauschii accessions TA2450 and TA2460 exhibited shorter mixing times and improved milling and baking characteristics when compared to the parental hexaploid line. The gluten proteins from the Ae. tauschii lines were examined using HPLC and capillary electrophoresis (CE). Separation of gliadins and glutenins revealed similar profiles to those from HRWW. CE showed that the HMW-glutenin subunits of Ae. tauschii had similar properties to those found in HRWW with the identification of two novel HMW-glutenin subunits, 43 and 44. The genes encoding the novel HMW-glutenin subunits were cloned and DNA sequencing is currently underway to further characterize the novel subunits. Analysis of soluble and insoluble polymeric proteins of the Ae. tauschii lines revealed patterns similar to that of HRWW, indicating that the Ae. tauschii gluten forms a large protein complex as observed in HRWW.


Prediction of gliadin and soluble/insoluble HMW-glutenin fractions in whole kernel wheat by near-infrared reflectance spectroscopy.

B.W. Seabourn, S.R. Bean, G.L. Lookhart, and O.K. Chung.

We confirmed in a recent study that gliadin and polymeric proteins (insoluble glutenins) could be predicted with sufficient accuracy from the near-infrared reflectance (NIR) spectra of wheat flour. In this study, we looked at the potential for predicting gliadin (G), soluble glutenin (SG), and insoluble glutenin (IG) contents from the NIR spectra of whole-kernel wheat. One-hundred hard winter wheats were obtained from the USDA-ARS Hard Winter Wheat Quality Laboratory (HWWQL), Manhattan, KS. The wheats had been grown at two federal regional breeding nurseries during the 1993-95 crop years. The wheats were selected using the HWWQL Relational Database based upon their aggregate milling and baking scores. The flours from these wheats were analyzed by HPLC for their G and SG contents, and by Leco Nitrogen Analyzer for their IG content. Using NIR spectra of the whole kernel wheats, we found that G and IG fractions could be predicted with an accuracy acceptable for screening purposes (r2 > 0.60). For IG, the standard error of cross-validation for the model was (r2 = 0.79), which was slightly lower than what was previously found in our study with flour r2 = 0.83. In both wheat and flour, we were unable to predict SG content. However, G content could be predicted in both wheat (r2 = 0.76) and flour (r2 = 0.79). Because IG has been confirmed in a number of studies to play an important role in bread making, particularly dough strength, these results indicate that NIR may be very useful in plant breeding programs and quality laboratories where rapid screening for dough strength of large numbers of wheat lines is needed.


Variability in a starch-isolation method and automated, digital image-analysis system used for the study of starch-size distributions in wheat flour.

D.B. Bechtel and J.D. Wilson.

A starch-isolation method and digital image-analysis system were developed to accurately measure size distributions of the starch populations in wheat. The image analysis system was coupled directly to a light microscope equipped with a computer-controlled step stage and automatic focus. Automation of data acquisition and processing eliminated some of the labor intensive steps previously required for analyzing starch size distributions. This system was used to standardize starch isolation methods and compare variation and reproducibility of the system. Operational variations were determined and assessed statistically. The number of fields of view required for low standard errors and acceptable speed of analysis was determined to be 50. A major advantage of the system has been the increased resolution. The use of higher magnifications and stage automation allowed the analysis of starch granules as small as 0.84 µm in diameter and thousands of starch granules per sample.


Can digital image analysis be developed into a reference method for determining starch-size distributions ?

D.B. Bechtel, C.R. Martin, and J.D. Wilson.

Light microscopy has long been the method of choice for measuring microscopic particles. However, the process is slow and labor-intensive, and limited numbers of particles can be analyzed. Automated image-analysis systems have been developed that address some of the concerns associated with these earlier methods. Other important issues have yet to be addressed, in particular, how particles that touch the edge of field of view are handled. Previously, image-analysis systems have either counted and measured the partial views of these particles or eliminated them from the analysis. Problems in analyzing starch granules are complex because of the wide range of sizes, from less than 1 µ to more than 30 µ in diameter. The larger the particle or higher the magnification used, the more likely it is that a particle will be touching the edge of the field of view. We found that the magnitude in which large type-A starch granules can touch the edge of field of view can approach 50 %. Although type A granules generally occur at less than a 7 % frequency, their large size contributes most of the total starch mass. Even small errors associated with counting the number of type-A granules, therefore, can greatly influence the total mass attributed to them. Using log normal distributions of the data, we have developed mathematical approaches to correct the errors associated with particles touching the edge. Image analysis can be used as a reference method for starch-size distribution determinations.


Registration of Harding wheat.

S.D. Haley, J.L. Gellner, M.A.C. Langham, Y. Jin, S. Kalsbeck, C. Stymiest, J. Rickertsen, R. Little, B.E. Ruden, O.K. Chung, B.W. Seabourn, D.V. McVey, and J.H. Hatchett.

Harding HRWW was developed by the South Dakota Agricultural Experiment Station and released to seed producers in the autumn of 1999. Harding is an awned, red-glumed, medium-late maturity, standard-height HRWW with excellent winter survival ability, a very broad disease resistance package, and superior yield performance compared to available cultivars in its maturity range. Harding was named after a county in northwest SD where winter survival ability is especially important for successful winter wheat production. Harding was selected as an F5:6 line from the cross 'Brule//Bennett/Chisholm/3/Arapahoe' made in 1986 by Dr. Jeffrey L. Gellner. Harding was identified as experimental line SD92107 in 1992 and was tested in the SD Crop Performance Testing (CPT) Variety Trial since 1996 and the Northern Regional Performance Nursery from 1996-98. In 4 years of statewide testing in the SD CPT (1996-99; 39 environments), Harding (3,903 kg/ha) was lower yielding than 2137, Alliance, and Arapahoe; similar in yield to Nekota; but higher yielding than cultivars with similar maturity and winter survival ability, including Crimson, Seward, Elkhorn, and Roughrider. Over this same testing period, at locations where winter injury was an important consideration for yield performance (13 locations), Harding (4,267 kg/ha) was the highest yielding entry, greater than Arapahoe, 2137, Nekota, Crimson, and Alliance. Composite milling and baking data (provided by the USDA-ARS Hard Winter Wheat Quality Laboratory, Manhattan, KS) from the SD Advanced Yield Trial (1995-98) identified Harding as a wheat with overall below-average milling characteristics and overall above-average baking characteristics.


Prediction of conventional wheat characteristics of hard winter wheats using single-kernel parameters.

O.K. Chung, J.B. Ohm, and B.W. Seabourn.

The single-kernel characteristics of 2,890 hard winter wheats that were collected from federal nurseries from 1990-97 were obtained from the Single Kernel Characterization System (SKCS). Flour yield showed a mean value of 68.8 % with a standard deviation of 2.9. Test weight (TW), % large kernels (% LK), 1,000-kernel weight (TKW), and near infrared hardness score (NIR-HS) showed means values of 60 lbs/bu, 28.6 g, 67 %, and 66 with standard deviations of 2.3, 4.7, 20.0, and 12, respectively. Single-kernel weight obtained from SKCS showed significant correlation coefficients (r) with TKW (r = 0.923), % lLK (r = 0.888), TW (r = 0.521), and flour yield (r = 0.384). SKCS hardness index had a significant correlation with NIR-HS (r = 0.557). To develop continuum regression prediction models of TW, % LK, TKW, and flour yield, eight SKCS characteristics and 12 machine parameters were used. Among 2,890 wheats, 1,200 and 300 wheats were selected as calibration and validation sets, respectively. The prediction model for flour yield showed R2 of 0.643 and root mean square error (RMSE) of 1.6 for the calibration set and R2 of 0.635 and RMSE of 1.6 for the validation set. Prediction models of TKW, % LK, NIR-HS, and TW showed R2 values of 0.898, 0.888, 0.625, and 0.472 for the calibration set and 0.893, 0.888, 0.609, and 0.473 for the validation set, respectively.


Effects of kernel-weight and hardness on end-use properties of hard winter wheats.

J.B. Ohm and O.K. Chung.

Hard winter wheat varieties showing contrast differences in single-kernel weight and near infrared hardness score (NIR-HS) were collected and blended to make '3 x 3' composites that had high (H), low (L), and medium (mixture of H and L in the same ratio) kernel weight and/or hardness, respectively. NIR-HS of composites were in the range of 74-87 for (H) and 29-48 for (L). Single-kernel weights of composites were in the range of 22-26 mg for (L) and 31-34 mg for (H). The composite of high NIR-HS and high kernel weight showed the highest flour yield among the 9 wheat composites. Blending of wheat of high hardness with low hardness significantly decreased flour yield: correlation coefficient (r) was significant between NIR-HS and flour yield (r = 0.91, P < 0.01). The largest loaf volume was obtained from flour milled from a wheat composite of high NIR-HS and low kernel weight probably because of its high protein content. The negative correlation between single-kernel weight and loaf volume (r = -0.92, P < 0.01) indicated that the blending of wheat of low kernel weight with high kernel weight could result in a decrease in loaf volume. These results showed that wheat kernel hardness and weight could be used to segregate hard winter wheats with good end-use properties for the wheat industry.


Experimental bake test for wheat breeding program.

M.S. Caley, O.K. Chung, and J.B. Ohm.

Wheat Quality Council flour samples from 1996-98 that included 73 winter and 81 spring wheats were baked by a pup straight-dough (S-D) method and a pound sponge and dough (Sp&D) method. The mean values of water absorption (WA) were 66.9 % for pup S-D and 59.9 % for pound Sp&D. The mix time (MT) of pup S-D and pound Sp&D were 5.5 and 5.3 min, respectively. Mean loaf volumes (LV) were 966 cc for pup S-D and 2,406 cc for pound Sp&D. The mean LV /100 g flour was 797 cc for pound Sp&D. The mean crumb grain scores (CGS) were 3.9 for both baking methods. The differences between mean values of pup S-D and pound Sp&P were significant in WA and LV per 100 g flour but not significant in MT and CGS (P < 5 %). Pup S-D showed a higher coefficient of variation (CV) in MT (36 %) than that of pound Sp&D (25 %). Pound Sp&D CV in WA (10 %) was higher than that of pup S-D (4 %). Significant (P < 0.1 %) correlation coefficients (r) occurred between the two baking methods for WA (r = 0.53), MT (r = 0.53), LV (r = 0.54), and CGS (r = 0.59). Flour protein contents were correlated significantly with WA (r = 0.57, P < 0.1 %), MT (r = 0.26, P < 1 %), and LV (r = 0.82, P < 0.1 %) in a pup S-D. Lower r values occurred between protein content and WA (r = 0.32, P < 0.1 %), MT (r = 0.13, P < 16 %), and LV (r = 0.54, P < 0.1 %) in a pound Sp&D. These results suggested that pup S-D would be better than pound Sp&D in breeding programs, not only because of the smaller quantity of flour required but also more sensitive responses to protein content.


Bread crumb grain study: wheat and flour characteristics.

S.H. Park, O.K. Chung, P.A. Seib, and M.S. Caley.

To investigate the relationships between crumb grain and wheat and flour characteristics, we used 61 hard winter wheats and flours with crumb grain scores varying from 1 (poorest) to 5 (best), loaf volumes from 795 to 1055 cc, and flour protein (FP) contents from 9.8 to 13.5 %. Among baking parameters, loaf volume and water absorption were not correlated significantly with crumb grain scores, whereas mixing time (r = -0.336, P < 0.01) showed a significant correlation. Crumb grain scores were correlated significantly with flour particle size (PS) distribution; they were correlated positively with the small particle-size fraction (<38 mm, 9.6-19.3 wt %) (r = 0.611, P < 0.001) and negatively with the large particle-size fraction (>75 mm, 37.5-64.0 wt %) (r = -0.450, P < 0.001). In addition, crumb grain scores showed significant correlations with rheological properties determined by a computerized mixograph; they were correlated positively with ascending slope (r = 0.403, P < 0.01) and negatively with peak area (r = -0.413, P < 0.001). A prediction equation developed without using baking data selected four parameters including flour protein (FP), wheat protein (WP), and flour particle size (PS) including < 38 mm and 38-53 mm. The equation is as follows: crumb grain score = -11.9 + 0.28FP + 0.07WP + 0.16PS (< 38 mm) - 0.20PS (38-53 mm) (R2 = 0.557).


Estimation of free lipids in wheat flour by near-infrared spectroscopy.

J.B. Ohm and O.K. Chung.

The absorbance (log 1/transmission) values of wheat flour free lipid (FL) in hexane were measured using an NIR Systems 6500 scanning spectrophotometer with a 2-mm cuvette. Results were used to develop calibration models for estimating flour FL contents. The best model for the estimation of FL content was obtained using modified partial least square (MPLS) and showed coefficients of determination (R2) of 0.95 for the calibration set and 0.89 for the validation set. Glycolipid contents could be estimated by a model developed by stepwise multiple regression, which had R2 values of 0.87 for the calibration set and 0.89 for the validation set. For digalactosyldiglycerides, the model obtained by MPLS had R2 values of 0.94 and 0.88 for the calibration and validation sets, respectively.


Cereal free lipid extraction: soxhlet versus supercritical fluid extraction (SFE) system.

J.D. Hubbard, O.K. Chung, J.M. Downing, and R.H. Lane.

The conventional extraction methods of free lipids (FL) or crude fats from cereals are using a soxhlet with nonpolar solvents. With a soxhlet using petroleum ether as a reference method, we developed the FL-extraction methods by an SFE system for seven cereal flours of rice, barley, rye, three wheats (hard red spring and winter and durum), oats, and corn meal. The optimum conditions varied somewhat depending on type of cereal, though all extraction pressures were 7,500 psi with a flow rate of 3 ml/min. The optimum SFE conditions were 60 ml CO^2^ without any modifier and a restrictor temperature (RT) of 80°C for rice; 60 ml CO^2^ with 12.2 % (vol) ethanol as modifier and RT of 80°C for barley, rye, and the three wheats; 120 ml CO^2^ with 15.0 % (vol) ethanol and RT of 120°C for oats; and 80 ml CO^2^ with 12.2 % (vol) ethanol and RT of 100°C for corn meal.


Nonstarch total-lipid extraction from lyophilized dough by a supercritical fluid extraction system.

J.D. Hubbard, O.K. Chung, and J.M. Downing.

In a previous study, we determined optimum conditions for nonstarch total lipid (NSTL) extraction from wheat flour by supercritical fluid extraction (SFE) system. We used water-saturated butanol (WSB) extraction as a reference. Conditions included 10,000 psi at 130°C for the extraction chamber pressure and temperature, respectively, and 60 ml extractant of CO^2^ and ethanol mixture (1:1 by volume) at 3 ml/min. Lipid binding during mixing is well known by cereal chemists; therefore, we studied optimum extraction conditions of NSTL from dough. For extraction of NSTL from lyophilized flour/water doughs mixed to optimum, the optimum SFE condition included 10,000 psi and 140 degrees C for the extraction chamber pressure and temperature, respectively, and 80 ml extractant of a mixture of CO^2^ and ethanol (1:1 by volume) at 3 ml/min. The NSTLs from flour and dough have been fractionated into nonpolar, glyco-, and phospholipids by a solid-phase extraction system. The amounts and compositions of NSTLs from flours and doughs were comparable.


Rapid extraction of genistein from soybean flours using enhanced-solvent extraction.

J.M. Downing, O.K. Chung, P.A. Seib, and J.D. Hubbard.

Genistein, the prominent isoflavone found in soybeans, may have several health benefits. Much interest in the analysis of isoflavones has been generated by the possibility of using these compounds in cancer therapy and prevention. In this investigation, genistein and its glucoside conjugates were extracted from ground soybeans in 20 min by an Enhanced Solvent Extraction (ESE) System using 80 % aqueous methanol. Possible applications of this methodology are an analytical method for research and development and isoflavone production. The ESE method pressurized the solvent to 2,000 psi for a 1-min static extraction at 80°C followed by a 10 ml dynamic extraction with a 5 ml/min flowrate. The ESE method was compared to a conventional extraction method that utilizes stirring in 80 % aqueous methanol for 1 hr. Genistein and its glucoside moieties were quantified by HPLC using a '4.6 x 150 mm' C8 column with a 1.0 % triflouroacetic acid/acetonitrile linear gradient. Results of the ESE of three soybean samples were comparable to those of the stirring method. Samples 1, 2, and 3 contained 1.7 mg, 1.4 mg, and 2.4 mg genistein/g sample by stirring, respectively, and had a corresponding genistein concentration of 1.9 mg, 1.3 mg, and 2.2 mg genistein/g sample extracted by ESE. An ESE method using 80 % aqueous ethanol as the extractant is being investigated.


Sensor technology for measuring grain volatiles and odors.

L.M. Seitz.

Odor is an important factor in grain grading, and an objective and safe method for classifying odors in grain is needed. To gain information relating to this problem, we used purge and trap (dynamic headspace) technology coupled with gas chromatography-mass spectroscopy to determine volatiles in several large sets of samples with known odors. Results from these tests have shown that specific compounds are associated with off-odors and that certain volatiles can indicate problems with insects, molds, heating, and other adverse conditions in grains. Sensors for rapid and convenient detection of volatiles probably will be required in instrumentation to be used routinely by grain inspectors or to provide capability for 'on-line' testing. Several types of sensors and sensor arrays recently have become available for detection of volatiles. Commercial 'electronic-nose' instruments are available that utilize sensors arrays and pattern recognition software. Our data on compound-odor relationships and appropriate groups of samples with known odors contribute to training these instruments to recognize normal and abnormal odors. Application of electronic-nose instruments for odor classification with grain samples is in an early stage of testing.


Comparison of volatile compounds in fresh, white bread crust and popcorn.

R. Rengarajan, L.M. Seitz, and M.S. Ram.

Volatiles from microwave popped popcorn and bread crust separated from freshly baked, white wheat bread were analyzed using two different methods. Ethylbenzene-d10, an internal standard, was added to each sample before the volatiles were purged with helium at 80°C,and collected on Tenax in a purge and trap instrument. Collected volatiles were desorbed thermally from the Tenax and transferred to a gas chromatograph-mass spectrometer for separation and identification. A supercritical fluid carbon dioxide extractor was used in the second method to collect the volatiles in a glass sparger kept at dry ice temperature. Internal standard, napthalene-d8, was added to the sample before the extraction. The sparger was connected to the purge and trap equipment and purged as mentioned above. Several compounds were observed in common between the two samples. Some pyrazines and other heterocyclics were found in popcorn but not in bread crust. However, 2-acetyl-1-pyrroline, a flavor impact compound that other researchers have reported for both popcorn and bread crust, was observed only in popcorn, possibly because of the low concentration and small sample size. Supercritical fluid carbon dioxide efficiently extracted carbonyls, pyrazines and some other compounds from 1-g samples of bread crust and popcorn.


Use of an autosampler for dynamic-headspace extraction of volatile compounds from grains and effect of added water on the extraction.

M.S. Ram, L.M. Seitz, and R. Rengarajan.

To aid routine analyses of grain samples for volatile compounds associated with off-odors, we used an autosampler attached to a purge and trap instrument. Trapped volatiles were transferred to a gas chromatograph- mass spectrometer instrument for separation and detection. Dynamic extraction of volatiles from about 18 g of whole grain at 80°C was accomplished by purging helium through a sample vial with a Teflon-lined septum on each end. The autosampler automatically added internal standard to the sample before purging began, which required the addition of 1 ml of water for complete transfer of the standard to the sample. The added water enhanced extraction of 1-octen-3-ol, 1-octen-3-one, and some other compounds from soybeans, but not from starchy grains like corn and wheat. Addition of a free radical scavenger, such as citric acid, greatly diminished the recovery of 1-octen-3-ol and 1-octen-3-one from soybeans.


Detecting vitreous and nonvitreous durum wheat kernels using near-infrared spectroscopy.

F.E. Dowell.

The vitreousness of durum wheat is used by the wheat industry as an indicator of milling and cooking quality. The current visual method of determining vitreousness is subjective and classification results between inspectors and countries vary widely. Thus, the use of NIR spectroscopy to classify vitreous and nonvitreous single kernels was investigated. Results showed that obviously vitreous or nonvitreous kernels were almost perfectly differentiated. When difficult-to-classify vitreous and nonvitreous kernels were included in the analysis, the classification accuracy was about 75 %. Classifications appear to be due, at least in part, to starch and protein differences between vitreous and non-vitreous kernels. NIR spectroscopy can provide the wheat industry with an objective means of determining the vitreousness of durum wheat samples.


Detection of waxy character and red/white wheat class using NIR spectroscopy.

F.E. Dowell, M. Tilley, S.R. Bean, G.L. Lookhart, D. Wang, F. Xie, and N. Rowley.

Wheat has three waxy proteins that affect the amylose content of starch. The presence or absence of these waxy proteins classifies the wheat as waxy (3 nulls or 0 alleles), partially waxy (1 or 2 nulls), or wild type (0 nulls or 3 alleles). Amylose content affects noodle quality, shelf life, and pulp quality of paper. NIR spectroscopy was used to determine if the number of alleles could be identified based on their NIR absorption characteristics. Results showed differences in absorption characteristics between all sample groups. Triple nulls were differentiated easily from all other groups. However, some overlap occurred when attempting to differentiate 0, 1, or 2 nulls from each other. Additional research used spectral information to classify red and white wheats in an attempt to remove subjectivity from class determinations. Most results showed greater than 99 % correct classification for single kernels when using the visible and NIR regions.


Prediction of quality parameters for soft wheat flour using near-infrared reflectance spectroscopy.

F.E. Dowell, N. Rowley, S.R. Bean, G.L. Lookhart, F. Xie, and P.L. Finney.

A diode-array, near-infrared spectrometer was used to determine what quality parameters of soft wheat flour could be correlated to their NIR absorption characteristics. The samples were analyzed using analytical, rheologial, milling, and baking methods to obtain more than 100 quality measurements. NIR spectra of the samples were well correlated to laboratory methods used to measure alkaline water, ETOH, pentosenase, carbonate, sulfite, histra, and sucrose retention capacities. Many of the absorption parameters including, bread absorption, Bohlin measurements, and mixograph absorption also were well correlated to the NIR spectra indicating that NIR may be a promising method for determining the water required for optimal mixing. The protein, hardness, and total gliadin and glutenin and some of their fractions also were well correlated to NIR. This technology may provide a means of rapidly analyzing flour for many quality parameters with one quick, nondestructive test when received at a bakery or leaving the mill.


Preliminary results of NIR spectroscopy to determine starch and protein quality in small samples.

F.E. Dowell, N.K. Rowley, F. Xie, M. Tilley, S.R. Bean, P.A. Seib, and G.L. Lookhart.

Most NIR instruments are designed to measure absorption or reflection characteristics of bulk samples. However, in many instances large amountsare not of material are available, such as when analyzing breeder samples or other aresmall-scale testing. In these instances, NIR analysis of a single kernel or a portion of flour from a single kernel may be desired. Procedures were developed utilizing fiber optics attached to a Perten Instrument DA7000 spectrometer (Springfield, Illinois) to collect absorbance spectra from these ultra-small samples. Small samples (< 20 mg) will be tested to determine waxy character (related to the presence or absence of GBSS proteins), amount of gliadin, amount of glutenin, and other quality attributes will be presented. The procedure is rapid (< 1 sec) and nondestructive and requires little sample preparation.


Development of a spectral-based weed sensor.

N. Wang, N. Zhang, Y. Sun, D. Peterson, and F.E. Dowell.

A spectral-based weed sensor was designed based on spectral characteristics of crops, weeds, and soil. Light-insensitive color indices were developed. The sensor was tested under laboratory conditions. The detection accuracies for wheat, soil, and weeds reached 98.68 %, 98.26 %, and 64.29 %, respectively.


Potential use of plant spectral characteristics in weed detection.

N. Wang, N. Zhang, F.E. Dowell, and J. Kurtz.

Plant and soil reflectance spectra were measured using a spectrometer. Significant wavelengths and feature values for weeds (leaf/stem), wheat (leaf/stem), and soil were determined using the mean difference and category contrast methods. Calibration models were developed on these wavelengths using partial least squares and discriminant analysis. All models classified soil with a rate of 100 %. Weed stems usually can be distinguished correctly from other classes. However, correctly identifying stems of specific weed species and differentiating between green objects, wheat leaf, wheat stem, and weed leaf were more difficult. This study demonstrated the potential of using spectral properties of plants and soil in weed detection.


The single kernel wheat characterization system (SKWCS).

C.R. Martin, F.E. Dowell, and J.L. Steele.

A brief description of the history and background for development of the Single Kernel Wheat Characterization System was presented. The chronology of the developmental steps which led to patenting, licensing and commercialization of the SKWCS was reviewed. The need and relevance of single kernel data for hardness and other quality measures were presented. The value of single-kernel data for any characteristic (distribution within a sample) was compared to sample mean values to further emphasize the general value of single kernel technology. Procedures to use single kernel data to estimate bulk mean values were reviewed. Applications other than wheat classification or hardness of mixtures were also reviewed. Some conceptual methodologies for using the single kernel data as a measure of product uniformity an prediction of other grain quality characteristics were presented. The operation, data collection, internal data processing, reliability and general maintenance requirements when using the Model 4100 to classify hard and soft wheat were reviewed. A general description of the software and computer interface which allows the system to rapidly assess single kernels (120 per minute) was presented. A summary of repeatability and reproducibility results for 10 SKWCS prototypes was presented. Recent developments and progress in adding single-kernel, NIR sensing to the Model 4100 were reported. The NIR interfacing was initiated with a Cooperative Research and Development Agreement with Perten Instruments North America in 1996. The integrated system extends the versatility of the system and can provide additional single-kernel quality measures such as protein, color class, damage, and vomitoxin.


Uniform procedures to determine wheat dockage and shrunken and broken kernels.

D.S. Chung, C.K. Spillman, J.L. Steele, S.K. Lee, C.H. Lee, and E. Maghirang.

Our objectives were to (1) develop a uniform procedure for wheat dockage determination and (2) develop a procedure for shrunken and broken kernels (SHBN) that can be combined with the dockage procedure. Six classes of wheat and three representative samples for each class were collected from various locations in the U.S. These samples were characterized for test weight, size distribution, geometric dimension, suspension velocity, and true density. These samples were characterized further for the amount and type of dockage and SHBN. The physical characteristics of each dockage component also were determined. A computer selection/prediction model was developed for determining the best combination of Carter-Day Dockage Tester (CDDT) settings for air velocity, riddle number, and location and number of sieves. Based on the outputs of the prediction model and the results of the model verification tests, two uniform procedures for combined dockage and SHBN determination using the CDDT were proposed and validated. One procedure was for all wheat classes except durum, A9 R2 T4 B2 (A9 = aspiration set at 9, R2 = #2 riddle, T4 = #4 top sieve, and B2 = #2 bottom sieve), and one for durum, A9 R25 T4 B2 (R25 = #25 riddle). The proposed procedures represent a feasible option for a combined uniform procedures using only the CDDT; efficiently removed dockage from wheat consistently lower than spike target but higher than that by the official methods; and were repeatable and simpler and required less time and equipment compared to the current official methods.


Energy efficiency of a new heat-pump, grain-drying concept.

J.L. Steele, D. Wang, and C.S. Chang.

An experimental closed-loop, heat-pump drying system was developed and evaluated for energy performance while drying shelled corn and sorghum. The system consisted of a stationary drying bed, a conventional heat pump, an air-to-air heat exchanger, a trim condenser, and a system to record grain and air conditions for performance evaluations. The experimental system was small in scale and designed to dry 10 bu/hr of shelled corn using recirculated forced air and projected equipment performances of COP = 4.0, air-air efficiency = 80 %, and fan efficiency = 50 %. Tests of three corn lots with initial moisture contents of 25, 20, and 18 % w.b. and one sorghum lot at 25 % w.b. were conducted during the 1998 autumn harvest season. Temperature of the heated air was controlled in the range of 110-120°F with airflow rates of 2,800 and 1,650 cfm for corn and sorghum, respectively. The average performance (ratio of input energy to mass of water removed) for the corn lots was 554 Btu/lb based on condensate collection and 510 Btu/lb based on grain lot weight differences before and after drying. The best energy performance observed in 4-6 hr periods was 440 Btu/lb. The average drying capacity was 9 bu/hr based on an equivalent moisture removal from 25 to 15.5 % w.b. per dry bu. The average thermal energy transfer rate of the air-air heat exchanger ranged from 550-718 Btu/min and averaged 28 % of the total (source-sink) thermal energy transfer rate. The average efficiency of the air-air heat exchanger was about 35 %, and the average COP was 6.4 including the air-air thermal transfer and 4.7 excluding that transfer. Refrigerant control during ambient air temperature swings (50-90°F) was difficult for the experimental system.


Representative sampling of first-break ground wheat.

M.C. Pasikatan, J.L. Steele, C.K. Spillman, E. Haque, and J.J. Higgins.

The sampling subsystem is a critical part of an on-line system for estimation of particle size distribution of ground materials. The representativeness of small sampling amounts (< 100g) of first-break ground wheat (ground wheat from first-break roller mill) and the samplers that could satisfy this requirement were evaluated in this study. The samplers used were Gamet rotary divider, spinning riffler, spinning disk divider, and Boerner divider with sampling amounts of 25, 50, 75, and 100 g. A follow-up experiment used Gamet rotary divider, spinning riffler, spinning disk divider with sampling amounts of 5, 15, 25, and 100 g. The split-split-plot experiments used wheat types (hard red winter and soft white winter) as main plots, samplers as subplots, and sampling amounts as subsubplots. The representativeness of a sampling amount obtained by a sampler was determined by comparing the geometric mean diameter and geometric standard deviation of a sample to those of the reference 100-g sample. The least sampling amount that was representative of the first-break ground wheat bulk was 5 g, provided the right procedure and sampler were used and the error was acceptable. Rotating samplers that obtain small fractions of the sample for each small time increment, such as a Gamet rotary divider and spinning riffler, satisfied the criteria for representative sampling of first-break ground wheat.


Particle-size distribution and sieving characteristics of first-break ground wheat.

M.C. Pasikatan, G.A. Milliken, C.K. Spillman, E.L. Haque, and J.L. Steele.

Eight particle-size distribution (PSD) models were tested with first-break ground wheat data from seven wheat classes (DRW, HRSW, HRWW, HWWW, SRWW, SWWW, WCB). PSDs were determined by sieving after first-break grinding of tempered 440 g samples using an experimental two-roll mill instrumented for grinding energy measurements. The distribution models tested were normal, lognormal, power, Schuhmann, Gaudin-Melloy, Rossin-Rammler, exponential, and exponential power. The normal distribution model described the experimental data best for hard wheats followed by the Rossin-Rammler distribution model. For soft wheats, the best model was soft wheat class specific, but the fits of the normal, Rossin-Rammler and Gaudin-Melloy distribution were generally better than that of the lognormal model.


Mixograph analysis guidelines based on mixograph dynamics.

J.L. Steele, M.D. Shogren, and D.L. Brabec.

Digitized mixogram analysis was investigated by simulating the moving pin positions and velocities as a function of input shaft rotation in a standard 10-g mixograph. The drag forces on the four moving pins were simulated as a function of moving pin velocity for a uniform viscous liquid. The drag force on each moving pin was converted to torque imposed on the bowl and summed to identify the basic cycles when flour-dough is mixed in a 10-g mixograph. These simulations were used as the basis for a number of conclusions regarding mixograph instrumentation and mixogram analysis in conjunction with the equations of motion for a spring-mass rotational system. These conclusions were validated with a number of flour-dough milling tests to form a basis for a simplified but adequate method of representing mixograms. Adequacy of the method was demonstrated through reconstruction of the mixogram and reproduction of mixograms via equivalent uniform viscous liquid simulations. Multipliers on mixing torque and time were used to demonstrate the effects of flour mass, mixing speed and mixer size.


News from the Grain Marketing and Production Research Center, Manhattan, KS.

D.E. Koeltzow.

This year has been exciting and productive at GMPRC. In a truly coöperative effort, a new wheat DNA-sequencing facility was established in our Plant Science and Entomology Research Unit with funding from the Kansas Wheat Commission, Kansas Agricultural Experiment Station, and the Wheat Genetics Resource Center at Kansas State University (KSU). This coöperation will continue throughout the life of this project as USDA-ARS scientists coöperate with university faculty to plan work supported by both state and federal funds. Initially, this facility will determine the DNA sequence of wheat markers associated with the defence responses to attacks by insects and diseases. Knowledge of such sequences will assist breeders with the deployment of resistance genes into commercial varieties. The ultimate goal for this facility is to determine the sequence of the entire genetic code for wheat.

Two new scientists were hired to replace two retirees. Dr. Catherine Katsar joined the Plant Science and Entomology Research Unit as a replacement for Dr. Jim Hatchett. She is a native of Brooklyn, NY and holds a B.S. degree in plant science from Cornell University. She received a M.S. degree in plant pathology and microbiology and a Ph.D. in entomology both from Texas A&M University. Her dissertation focused on greenbug resistance in sorghum. She continued further genetic studies of the greenbug as a USDA postdoctoral research associate in the Plant Protection Unit in Ithaca, NY. Dr. Katsar will be working on the mechanisms of wheat resistance to Hessian fly. Her work will include the development of more resistant wheat germ plasm.

Dr. Mark Casada joined the Engineering Research Unit in December, 1999, to fill the position vacated by Dr. Jack Chang. Dr. Casada received his B.S. in mechanical engineering and his M.S. in agricultural engineering from the University of Kentucky. After receiving his Ph.D. in biological and agricultural engineering from North Carolina State University, Dr. Casada joined the faculty of the Department of Biological and Agricultural Engineering at the University of Idaho in Moscow. At the University, Dr. Casada taught both graduate and undergraduate courses and conducted a research program focused on the storage and aeration conditions for wheat and corn. His main responsibilities at GMPRC will include leading and redefining the research program responsible for developing methods to maintain grain quality in storage bins and during handling by elevators and other components of the grain delivery system.

Finally, with support from U.S. Wheat Associates; GMPRC, KSU, and the American Institute of Baking (AIB) are coöperating in a new program designed to evaluate the wheat quality trait preferences of foreign buyers of U.S. HRWW. Under this program, various state wheat commissions and organizations provided representative samples of the major HRWW varieties that are currently being grown in the U.S. Samples also were segregated according to shipping ports including the Pacific Northwest, the Gulf, and California. They have been analyzed for quality by GMPRC and AIB scientists and milled into flour using KSU facilities. Flour, and in certain instances whole wheat, will be shipped to foreign coöperators who will evaluate these samples for quality in their own production facilities. Information on the desired quality traits will be shared with members of the U.S. wheat industry including breeders, producers, and grain handlers. Plans are to continue this program for multiple years, and the goal is to design improved varieties that can meet more of the quality needs by our foreign customers.