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CROP BREEDING & GENETICS

Milling and Baking Quality of Low Phytic Acid Wheat

^a Univ. of Idaho Research and Extension Ctr., P.O. Box 870, Aberdeen, ID 83210
^b USDA-ARS, 1680 Madison Ave, Wooster, OH 44691

^* Corresponding author (souza.6{at}osu.edu)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Low phytic acid (LPA) wheat (Triticum aestivum L.) is one approach to improving nutritional quality of wheat by reducing the major storage form of phosphorus and increasing the level of inorganic phosphorus, which is more readily absorbed by humans and other monogastric animals. Milling and baking quality evaluations were conducted on hard red, hard white, and soft white spring wheat grain from field trials to evaluate the effects of the LPA genotype on the end-use quality of wheat. In hard wheat backgrounds, the LPA genotypes were not associated with detrimental effects on flour protein concentration, dough mixing properties, or bread loaf volume. LPA wheats had consistent, substantial increases (up to 0.93 g kg^–1) in flour ash concentration relative to wild-type (WT) wheats. Higher flour ash in WT wheats is often a sign of higher aluerone and bran fragments which are visually evident in dulling of Asian noodles color. However, initial alkaline noodle brightness (L* = 86.8–87.5) from hard white LPA flours was at least as high as from hard white wild-type flours (L* = 86.1–87.9). LPA genotypes have demonstrated a significant redistribution of minerals from the bran to the endosperm; this redistribution of minerals most likely caused the increase in flour ash rather than greater partitioning of bran into the flour. In the soft wheat background, LPA genotypes had greater sodium carbonate and sucrose SRC (31 and 43 g kg^–1 greater than wild type, respectively), suggesting that LPA wheats milled with greater apparent starch damage and/or pentosan content than WT sib lines.

Abbreviations: HIP, high inorganic phosphorus • LPA, low phytic acid • RVA, rapid visoanalyzer • WT, wild type

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
WE HAVE IDENTIFIED low phytic acid (LPA) mutations in wheat (Guttieri et al., 2004). Low phytic acid mutants of other crops, including barley (Hordeum vulgare L.; Larson et al., 1998), rice (Oryza sativa L.; Larson et al., 2000), soybean [Glycine max (L.) Merr.; Wilcox et al., 2000], and maize (Zea mays L.; Raboy et al., 2000) were identified earlier. Although the biochemical characteristics of the seed of these mutants are well described, reports of the effect of the LPA trait on manufacturing quality are limited. LPA wheat is of interest as one approach to improving the nutritional quality of wheat fed to humans and livestock. Animals fed diets with LPA corn and barley have demonstrated greater feed efficiency, improved digestibility, better retention of P, Ca, and N, and significant decrease in P excretion compared with animals fed WT grain (reviewed by Mendoza 2002). Human diets high in phytic acid (PA) can lead to zinc deficiency, since PA is negatively correlated with zinc absorption. PA does not affect copper absorption in humans but slightly inhibits manganese absorption. PA forms insoluble complexes with iron that are nutritionally unavailable at the pH of the small intestine. Diets high in PA and low in iron can lead to iron deficiency (reviewed by Lönnerdal, 2002).

Previous research with this mutant concluded that the LPA mutants identified in wheat reduce seed phytate by approximately 33% to only nominal changes, depending on the environment and genetic background. Yet, in multiple genetic backgrounds and environments, significant increases of approximately 100 to 300% in inorganic P were observed in seed of LPA wheat when compared with similar (WT) selections (Guttieri et al., 2006a). The distribution of P within the kernel also was affected by the LPA trait with mutant lines having three times the concentration of P in the endosperm than was observed for similar WT lines, even though total P per seed was similar for both LPA and WT lines (Guttieri et al., 2006b). Similarly, LPA mutations elevated the concentration of magnesium in the endosperm relative to paired WT genotypes (Guttieri et al., 2006b). In earlier work, we were able to demonstrate that the LPA mutations can affect yield and plant development. However, those effects appear dependent on genetic background and are difficult to generalize into a simple relationship (Guttieri et al., 2006a). This led us to conclude that it was possible through prebreeding to develop LPA wheats that were agronomically competitive with WT genotypes.

We have undertaken studies to address the effects of the LPA phenotype on wheat quality in three genetic backgrounds: soft white spring, hard red spring, and hard white spring. Flour extraction and flour ash concentration are key quality considerations in all three classes of wheat (Souza et al., 2002). Flour protein concentration and dough rheological properties are critical quality considerations in hard wheat classes. Bread loaf volume is of particular importance in hard red wheat, and noodle brightness and color stability are of particular importance in hard white wheat. Water absorption properties are a primary consideration for soft wheat pastry quality, and flour pasting parameters are important considerations for Asian noodle products. The studies presented in this manuscript describe the effects of the LPA phenotype on these end-use quality parameters.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Generation of Experimental Materials
Hard Red Spring
‘Grandin’ hard red spring wheat was used as a recurrent parent in crosses with the low phytic acid source Js-12 LPA (Guttieri et al., 2004). Two families of F₂ plants derived from of two BC₃F₁ plants with the pedigree ‘Grandin’*4/Js-12 LPA were grown in the greenhouse. The two BC₃ F₁ plants were designated "B" and "C" with progeny families derived from the two plants named after the two BC₃ F₁ plants. A preliminary evaluation to eliminate heterozygous genotypes was conducted on F₃ seed from each BC₃F₂ plant using a high inorganic phosphorus (HIP) phenotype of individual kernels as described previously (Guttieri et al., 2004). The BC₃F_2:3 seed from BC₃F₂ plants determined to be homozygous for HIP phenotype or WT phenotype were hand-planted in the field near Aberdeen, ID, in 2002 with progeny rows tracing to each BC₃F₂ plant kept separate. Field-grown seed was evaluated for uniformity of HIP phenotype. Fourteen BC₃F_2:4 families were advanced into replicated yield trials in 2003, including three strong HIP phenotype LPA selections and three wild-type HIP selections derived from the "C" BC₃ F₁ plant (designated as "C" families), and five moderate HIP phenotype LPA selections and three WT HIP selections from the "B" BC₃ F₁ plant (designated as B families).

Hard White Spring
Similarly, BC₂F₂ plants derived from a BC₂F₁ plant with the pedigree ‘Lolo’*3/Js-12 LPA were grown in the greenhouse. Lolo is a high-yielding, hard white spring wheat adapted to irrigated and rain-fed production in the Pacific Northwest (Souza et al., 2003). Preliminary evaluation of HIP phenotype was conducted on BC₂F_2:3 seed from each BC₂F₂ plant to eliminate heterozygous genotypes. BC₂F_2:3 families identified as homozygous HIP phenotype and BC₂F_2:3 families identified as homozygous WT phenotype were hand-planted in the field at Aberdeen in 2002. Field-grown BC₂F_2:4 seed harvested in 2002 was evaluated for uniformity of HIP phenotype. Seed harvested in 2002 of twelve BC₂F_2:4 families from the single BC₂F₁ plant selection were advanced into replicated yield trials: five WT selections and seven HIP selections.

Soft White Spring Wheat
A population derived from the cross Js-12-LPA/IDO563 was advanced in the greenhouse by single seed descent through the F₄ generation. IDO563 is a sib-selection to the line that was mutagenized to produce Js-12-LPA. Eighty-two F_4:5 families were planted in the field at Aberdeen in 2002. Seed from each family was tested for uniformity of HIP phenotype. Twelve WT and 10 HIP phenotype F_4:6 families were advanced into replicated yield trials.

Field Trials
Trials were grown at the University of Idaho Aberdeen Research and Extension Center near Aberdeen, ID, in 2003 and 2004 and at the University of Idaho Tetonia Research and Extension Center near Tetonia, ID, in 2004. The hard red spring, hard white spring, and soft white spring populations were evaluated as separate experiments, each including the parental genotypes. Experiments were randomized arrangements of incomplete block designs with three replications. Soil test P in the first 30 cm of soil at Aberdeen was 26 mg kg^–1 in 2003 and 13 mg kg^–1 in 2004, and at Tetonia was 21 mg kg^–1 in 2004. Trials were planted at Aberdeen on April 8, 2003 and April 9, 2004. Trials were planted at Tetonia on 3 May 2004. Plot size was 1.4 m x 3 m. The experimental area was fertilized with ammonium nitrate before planting on the basis of University of Idaho soil test recommendations (Brown et al., 2001). The experimental area at Aberdeen was irrigated with overhead sprinklers to replace estimated evapotranspiration. Weeds were controlled with labeled applications of registered small grain herbicides. Trials were harvested at Aberdeen on 18 Aug. 2003 and 30 Aug. 2004 and at Tetonia on 23 Sep. 2004 with a small plot combine equipped with a weighing system (Harvestmaster, Juniper Systems, Logan, UT).

Phosphorus Composition
Whole meal wheat samples were prepared by grinding on a UDY cyclone mill (UDY Co., Fort Collins, CO). Samples were dried in a 70°C drying oven for a minimum of 3 d before extraction or digestion. Sodium phytate (phytic acid, dodecasodium salt) was purchased from Sigma (St. Louis, MO) and checked for purity on HPLC before use as a standard for phytic acid phosphorus experiments. All other reagents were purchased from VWR (West Chester, PA). Data were analyzed using Microsoft Excel (Microsoft, Redmond, WA).

Inorganic phosphorus was determined by a variation of the Chen method (Chen et al., 1956) modified for use on microtiter plates. Briefly, dried samples (0.5 g) of whole meal or milling fractions were extracted in 10 mL of 12.5% (w/v) trichloroacetic acid (TCA) containing 25 mM magnesium chloride at 4°C overnight with continuous shaking, followed by centrifugation at 4°C and 5000 g for 15 min. The supernatant was removed and brought to a standard volume of 25 mL with distilled water. The extracts were then assayed with equal volumes of Chen's reagent along with prepared potassium phosphate standards and reagent blank on microtiter plates. After 1 h incubation at room temperature, plates were read at 820 nm on a Dynatech Laboratories MRX microplate reader (Chantilly, VA). Each sample was plated in quadruplicate, and each analysis performed in duplicate.

Total phosphorus was determined by digestion of dried samples (0.15–0.3 g) in 2 mL concentrated sulfuric acid and 30% (v/v) hydrogen peroxide at 120°C until solutions were clear and colorless and all traces of peroxide were gone. Digested samples were cooled to room temperature and diluted to a standard volume of 25 mL. Samples were then assayed for P content on microtiter plates as described above for inorganic P content but with a modification of the Chen's reagent. Specifically, because of the sulfuric acid content of the samples, Chen's reagent was prepared as described (Chen et al., 1956) but with substitution of water for sulfuric acid. Sulfuric acid was added to standards and to samples requiring further dilution in appropriate volumes to provide the appropriate concentration of 0.3 M sulfuric acid in all wells. Microtiter plates were read at 820 nm on a Dynatech Laboratories MRX microplate reader after 1 h at room temperature. Each digested sample was plated in quadruplicate, and each analysis was performed in duplicate.

Phytic acid content was determined by the method of Haug and Lantzsch (1983), as adapted for use on microtiter plates. Briefly, dried samples were extracted at 4°C overnight with continuous shaking in 0.2 M HCl. Samples were centrifuged at 5000 g at 4°C for 15 min, and the extract was brought to a standard volume with 0.2 M HCl. (Any further dilution necessary to bring the samples into the detection range of 1.5–24 µg/mL was accomplished in 0.2 M HCl.) Sample extracts and prepared standards were then treated as follows: 1.0 mL of extract (or known standard sodium phytate solution) was incubated in a boiling water bath for 15 min with 1.0 mL of 415 µM ferric ammonium chloride prepared in 0.2 M HCl, cooled in an ice bath for 15 min and mixed well. Samples were then assayed directly on microtiter plates in a ratio of 120-µL sample (or prepared sodium phytate standard) to 180 µL 2,2'-bipyridine: thioglycolic acid solution, and read without delay at 530 nm on a Dynatech Laboratories MRX microplate reader. Each sample was plated in quadruplicate, and each experiment performed in duplicate.

Quality Evaluations
Samples from each plot were tempered and milled on a Brabender Quadrumat Senior mill (Brabender Instruments Inc., S. Hackensack, NJ) according to American Association of Cereal Chemistry (AACC, 2000) procedures (methods 26–10 and 26–21A). Flour protein concentration was determined with a near-infrared analyzer (Instalab 600, Dickey-John Corp., Auburn, IL, AACC method 39–10A), calibrated by automated combustion analysis of total N content (LECO Model NFP-428, LECO Corp., St. Joseph, MO), and corrected to 120 g kg^–1 moisture. Mixograph analyses (AACC method 54–50) and bread baking (AACC method 10–10B) were as described in Souza et al. (1993). Alkaline noodles were prepared as described previously (Guttieri et al., 2001a). Noodle sheet color was measured initially at the time of sheeting and after 24-h incubation in resealable plastic bags in Commission Internationale de l'Eclairage (CIE) tristimulus color space (L*, a*, b*) with a Minolta CM-2002 spectrophotometer (Minolta Camera, Chuo-Ku, Osaka, Japan) with a 50-mm measurement aperture. Flour pasting viscosity was determined on a Foss Super 3 Rapid Viscoanalyzer (RVA, Newport Scientific Pty. Ltd., Warriewood NSW, Australia) as described in Guttieri et al. (2001b). Solvent retention capacity (SRC) of soft wheat flour was measured in accordance with AACC 56–11, as modified in Guttieri et al. (2001b).

Statistical Analysis
Analyses of variance were conducted by PROC MIXED in SAS (Release 8.02, SAS Institute, Cary, NC). Experiments at Aberdeen initially were analyzed with year as a fixed effect to test the interaction of year with entry. Significant year x selection interactions were uncommon and when observed were relatively small compared with the trial main effects and did not alter conclusions. Therefore, data were combined over years for analysis. In the hard red spring (HRS) study, family and genotype (WT vs. LPA) were treated as fixed effects; year replication(year) and family x genotype(selection) were treated as random effects. In the hard white experiment, genotype was treated as fixed effect, and year, replication(year), and genotype(selections) were treated as random effects. Single-degree of freedom contrasts were used to test the effect of genotype (WT vs. LPA) (within the 12 selections of the K family). In the soft white experiment, data were analyzed with genotype (WT vs. LPA) as a fixed effect; year, replication(year), and selection(genotype) were analyzed as random effects. The Tetonia 2004 trials were analyzed analogously to the Aberdeen trials without year effects.

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Hard Red Spring Study
In this study, the LPA trait significantly reduced phytic acid in HRS grain with a resulting increase in inorganic phosphorus (Fig. 1). The magnitude of the reduction in PA was greater in 2003 than in 2004. The LPA trait had no effect on flour protein concentration (Table 1). In the Aberdeen trials, WT selections had greater flour yield than LPA selections. This effect of genotype on flour yield was not apparent at Tetonia (Table 2), perhaps because flour yields from Tetonia were lower than from Aberdeen and this location effect masked a genotype effect. Ash concentration was significantly greater in flours from LPA wheats than in flours from WT wheats. This effect was observed at both locations and was more pronounced in the C family, in which the LPA effect on P distribution was greatest.

View larger version (64K): [in this window] [in a new window]   Fig. 1. Phosphorus distribution in whole grain of low phytic acid (LPA) and wild-type (WT) selections of two BC3–derived families of the cross Grandin*4/Js-12-LPA. Grain was produced under irrigation at Aberdeen, ID, in 2003 and 2004 and under rain-fed conditions at Tetonia, ID, in 2004 (adapted from Guttieri et al., 2006a).

Hard White Spring Study
The LPA trait reduced PA content while increasing inorganic phosphorus in hard white spring selections (Fig. 2). As in the HRS trial, flour yield from LPA HWS selections was significantly lower than flour yield from WT HWS selections when grown at Aberdeen (Table 3). However, flour yields from Tetonia production were similar (Table 4). Again, flour yields from Tetonia were low and may have masked effects of the LPA trait. Ash concentration was 0.91 g kg^–1 greater in LPA flours than in WT flours in Aberdeen trials and 0.33 g kg^–1 greater in LPA flours than in WT flours in the Tetonia trial. LPA and WT flours produced similar mixographs in the Aberdeen trials. However, in the Tetonia trial, time to mixograph peak was 0.6 min longer for LPA flours than for WT flours, and mixograph tolerance of LPA flours was greater. Protein concentrations of LPA and WT flours were not significantly different.

View larger version (62K): [in this window] [in a new window]   Fig. 2. Phosphorus distribution in whole grain of low phytic acid (LPA) and wild-type (WT) selections of the cross Lolo*3/Js-12-LPA. Grain was produced under irrigation at Aberdeen, ID, in 2003 and 2004 and under rain-fed conditions at Tetonia, ID, in 2004 (adapted from Guttieri et al., 2006a).

View larger version (61K): [in this window] [in a new window]   Fig. 3. Phosphorus distribution in whole grain of low phytic acid (LPA) and wild-type (WT) selections of the cross IDO563/Js-12-LPA. Grain was produced under irrigation at Aberdeen, ID, in 2003 and 2004 (adapted from Guttieri et al., 2006a).

	CONCLUSIONS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
The accuracy of the milling and baking measurements in these trials allowed us to measure genetic changes that were below the level of variation that would be detectable in an industrial setting. For example, the initial brightness of the LPA Lolo alkali noodles was greater than for the WT sib lines, yet they visually indistinguishable to the naked eye (Table 5). For a number of traits, the mutant and sib lines had the same measured value down to the last significant decimal. Our primary concern was to identify gross variation due to the LPA trait that may be difficult to overcome with conventional breeding methods.

The power of the sib-line experimental design allows us to make several general conclusions about the effect (or lack of effect) of the LPA trait on milling and baking quality. In the hard wheat backgrounds, the LPA trait was not associated with detrimental effects on dough mixing properties, bread loaf volume, or alkaline noodle color. The primary concern in producing and milling LPA wheats will be the consistent, substantial increase in flour ash concentration. This appears to be a problem of perception rather than function, since noodle color of LPA flours was excellent. Previous work with these genotypes (Guttieri et al., 2006a,2006b) has shown that LPA genotypes have a significant redistribution of phosphorous and other minerals (primarily magnesium) from the bran to the endosperm. The redistribution of minerals likely is the cause for the increase in flour ash rather than greater partitioning of bran into the flour. If the latter were a factor, we would have measured greater discoloration of Asian noodles in LPA selections of the hard white study than in WT selections.

In the soft wheat background, however, the effects of the LPA trait on sodium carbonate and sucrose SRC suggest that LPA wheats milled with greater starch damage and/or pentosan content. This observation needs to be tested in additional soft wheat backgrounds and additional biochemical evaluations.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
This study was conducted while the third author was a professor at the University of Idaho.

Received for publication March 13, 2006.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

• American Association of Cereal Chemists. 2000. Approved methods of the AACC. 10th ed. AACC, St. Paul, MN.
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• Souza, E., R. Graybosch, and M.J. Guttieri. 2002. Breeding for improved milling and baking quality in wheat; a review. J. Crop Prod. 5:39–74.[CrossRef]
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