Published online November 21, 2006
Agronomic Performance of Low Phytic Acid Wheat M. J. Guttieri, K. M. Peterson, and E. J. Souza* tion (reviewed by Lo¨nnerdal, 2002). Phytic acid forms insoluble complexes with Fe that are nutritionally unavailable at the pH of the small intestine. Diets high in PA and low in Fe can lead to Fe deficiency. However, in human populations with high-Fe diets, the formation of PA–Fe complexes may provide protection against colon cancer by reducing Fe-induced oxidative injury (reviewed by Minihane and Rimbach, 2002). Although the biochemical characteristics of these mutant seed are well described, published reports of the agronomic performance of LPA crops are limited. The germplasm registration notice for KBNT lpa1-1, an LPA rice, noted that the yield of the LPA mutant genotype was about 90% of standard Arkansas rice cultivars (Rutger et al., 2004a). The germplasm registration notice for Goldhull Low Phytic Acid (GLPA) rice included limited yield trial data (Rutger et al., 2004b). However, the GLPA and KBNT lpa1-1 rice germplasms yielded 95 to 98% of the check cultivar, ‘Kaybonnet’, from which KBNT lpa1-1 was derived. There are no published reports of replicated agronomic trials with LPA barley or maize. Low phytic acid soybean lines with the pha1pha1pha2pha2 genotype had significantly lower seedling emergence than WT lines (Oltmans et al., 2005). Other agronomic traits were not consistently different among the three soybean populations studied. The LPA soybeans with the mips allele also had significantly lower seedling emergence than WT lines with the Mips allele (Meis et al., 2003). The LPA wheat mutant, Js-12 LPA, was described as “agronomically unacceptable” because of its reduced stature, markedly weak straw, and dramatically reduced grain yield (Guttieri et al., 2004). However, mutagenesis often induces undesirable traits that segregate independently of the target trait. Within our breeding program we have observed that backcross progeny of crosses to transfer the LPA trait from Js-12 LPA to non-LPA cultivars has resulted in LPA germplasm that is phenotypically similar to the non-LPA parent in all aspects other than P partitioning in the seed. In this study, we report on the assessment of the agronomic effects of the LPA genotype from Js-12-LPA in backcross-derived wheat lines.
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
ABSTRACT Low phytic acid (LPA) genotypes of wheat (Triticum aestivum L.) improve the nutritional quality of wheat by reducing the concentration of phytic acid (PA) in the aleurone layer, thus reducing the chelation of nutritionally important minerals and improving the bioavailability of phosphorus. Field studies were conducted at Aberdeen and Tetonia, ID, in 2003 and 2004 to evaluate the effects of the LPA genotype on the agronomic performance of wheat. These studies included wildtype (WT) and LPA genotypes in hard red spring, hard white spring, and soft white spring wheat genetic backgrounds. In the hard red spring genetic background, LPA genotypes had delayed development and reduced grain yield (8–25%) in the high yield environment, in part due to reduced kernel size (up to 3 mg kernel21). In the hard white spring genetic background, differences in crop development and grain yield were not observed; however, in the high yield environment LPA genotypes produced smaller kernels (2.0–2.4 mg kernel21). In the soft white spring genetic background, LPA genotypes developed earlier, but the grain yield of LPA genotypes was reduced 20 to 24% in the high yield environment. However, LPA kernels, on average, were heavier and larger in diameter than WT kernels. The absence of consistent effects of the LPA genotype across the three genetic backgrounds suggests that deleterious effects of the LPA genotype may be mitigated by plant breeding.
P
storage form in cereal grains of seedborne P. Most surveys of native variation in cereals for PA have identified only limited variation in the fraction of seed P stored as phytate. Therefore, mutagenesis has been used to develop LPA mutants. We identified LPA mutants of wheat that reduce seed phytate by about 30 to 40% (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, with most mutants reducing seed phytate and elevating seed inorganic P concentration. Low phytic acid wheat may improve the nutritional quality of wheat fed to livestock and humans. Animals fed diets with LPA corn and barley have demonstrated greater feed efficiency, improved digestibility, better retention of P, Ca, and N, and a significant decrease in P excretion (Mendoza, 2002). Human diets high in PA can lead to Zn deficiency, as phytate is negatively correlated with Zn absorption. Phytic acid does not affect Cu absorption in humans, but slightly inhibits Mn absorpHYTATE IS THE PRIMARY
MATERIALS AND METHODS Generation of Experimental Materials Hard Red Spring
M.J. Guttieri and K.M. Peterson, Univ. of Idaho Research and Extension Center, P.O. Box 870, Aberdeen, ID 83210; E.J. Souza, 105AWilliams Hall, OARDC, 1680 Madison Ave., Wooster, OH 44691. Received 6 Jan. 2006. *Corresponding author (
[email protected]).
Two families of F2 plants derived from 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 of a high
Published in Crop Sci. 46:2623–2629 (2006). Crop Breeding & Genetics doi:10.2135/cropsci2006.01.0008 ª Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA
Abbreviations: HIP, high inorganic phosphorus; LPA, low phytic acid; PA, phytic acid; PAP, phytic acid phosphorus; WT, wild-type.
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Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
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CROP SCIENCE, VOL. 46, NOVEMBER–DECEMBER 2006
inorganic phosphorus (HIP) phenotype to eliminate heterozygous genotypes was conducted on F2:3 seed from each BC3F2 plant. On the basis of progeny phenotyping, the B family will be designated as Moderate lpa family and the C family as Strong lpa family for purposes of this manuscript. The HIP phenotype of individual kernels was evaluated as described previously (Guttieri et al., 2004). The BC3F2:3 seed of BC3F2 plants determined to be homozygous for HIP 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 BC3F2:4 families were advanced into replicated yield trials in 2003, including three strong HIP phenotype LPA selections and three WT 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 BC2F2:3 seed from each BC2F2 plant to eliminate heterozygous genotypes. BC2F2:3 families identified as homozygous HIP phenotype and BC2F2:3 families identified as homozygous WT phenotype were hand-planted in the field in 2002. Field-grown BC2F2:4 seed harvested in 2002 was evaluated for uniformity of HIP phenotype. Seed harvested in 2002 of 12 BC2F2:4 families from the single BC2F1 plant selection were advanced into replicated yield trials: five WT selections and seven HIP selections. Soft White Spring 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 F4:5 families were planted in the field in 2002. Seed from each family was tested for uniformity of HIP phenotype. Twelve WTand 10 HIP phenotype F4:6 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 in 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 kg21 in 2003 and 13 mg kg21 in 2004, and at Tetonia was 21 mg kg21 in 2004. Trials were planted at Aberdeen on 8 Apr. 2003 and 9 Apr. 2004. Trials were planted at Tetonia on 3 May 2004. Plot size was 1.4 by 3 m and target seeding density was 2 million seeds ha21. The soil type at Aberdeen was a Declo sandy loam (coarse-loamy, mixed, superactive, mesic Xeric Haplocalcids); the soil type at Tetonia was a loess, coarse silt loam (Pachic Cryoboroll, predominantly of the Tetonia series [coarse-silty, mixed, superactive Calcic Pachic Haplocryolls]). The experimental area was fertilized with ammonium nitrate before planting based on University of Idaho soil test recommendations. The experimental area at Aberdeen was irrigated using
overhead sprinklers to replace estimated evapotranspiration. The experimental area at Tetonia was rain fed. Weeds were controlled with registered small grain herbicides per standard procedures. Trials were harvested at Aberdeen on 18 Aug. 2003 and 30 Aug. 2004, and at Tetonia on 23 Sept. 2004 using a small plot combine equipped with a weighing system (Harvestmaster, Juniper Systems, Logan, UT). Harvested grain was cleaned on a standard air–screen cleaner and the test weight was recorded. Kernel characteristics were evaluated on 300kernel samples from each plot using a Single Kernel Characterization System 4100 (Perten Instruments, Reno, NV).
Phosphorus Composition Whole meal wheat samples were prepared by grinding on a UDY cyclone mill. Samples were dried in a 708C drying oven for a minimum of 3 d before extraction or digestion. Sodium phytate as a dodecasodium salt (Sigma Chemical Co., St. Louis, MO) was checked for purity by high performance liquid chromatography before use as a standard for PAP experiments. Data were analyzed using the software Microsoft Excel. Inorganic P 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 containing 25 mM magnesium chloride at 48C overnight with continuous shaking, followed by centrifugation at 48C and 5000 g for 15 min. The supernatant was removed and brought to a standard volume of 25 mL with distilled water. Then, the extracts were 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 (DYNEX Technologies, Chantilly, VA). Each sample was plated in quadruplicate, and each analysis performed in duplicate. Total P was determined by digestion of dried samples (0.15– 0.30 g) in 2 mL concentrated sulfuric acid and 30% hydrogen peroxide at 1208C 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, due to 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 0.6 M sulfuric acid in all wells. Microtiter plates were read at 820 nm on a Dynatech Laboratories (Chantilly, VA) 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 48C overnight with continuous shaking in 0.2 M HCl. Samples were centrifuged at 5000 g at 48C 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 to 24 mg mL21 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 mM 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-mL
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sample (or prepared sodium phytate standard) to 180 mL 2,29bipyridine–thioglycolic acid solution, and read without delay at 530 nm on an MRX microplate reader (Dynatech Laboratories, Chantilly, VA). Each sample was plated in quadruplicate, and each experiment assayed in duplicate.
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
Statistical Analysis Analyses of variance were conducted using PROC MIXED in SAS (SAS Institute, 2000). The Aberdeen 2003 and 2004 experiments initially were analyzed with year as a fixed effect to test the interaction of year with entry. Significant year 3 selection interactions were observed in all trials. Therefore, years were analyzed separately. In the HRS experiments, data were analyzed with family and genotype (WT vs. LPA) as fixed effects; replication and selection|(family 3 genotype) were treated as random effects. In the HWS experiment, family 3 genotype was treated as fixed effect and replication and selection (family 3 genotype) as random effects; single-degree of freedom contrasts were used to test the effect of genotype (WT vs. LPA) within the 12 selections. In the SWS experiment, data were analyzed by year with genotype (WT vs. LPA) as a fixed effect and selection within genotype and replication estimated as random effects.
RESULTS Hard Red Spring Study In both years of the study at Aberdeen, the heading date of LPA selections was delayed significantly relative
to WT selections (Table 1). The LPA selections were significantly shorter than WT selections at Aberdeen on 10 June, but LPA and WT selections achieved similar final stature at both Aberdeen and Tetonia. In 2003 at Aberdeen, growth stage on 10 June was significantly delayed for LPA selections of both families. In 2003 Aberdeen and 2004 Tetonia, grain yield of selections in the Moderate lpa family was significantly greater than the Strong lpa family (Table 1). But in 2004 Aberdeen, the two families produced similar grain yields. In both years, WT selections significantly outyielded LPA selections by 8 to 25% at Aberdeen. Genotype did not significantly affect yield at Tetonia in 2004. Test weight of LPA selections grown at Aberdeen in 2004 was lower than WT selections, but test weights of LPA and WT selections were similar at Aberdeen in 2003 and Tetonia in 2004. And in both 2003 and 2004 at Aberdeen, kernel weights of LPA selections were 1.6 mg and 3.0 mg smaller than that of WT selections. Yet at Tetonia, kernel weight was not affected by genotype. Kernel weights of WT selections from the Tetonia trial were about 4 mg lower than from the Aberdeen trials; this environmental effect may have masked the differences between the LPA and WT genotypes. Kernel hardness was not affected by genotype, and kernel diameter was reduced 0.06 to 0.08 mm only in the Aberdeen 2004 trial (Table 1).
Table 1. Agronomic performance of wild-type (WT) and low phytic acid (LPA) Grandin*4/Js-12 low phytic acid (LPA) genotypes in the hard red spring wheat study grown at Aberdeen (irrigated) in 2003 and 2004 and at Tetonia (rain-fed) in 2004. Moderate LPA family
Height (10 June) 2003 Aberdeen 2004 Aberdeen Growth stage (10 June), 2003 Aberdeen 2004 Aberdeen Heading date, DOY‡ 2003 Aberdeen 2004 Aberdeen Final height, cm 2003 Aberdeen 2004 Aberdeen 2004 Tetonia Yield, kg ha21 2003 Aberdeen 2004 Aberdeen 2004 Tetonia 21 Test weight, kg hL 2003 Aberdeen 2004 Aberdeen 2004 Tetonia SKCS§ hardness 2003 Aberdeen 2004 Aberdeen 2004 Tetonia SKCS weight, mg 2003 Aberdeen 2004 Aberdeen 2004 Tetonia SKCS diam., mm 2003 Aberdeen 2004 Aberdeen 2004 Tetonia
Strong LPA family
Mixed effects analysis of variance F value
WT (N 5 3)
LPA (N 5 5)
WT (N 5 3)
LPA (N 5 3)
Family
Genotype
Family 3 genotype
38.0 6 0.7 43.1 6 1.5 Zadoks scale 44 6 1 38 6 1
360 6 0.5 36.3 6 1.2
37.1 6 0.7 44.7 6 1.5
35.3 6 0.7 37.6 6 1.5
1.5 ns† 1.2 ns
8.4* 28.0**
0.0 ns† 0.0 ns
42 6 1 37 6 0
44 6 1 39 6 1
42 6 1 38 6 1
0.1 ns 3.4 ns
14.3** 3.4 ns
0.0 ns 0.7 ns
169 6 0 176 6 0
170 6 0 179 6 0
169 6 0 176 6 0
170 6 0 179 6 0
0.2 ns 2.4 ns
20.5** 61.5**
0.0 ns 0.6 ns
104 6 2 109 6 1 76.2 6 1.8
106 6 1 110 6 1 78.4 6 1.6
102 6 2 107 6 1 77.6 6 1.8
103 6 2 109 6 1 77.0 6 1.8
2.3 ns 1.5 ns 0.1 ns
1.1 ns 0.8 ns 0.7 ns
0.2 ns 0.1 ns 1.9 ns
7980 6 330 6090 6 320 2620 6 240
7310 6 260 6090 6 320 2620 6 240
7000 6 330 6520 6 320 2290 6 240
6140 6 340 4980 6 320 1770 6 240
10.3** 0.1 ns 7.4*
6.6* 25.7** 3.8 ns
0.2 ns 2.1 ns 0.8 ns
92.6 6 0.4 90.4 6 0.5 83.9 6 0.8
92.2 6 0.3 89.5 6 0.5 83.4 6 0.7
92.7 6 0.4 91.7 6 0.5 86.4 6 0.8
91.9 6 0.4 89.5 6 0.5 85.0 6 1.0
0.1 ns 3.9 ns 9.2**
3.3 ns 20.8** 2.4 ns
0.3 ns 3.7 ns 0.5 ns
80.9 6 0.9 76.4 6 1.5 65.1 6 1.9
82.7 6 0.7 75.2 6 1.2 65.5 6 1.6
79.7 6 0.9 74.1 6 1.5 67.0 6 1.9
81.9 6 0.9 74.8 6 1.5 65.1 6 2.4
1.4 ns 1.0 ns 0.1 ns
6.1* 0.0 ns 0.1 ns
0.1 ns 0.6 ns 0.3 ns
37.6 6 0.5 37.7 6 0.6 33.2 6 0.5
35.4 6 0.4 35.2 6 0.5 33.6 6 0.4
37.3 6 0.5 39.7 6 0.6 33.7 6 0.5
36.2 6 0.5 36.2 6 0.6 33.9 6 0.7
0.2 ns 9.0* 0.6 ns
11.8** 36.3** 0.4 ns
1.3 ns 1.0 ns 0.1 ns
2.69 6 0.03 2.65 6 0.03 2.55 6 0.02
2.65 6 0.02 2.59 6 0.02 2.63 6 0.02
2.65 6 0.03 2.70 6 0.03 2.57 6 0.03
2.68 6 0.03 2.62 6 0.03 2.59 6 0.03
0.1 ns 4.5 ns 0.2 ns
0.1 ns 8.7* 4.2 ns
0.6 ns 0.3 ns 1.3 ns
* F value significant at P , 0.05. ** F value significant at P , 0.01. † ns, nonsignificant. ‡ DOY, day of year. § SKCS, Single Kernel Characterization System (Perten Instruments, Reno, NV).
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Table 2. Phosphorus distribution in wild-type (WT) and low phytic acid (LPA) Grandin*4/Js-12 LPA genotypes in the hard red spring wheat study grown at Aberdeen in 2003 and 2004 and at Tetonia in 2004. Inorganic P 2003 Aberdeen
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
Genotype Moderate lpa family WT (N 5 3) LPA (N 5 5) Strong lpa family WT (N 5 3) LPA (N 5 3) Family Genotype Family 3 genotype
Phytic acid P
2004 Aberdeen
2004 Tetonia
2003 Aberdeen
2004 Aberdeen
2.93 6 0.14 2.51 6 0.11
mg g 1.47 6 0.09 1.24 6 0.07
Total P 2004 Tetonia
2003 Aberdeen
2004 Aberdeen
2004 Tetonia
1.90 6 0.11 1.76 6 0.09
4.09 6 0.13 4.00 6 0.12
3.01 6 0.11 2.98 6 0.10
2.36 6 0.09 2.32 6 0.08
1.86 6 0.11 1.83 6 0.11
4.32 6 0.13 4.18 6 0.14
3.05 6 0.11 3.15 6 0.11
2.31 6 0.09 2.49 6 0.09
0.1 ns 0.6 ns 0.3 ns
4 ns 1.2 ns 0.1 ns
2.2 ns 0.2 ns 0.9 ns
0.6 ns 0.9 ns 2.3 ns
21
0.23 6 0.07 0.38 6 0.06
0.30 6 0.07 0.39 6 0.05
0.16 6 0.03 0.29 6 0.02
0.28 6 0.07 0.96 6 0.07
0.32 6 0.07 0.77 6 0.07
0.19 6 0.03 2.75 6 0.14 1.47 6 0.09 0.46 6 0.03 1.77 6 0.14 0.94 6 0.09 Mixed effects analysis of variance F value
21.9** 38.6** 15.5**
12.0** 22.9** 10.4**
12.1** 52.1** 5.9*
11.4** 26.8** 4.4 ns
3.4 ns† 21.1** 3.4 ns
* F value significant at P , 0.05. ** F value significant at P , 0.01. † ns, nonsignificant.
Total P concentration was 1.3 mg g21 greater (»36% greater) in the 2003 Aberdeen trial than in the 2004 Aberdeen trial (Table 2), and may be related to greater soil P concentration in 2003. Total grain P concentration in the Tetonia 2004 trial was 2.37 mg g21, »24% less than the Aberdeen 2004 trial. The PAP concentration was 0.75 mg g21 (36%) lower in LPA selections of the Strong lpa family and 0.32 mg g21 (15%) lower in LPA selections of the Moderate lpa family, relative to the WT sibs, in the Aberdeen trials. However, PAP concentration was not significantly affected by LPA genotype in the Tetonia trial. Inorganic P concentration was greatest in LPA selections of the Strong lpa family in all trials. The LPA selections of the Strong lpa family had 2.4 to 3.4 times greater inorganic P concentration than WT selections; LPA selections of the Moderate lpa family had 1.3 to 1.8 times greater inorganic P concentration than WT selections.
Hard White Spring Study In contrast to the HRS study, plants of HWS LPA selections developed at similar rates to HWS WT selections at Aberdeen as indicated by height at 10 June and heading date (Table 3). At Tetonia, HWS LPA selections were taller at maturity than WT selections, a difference not observed at Aberdeen in 2003 or 2004. Also in contrast to the HRS study, HWS LPA selections yielded similar to WT selections in all three trials (Table 3). Test weight of LPA selections was lower than WT selections only in the Aberdeen 2004 trial. However, kernel weight and kernel diameter of LPA selections grown at Aberdeen were 2.0 to 2.5 mg kernel21 and 0.07 to 0.08 mm smaller, respectively, than weights and diameters of WT selections; this effect was not observed at Tetonia, where kernel weight and diameter were smaller. Kernel hardness was not affected by genotype. Total P concentration did not vary as widely among the three trials of the hard white study as among the three trials of the hard red study (Table 4). Among the WT selections, total P concentration ranged from 2.30 mg g21 in Tetonia 2004 to 2.96 mg g21 in Aberdeen 2003. The PAP concentrations were 0.65 mg g21 to 0.41 mg g21 lower (»25% decrease) in LPA selections
than WT selections in every trial. Inorganic P concentration was 2.4 to 3 times greater in LPA selections than WT selections in all trials.
Soft White Spring Study In contrast to the HRS and HWS studies, in both years of the soft white spring study at Aberdeen, the plant development of LPA selections was early relative to WT selections from these families; the Zadoks stage Table 3. Agronomic performance of wild-type (WT) and low phytic acid (LPA) Lolo*3/Js-12 LPA genotypes in the hard white spring wheat study grown at Aberdeen in 2003 and 2004 and at Tetonia in 2004. WT (n 5 5) Height (10 June) 2003 Aberdeen 45 6 1 2004 Aberdeen 58 6 2 Growth stage (10 June), Zadoks scale 2003 Aberdeen 48 6 0 2004 Aberdeen 43 6 1 Heading date, DOY‡ 2003 Aberdeen 167 6 0 2004 Aberdeen 174 6 0 Height at maturity, cm 2003 Aberdeen 109 6 3 2004 Aberdeen 112 6 1 2004 Tetonia 87 6 3 21 Grain yield, kg ha 2003 Aberdeen 6570 6 380 2004 Aberdeen 5730 6 310 2004 Tetonia 2810 6 200 21 Test weight, kg hL 2003 Aberdeen 92.3 6 0.5 2004 Aberdeen 91.8 6 0.3 2004 Tetonia 87.2 6 0.8 Kernel hardness 2003 Aberdeen 68.9 6 1.3 2004 Aberdeen 66.2 6 2.0 2004 Tetonia 56.4 6 1.4 Kenel weight, mg 2003 Aberdeen 40.2 6 0.7 2004 Aberdeen 40.3 6 0.9 2004 Tetonia 34.7 6 0.7 Kernel diam., mm 2003 Aberdeen 2.89 6 0.02 2004 Aberdeen 2.85 6 0.04 2004 Tetonia 2.66 6 0.02 * Contrast significant at P , 0.05. ** Contrast significant at P , 0.01. † ns, nonsignificant. ‡ DOY, day of year.
LPA (n 5 7)
Contrast F (LPA vs. WT)
45 6 1 59 6 1
0.0 ns† 0.2 ns
48 6 0 43 6 1
0.4 ns 0.1 ns
166 6 0 173 6 0
0.4 ns 7.8*
111 6 3 114 6 1 94 6 3
0.4 ns 2.2 ns 5.1*
6470 6 340 5720 6 370 2810 6 180
0.1 ns 0.0 ns 0.0 ns
91.8 6 0.4 90.4 6 0.2 86.4 6 0.8
1.0 ns 14.1** 3.0 ns
70.7 6 1.2 65.7 6 1.7 57.5 6 1.2
1.3 ns 0.0 ns 0.6 ns
38.2 6 0.6 37.9 6 0.8 34.1 6 0.6
4.6 ns 6.9* 0.3 ns
2.82 6 0.02 2.77 6 0.04 2.66 6 0.02
4.5* 4.7* 0.1 ns
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Table 4. Phosphorus distribution in wild-type (WT) and low phytic acid (LPA) Lolo*3/Js-12 LPA genotypes in the hard white spring wheat study grown at Aberdeen in 2003 and 2004 and at Tetonia in 2004. Inorganic P Genotype
2003 Aberdeen
2004 Aberdeen
Phytic acid P 2004 Tetonia
2003 Aberdeen
2004 Aberdeen
Total P 2004 Tetonia
2003 Aberdeen
2004 Aberdeen
2004 Tetonia
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
mg g21 WT (n 5 5) 0.33 6 0.09 0.34 6 0.06 0.19 6 0.04 2.40 6 0.08 1.96 6 0.07 1.68 6 0.05 2.96 6 0.11 2.71 6 0.07 2.30 6 0.12 LPA (n 5 7) 1.00 6 0.07 0.85 6 0.05 0.46 6 0.04 1.75 6 0.07 1.51 6 0.06 1.27 6 0.04 3.37 6 0.09 3.00 6 0.06 2.43 6 0.11 Contrast F (LPA vs. WT) 37.6** 43.5** 33.1** 38.9** 22.8** 40.1** 8.6** 10.1** 2.1 ns† ** Contrast significant at P , 0.01. † ns, nonsignificant.
of LPA selections on 10 June was 2 to 3 stages more advanced than WT selections; and LPA selections headed 2 to 3 d earlier than WT selections (Table 5). At Aberdeen, LPA selections and WT selections had similar height on 10 June and at maturity. But at Tetonia, LPA selections were taller than WT selections at maturity. Grain yield of LPA selections grown at Aberdeen averaged 24 and 20% lower than WT selections in 2003 and 2004, respectively (Table 5). However, at Tetonia the yields of WTand LPA selections were similar. Test weight of LPA grain was 4.1 and 2.5 kg hL21 lower than WT selections in 2003 and 2004 at Aberdeen, respectively. However, test weights at Tetonia in 2004 were similar. In 2004, LPA kernels were 2 mg heavier than WT kernels Table 5. Agronomic performance of wild-type (WT) and low phytic acid (LPA) F4:6 Js-12-LPA/IDO563 genotypes in the soft white spring wheat study grown at Aberdeen in 2003 and 2004 and at Tetonia in 2004. WT lines (n 5 12) Height (10 June) 2003 Aberdeen 47 6 1 2004 Aberdeen 42 6 1 Growth stage (10 June), Zadoks scale 2003 Aberdeen 48 6 1 2004 Aberdeen 40 6 1 Heading date, DOY‡ 2003 Aberdeen 167 6 0 2004 Aberdeen 174 6 1 Height at maturity, cm 2003 Aberdeen 87 6 1 2004 Aberdeen 92 6 2 Grain yield, kg ha21 2003 Aberdeen 6924 6 325 2004 Aberdeen 6363 6 278 2004 Tetonia 2040 6 220 21 Test weight, kg hL 2003 Aberdeen 90.6 6 0.5 2004 Aberdeen 88.9 6 0.4 2004 Tetonia 85.4 6 0.5 Kernel hardness 2003 Aberdeen 45.2 6 1.6 2004 Aberdeen 37.0 6 1.8 2004 Tetonia 39.7 6 1.7 Kenel weight, mg 2003 Aberdeen 34.5 6 0.5 2004 Aberdeen 31.9 6 0.6 2004 Tetonia 27.1 6 1.2 Kernel diam., mm 2003 Aberdeen 2.54 6 0.01 2004 Aberdeen 2.42 6 0.02 2004 Tetonia 2.25 6 0.07 * Contrast significant at P , 0.05. ** Contrast significant at P , 0.01. † ns, nonsignificant. ‡ DOY, day of year.
LPA lines (n 5 10)
Mixed effect ANOVA F value LPA vs. WT
48 6 1 43 6 1
0.4 ns† 0.8 ns
50 6 1 42 6 1
8.9** 6.1*
165 6 0 172 6 1
8.2** 6.2*
88 6 1 95 6 2
0.2 ns 0.7 ns
5281 6 356 4973 6 305 2020 6 230
11.6** 11.4** 0.0 ns
86.5 6 0.6 86.4 6 0.4 85.1 6 0.5
24.5** 21.0** 0.2 ns
46.5 6 1.7 37.3 6 2.0 41.4 6 1.8
0.3 ns 0.0 ns 0.6 ns
35.3 6 0.5 33.6 6 0.6 29.6 6 1.2
1.0 ns 4.6* 14.7**
2.65 6 0.01 2.54 6 0.02 2.40 6 0.07
41.4** 21.9** 20.1**
at Aberdeen and 2.5 mg heavier than WT kernels at Tetonia, but were similar in weight in 2003 at Aberdeen. Interestingly, LPA kernels were 0.11 and 0.13 mm wider in diameter than WT kernels in 2003 and 2004 at Aberdeen, respectively, and 0.15 mm wider at Tetonia. Kernel hardness was not affected by genotype (Table 5). Total P concentration in LPA selections was 13 to 17% greater than in WT selections in all three trials (Table 6). However, total P yield of LPA and WT selections was similar in both years (data not shown). In contrast to results with HRS wheats, total P concentration was similar in both years of the soft white study at Aberdeen. Total P concentration in soft white grain produced at Tetonia was 66% of the concentration in grain produced at Aberdeen. The PAP concentration in LPA selections was 0.80 to 0.38 mg g21 lower (»33% lower) than in WT selections in grain produced at Aberdeen, similar to the Strong lpa family in the hard red spring study. In grain produced at Tetonia, PAP concentrations in LPA selections was 27% lower than in WT selections. Inorganic P concentration in LPA selections grown at Aberdeen was 5.3 and 3.9 times the concentration in WT selections in 2003 and 2004, respectively. Inorganic P concentration of LPA selections grown at Tetonia in 2004 was 3.7 times the concentration in WT selections.
DISCUSSION From previous studies with other LPA crops, we anticipated a number of possible changes in plant phenotype, including reduced seedling emergence, lower grain yield, altered seed anatomy (smaller kernels), and, of course, reduced PA concentration (Ertl et al., 1998; Rutger et al., 2004a, 2004b; Oltmans et al., 2005). We observed no seedling emergence or final stand differences between LPA and WT selections in any of the three genetic backgrounds or at any of the three locations. The changes in plant development in our experiments appeared to be more subtle and varied across populations than in previous reports for LPA crops. Plant growth was either accelerated or delayed, depending on genetic background (soft white and hard red populations, respectively). Similarly, LPA phenotype was associated with increased plant height in two of the three backgrounds (hard white and soft white) in the rain-fed environment, but was unchanged in all backgrounds in the irrigated environment, where we typically expect greater height differences. The interaction of background and LPA phenotype on plant development suggests two pos-
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Table 6. Phosphorus distribution in wild-type (WT) and low phytic acid (LPA) F4:6 Js-12-LPA/IDO563 genotypes in the soft white spring wheat study grown at Aberdeen in 2003 and 2004 and at Tetonia in 2004. Inorganic P
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
Entry WT lines (n 5 12) LPA lines (n 5 10) ANOVA F (LPA vs. WT)
2003 Aberdeen 0.25 6 0.04 1.37 6 0.04 340.9**
2004 Aberdeen 0.26 6 0.03 1.08 6 0.03 372.7**
Phytic acid P 2004 Tetonia 0.16 6 0.03 0.59 6 0.05 91.4**
Total P
2003 Aberdeen
2004 Aberdeen
2004 Tetonia
2003 Aberdeen
2004 Aberdeen
2004 Tetonia
2.45 6 0.08 1.65 6 0.09 42.5**
mg g21 1.98 6 0.05 1.30 6 0.06 75.3**
1.43 6 0.07 1.05 6 0.08 12.3**
2.96 6 0.09 3.48 6 0.09 16.6**
3.12 6 0.06 3.56 6 0.06 28.8**
1.89 6 0.10 2.22 6 0.11 7.8**
** Contrast significant at P , 0.01.
sibilities: (i) the populations in this study differ for genes affecting growth and development that derive from the mutagenized donor, Js-12, for the LPA trait (Guttieri et al., 2004); or (ii) the LPA trait interacts with genetic background in an epistatic manner to alter the rate of development and final plant height. These alternative hypotheses suggest the need for future work, particularly as the LPA trait affects growth and development. Grain yield is the economic summation of a wheat plant’s growth. In these experiments, LPA was associated with yield penalties in two of the three populations in the higher yielding irrigated location, yet no differences in yield were observed in the rain-fed location for any of the three populations. Yield differences in the irrigated Aberdeen site were of similar magnitude to those previously reported for rice (Rutger et al., 2004a, 2004b). Yield reductions in the soft white background can be partly attributed to the earlier heading date (»2 d), which generally is associated with decreased yield, particularly in an early maturing background such as Js-12 LPA/IDO563 (WT lines headed 2 d earlier than WT Grandin lines). The nonuniform yield response suggests the possibility of selecting LPA wheat genotypes that are competitive with WT lines. Our results in wheat differ from those in rice and maize (Rutger et al., 2004a, 2004b; Ertl et al., 1998) possibly due to greater genetic redundancy (polyploidy) and genetic buffering in wheat relative to the other crops. Genetic buffering appears to have limited the expression of LPA in wheat. Previous segregation data suggested that Js-12 carried a minimum of two mutations for LPA and possibly a third. The phenotypes of the backcross families are consistent with the Grandin Moderate family carrying a single mutation as it is intermediate in seed PA concentration between the WT and the Strong family. The LPA C families of Grandin backcross progeny have greater seed PA concentration than the Js-12-LPA line. For example the mean PA concentration of the lpa selections of the Strong lpa family grown in 2003 at Aberdeen was 1.77 mg g21, while the mean PAP concentration of Js-12-LPA was 1.32 mg g21, suggesting that the Strong lpa families may carry multiple LPA genes, but still have fewer than the original LPA source. Despite multiple genes conferring LPA phenotypes, the relative reduction in PA concentration in wheat is modest when compared with reductions observed in single gene mutations of barley and rice, which were 45% (Larson et al., 1998; Rutger et al., 2004a, 2004b). The genetic buffering offers both the potential
to modulate the reduction of PA as well as changes that may be genetically correlated to the LPA trait. Previous research has suggested that environment can modulate PA levels in Glycine spp. (Raboy and Dickinson, 1993), but previous studies with LPA mutants of other crop species have emphasized the effect of genotype over environment. In our study, the effect of the three environments is nearly as great as the effect of genotype, with the PA concentration in the Grandin background having the greatest sensitivity to the effects of environment. We found no evidence for crossover interactions between LPA genotype and environment, but did find the greatest effect of genotype in the environment with the greatest concentration of PA in the WT seed (Aberdeen 2003). In this case, as in previous reports (Raboy and Dickinson, 1993), it is interesting to speculate whether the lower soil P levels of Aberdeen 2004 caused the lower PA concentrations relative to Aberdeen 2003. Raboy and Dickinson (1993) demonstrated that WT soybean genotypes grown in environments with increasing P fertility produced increased PAP and decreased non-PAP. The total P concentration in seed within the soft white background varied little with the environment (Table 6), in contrast to the hard red population where total P averaged for WT in an environment ranged from 2.4 6 0.1 in Tetonia 2004 to 4.1 6 0.1 in Aberdeen 2003. All three populations had differences in their measured seed PA concentration among environments. Taken together, it appears that it may be possible in wheat to manipulate LPA genotype, background genotype, and the environment to lower total P and PAP, as has been previously proposed in maize (Wardyn and Russell, 2004). ACKNOWLEDGMENTS This research was supported by USDA-CSREES National Research Initiative Grant No. 2002-35503-12546, Project No. IDA00204-CG.
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