Published online 20 May 2008
Published in Crop Sci 48:929-932 (2008)
© 2008 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Seed Source Affects Field Emergence of Low-Phytate Soybean Lines
Brian P. Anderson and
Walter R. Fehr*
Dep. of Agronomy, Iowa State Univ., Ames, IA 50011. This journal paper of the Iowa Agric. and Home Econ. Exp. Stn., Ames, IA, Project No. 5103 was supported by the Hatch Act, State of Iowa, Iowa Soybean Association, Raymond F. Baker Center for Plant Breeding, and the United Soybean Board
* Corresponding author (wfehr{at}iastate.edu).
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ABSTRACT
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Seed production in subtropical environments is commonly used by soybean [Glycine max (L.) Merr.] breeders to reduce the number of years for cultivar development. Low-phytate (LP) soybean lines with the mips allele had substantially reduced field emergence when planted with seed from a subtropical source. The objective of this study was to determine if seed source also would impact the field emergence of low-phytate lines with the pha1 and pha2 alleles. Seed of six BC3F4-derived LP lines, the LP donor parent CX1834-1-6 (CX1834), and the normal-phytate (NP) recurrent parent B01769B019 (B019), was harvested from the field at Ames, IA, in 2005 (IA-2005), at Ponce, PR, in January 2007 (PR-Jan), and at Ponce, PR, in May 2007 (PR-May). The three seed sources of the eight lines were evaluated at three Iowa locations in 2007. The mean field emergence of LP lines was 77.6% for the IA-2005 source, 70.1% for the PR-Jan source, and 25.4% for the PR-May source while that of B019 ranged from 80.3 to 82.0% for the three sources. The seed source used to plant lines with the pha1 and pha2 alleles can have a significant influence on their field emergence, which can impact the development and evaluation of LP lines and on seed increases for commercial production.
Abbreviations: B019, B01769B019 • CX1834, CX1834-1-6 • IA-2005, Iowa 2005 harvest • LP, low-phytate • NP, normal-phytate, PR-Jan, Puerto Rico January 2007 harvest • PR-May, Puerto Rico May 2007 harvest
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INTRODUCTION
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PHYTATE (myo-inositol 1,2,3,4,5,6-hexakisphosphate) is the major storage form of phosphorus (P) in normal-phytate (NP) soybean lines (Oltmans et al., 2005). Non-ruminant animals, such as swine and poultry, are unable to fully utilize the P in phytate due to their inability to produce sufficient phytase enzyme (Cromwell et al., 1995). Livestock producers add inorganic P and phytase enzyme to the rations of non-ruminants to meet their mineral requirements (Powers et al., 2006). Phytate also forms salts with divalent cations, such as zinc and iron, rendering them unavailable (Erdman and Poneros-Schneier, 1989). Both phytate P and bound cations are excreted in animal waste, potentially causing negative environmental impact (Daverede et al., 2004).
The four genetic sources of the low-phytate (LP) trait reported in soybean are the mips allele, the combination of the pha1 and pha2 alleles, the Gm-lpa-TW-1 allele, and the Gm-lpa-ZC-2 allele (Sebastian et al., 2000; Oltmans et al., 2004; Yuan et al., 2007). The mips, pha, and Gm-lpa-TW-1 genetic sources of the trait can reduce the field emergence of soybean (Meis et al., 2003; Hulke et al., 2004; Oltmans et al., 2004; Yuan et al., 2007). By backcrossing the pha1 and pha2 alleles from CX1834 into the NP line B019, some LP lines were recovered that had similar field emergence to the recurrent parent when planted with seed harvested in Puerto Rico during January 2005 (Spear and Fehr, 2007). When the LP lines with normal field emergence that they identified were planted at Ames, IA, in 2006 with seed harvested in Puerto Rico during May, all of the LP lines had significantly lower field emergence than the NP recurrent parent. This suggested that the influence of seed source on the field emergence of LP lines with the mips and Gm-lpa-TW-1 alleles also may be a factor in the field emergence of LP lines with the pha1 and pha2 alleles (Meis et al., 2003; Yuan et al., 2007). The objective of this study was to determine if the source of seed used to plant LP lines with the pha1 and pha2 alleles can influence their field emergence.
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MATERIALS AND METHODS
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The six BC3F4-derived LP lines used in this study had normal field emergence when evaluated by Spear and Fehr (2007) in Iowa during 2005. They were developed by crossing the LP line CX1834 to the NP line B019, backcrossing to B019 for three generations, and selecting BC3F4 plants from the population that were homozygous for the pha1 and pha2 alleles. CX1834 was developed by the USDA-ARS and Purdue University, and B019 was developed jointly by Iowa State University and Pioneer Hi-Bred International, Inc.
Seed of the six LP lines, CX1834, and B019 harvested at Ames in 2005 by Spear and Fehr (2007) was used to plant two seed increases near Ponce, PR, at the research station of the Illinois Crop Improvement Association where the soil type is a San Antón sandy clay loam (fine-loamy, mixed, superactive, isohyperthermic Cumulic Haplustoll). One seed increase was planted on 16 Oct. 2006 under natural daylength conditions and harvested in Jan. 2007. The second seed increase was planted on 30 Jan. 2007 under natural daylength conditions and harvested in May 2007. For both increases, each line was planted in a single two-row plot 7.6 m long with a seeding rate of 13 seeds m–1.
In the summer of 2007, the eight lines were planted with the IA-2005, PR-Jan, and PR-May sources as a factorial arrangement in a randomized complete-block design with two replications at Ames, Carlisle, and Lewis, IA. The soil type at Ames is a Nicollet loam (fine-loamy, mixed, mesic Aquic Hapludoll), at Carlisle it is a Tama silty clay loam (fine-silty, mixed, superactive, mesic Typic Agriudoll), and at Lewis it is a Marshall silty clay loam (fine-silty, mixed, mesic Typic Hapludoll). The plots were two rows 3.05 m long with a spacing of 0.69 m between rows within a plot and 1.02 m between rows of adjacent plots. The seeding rate was 30 seeds m–1. The planting dates were 6 June at Ames, 19 May at Carlisle, and 17 May at Lewis.
Seedling emergence percentage was determined for all plots by counting the number of plants, dividing the number by the 200 seeds planted in each plot, and multiplying the quotient by 100. The plants were counted at the V3 stage when there were three nodes on the main stem with fully developed leaves (Fehr and Caviness, 1977).
The data were analyzed as a factorial arrangement of a randomized complete-block design by the generalized linear model procedure of the SAS statistical software (release 9.1.3) (SAS Institute, 2006). The eight lines, three sources, and three locations were considered fixed effects, and replications were considered a random effect. An F-test was used to determine the significance of the main effects.
When the field emergence data indicated that there were substantial differences among seed sources, a warm germination test was conducted to make a visual assessment of seed germination from the seed sources. The test was conducted in the Seed Science Center of Iowa State University at Ames with two replications of the eight entries. Two sheets of 19-ply Kimpak (Neenah, WI) were moistened with 825 mL of water and placed on a fiberglass food service tray that measured 45 x 66 cm. Four random entries were planted with 100 seeds each on a tray. The two trays for each replication were placed in a germination cart 0.5 m wide x 0.7 m deep x 1.6 m high. The cart was made of aluminum, except for the rear panel that was made of Plexiglass to permit light from a wall of fluorescent lamps into the cart. The two carts were placed in a growth room at 25°C for 7 d. The classification of seeds for germination was based on standards provided by the Association of Official Seed Analysts (AOSA, 2005).
The inorganic P and phytic acid P contents of the seed of the eight lines from the three sources were determined. A random sample of 50 seeds of each entry from each source was ground to pass through a 1-mm screen using a UDY Cyclone sample mill (UDY Corporation, Fort Collins, CO). Inorganic P was determined on two replications by a modification of the technique described by Chen et al. (1956). Two replications of 0.5 g of ground seed were independently extracted in 20 mL of 12% trichloroacetic acid that contained 2.6 mM magnesium chloride. The samples were stirred overnight at 4°C. Following extraction, the samples were centrifuged at 14000 x g for 20 min. A volume of 100 µL of the solution was added to 3.9 mL of ddH20 and 4 mL of Chen's reagent was added to the solution. Samples were allowed to react for 2 h at room temperature before they were analyzed at 820 nm on a Varian Cary 50 Bio UV-Visable spectrophotometer (Palo Alto, CA).
Phytic acid P was determined by capillary zone electrophoresis (CZE) as developed by Nardi et al. (1992) and described in detail by Spear and Fehr (2007). A 20-mg sample of ground seed was placed into a scintillation vial and extracted in 10 mL of 0.5 mM L-aspartic acid. The solution was stirred at room temperature for 20 min using a magnetic stir plate. Following extraction, a 750-µL sample was placed in a 0.22 µm Spin-X centrifugal filter (Costar Corning, NY), and centrifuged with a benchtop microfuge for 10 min. A 250-µL aliquot of the filtered sample was loaded on a 96-well plate and analyzed with a Beckman-Coulter P/ACE MDQ capillary electrophoresis system (Fullerton, CA).
The phytate P contents of soybean as measured by CZE are approximately 10-fold less than the values reported for high-performance liquid chromatography (HPLC) analysis (Wilcox et al., 2000; Spear and Fehr, 2007). The difference is due at least in part to the acid used to extract phytate P for the two methods. The phytate P for CZE is extracted by L-aspartic acid, which is a weaker acid than the HCl used for extraction of phytate P for HPLC. As a result, less of the phytate P in the seed is solubilized in the CZE procedure. Although the absolute values for phytate P differ for CZE and HPLC, the relative differences between LP and NP lines are similar for the two methods.
The data from the warm germination, inorganic P, and phytic acid P tests were analyzed as a factorial arrangement in a randomized complete-block design. Genotypes and sources were considered fixed effects and replications were considered a random effect. An F-test was used to determine the significance of the main effects.
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RESULTS AND DISCUSSION
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Significant differences in mean field emergence were observed among the three seed sources (Table 1
). The mean field emergence of all lines was 75.4% for IA-2005, 65.3% for PR-Jan, and 37.8% for PR-May. The differences among sources were due to the major reduction in field emergence of the six A05 lines from the PR-May harvest. In contrast, the emergence of the NP parent B019 was greater than 80% for the three sources. The six LP lines had significantly better emergence than CX1834 when grown from seed harvested from IA-2005 and PR-Jan, but none of the A05 lines were significantly different than CX1834 when grown from the PR-May source. The poor field emergence of the LP lines harvested in PR-May was consistent with the poor field emergence of LP lines and CX1834 harvested from the same location in May 2006 that led to this study.
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Table 1. Entry means for agronomic and seed traits of six low-phytate lines, the low-phytate donor line, and the normal-phytate recurrent parent from three sources.
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The number of seeds of the LP lines with normal growth in the warm germination test was significantly less for PR-May than for the other two sources (Table 2
). In contrast, the NP parent B019 had a higher number of normal seeds for PR-May than the other two sources. Spear and Fehr (unpublished data, 2006) found in a warm germination test that CX1834 had 14% units fewer normal seeds compared with B019 for seed from a PR-Jan harvest in 2004 compared with 61% units fewer normal seeds than B019 for seed from a PR-May harvest in 2005. The reduced frequency of seeds of the LP lines with normal growth in our study was associated with the same infection of seed storage fungi reported by Spear and Fehr (2007) for CX1834 in their warm germination test. Our results support their suggestion that susceptibility of LP lines to seed storage fungi may be associated with their reduced field emergence.
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Table 2. Mean germination percentages from a warm germination test of six low-phytate lines, the low-phytate donor line, and the normal-phytate recurrent parent from three sources.
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The decreased emergence of LP lines from PR-May could not be attributed to differences in the harvest procedures because the seed from all sources was harvested promptly after maturity when the plants were dry enough to thresh. One environmental factor that merits consideration in exploring the cause of the decreased emergence is the temperature during seed fill, which commonly is considered to be the 30 d before physiological maturity. The average daily high temperature during seed fill was 32.4°C for PR-May, 31.1°C for PR-Jan, and 27.9°C for IA-2005. There were 18 d in PR-May, 3 d in PR-Jan, and 1 d in IA-2005 when the high daily temperature during seed fill was 32.2°C or greater. The relative humidity for PR-May was less than that of IA-2005. During seed fill, the average high relative humidity was 86.8% in IA-2005, 74.6% in PR-Jan, and 76.3% in PR-May.
The phytate P and inorganic P content of the A05 lines was significantly different for the three sources (Table 1). The mean phytate P content of the six A05 lines was 0.18 mg g–1 for IA-2005, 0.41 mg g–1 for PR-Jan, and 0.61 mg g–1 for PR-May, while their mean inorganic P content was 2.36 mg g–1 for IA-2005, 3.97 mg g–1 for PR-Jan, and 3.92 mg g–1 for PR-May. It is not known if the greater phytate P in seed from PR-May could be associated with the decreased emergence of that source. The results indicated that the environment of seed production can have a major influence on the phytate P and inorganic P content of seed from LP lines with the pha1 and pha2 alleles. Based on the research of Israel et al. (2007), differences in the P content of the soils of the three seed production environments probably would not be responsible for the significant differences in phytate P among seed sources observed in our study. Additional research will be needed to determine the factors that are responsible for causing variation in phytate P among seed sources.
The results have important implications for the choice of environments that are most suitable for breeding low-phytate cultivars with the pha1 and pha2 alleles and for commercial production of their seed. It is common in soybean breeding programs to grow segregating populations and seed increases in subtropical environments during the winter in North America. If segregating populations are grown in environments comparable to PR-May, there could be a substantial decrease in the frequency of the LP segregates that would be recovered in subsequent generations. The PR-May type environments also would not be useful for obtaining seed of experimental LP lines for evaluation of field emergence when the goal is to develop LP lines that would have normal field emergence when grown from a source comparable to IA-2005 or PR-Jan.
The field emergence of the A05 lines when grown from the IA-2005 source was similar to that reported by Spear and Fehr (2007). Our study supports their results that indicated it is possible to develop LP lines with field emergence similar to NP lines, but only when they are grown from appropriate seed sources. The two studies indicate that it may be possible to grow LP cultivars with adequate field emergence as long as the seed for commercial plantings is produced in temperate climates and seed lots are adequately tested for germination before planting. Additional research will be needed to determine the range of environments in which seed of LP cultivars can be produced successfully, and the types of germination tests that should be used to identify acceptable seed lots (Meis et al., 2003; Spear and Fehr, 2007).
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ACKNOWLEDGMENTS
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The authors thank Grace A. Welke, Susan L. Johnson, Kevin O. Scholbrock, Raechel M. Baumgartner, Loren A. Trimble, and Joel D. Nott for their assistance conducting the field and laboratory tests.
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NOTES
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All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication December 4, 2007.
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ABSTRACT
INTRODUCTION
