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a Pioneer Hi-Bred International, Inc., P.O. Box 177, Johnston, IA 50131-0177
b Pioneer Hi-Bred International, Inc., P.O. Box 1004, Johnston, IA 50131-1004
c Dep. of Agronomy, Iowa State University, Ames, IA 50011-1010
* Corresponding author (wfehr{at}iastate.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Accessions in the USDA soybean germplasm collection have been screened by polyacrylamide gel electrophoresis for the presence or absence of electrophoretic forms of soybean trypsin inhibitors (SBTI). Five electrophoretic forms have been discovered (Hymowitz and Hadley, 1972; Orf and Hymowitz, 1977; Orf and Hymowitz, 1979; Singh et al., 1969; Quigyan et al., 1995). The genetic control of four forms, Tia, Tib, Tic, and Tid, has been reported as a codominant multiple allelic series at a single locus (Singh et al., 1969; Hymowitz and Hadley, 1972; Orf and Hymowitz, 1977; Quigyan et al., 1995). Orf and Hymowitz (1979) found that the fifth form does not exhibit a SBTI-A2 band and is inherited as a recessive allele designated ti. They also found that crude seed protein from seeds that lacked the SBTI-A2 band had a 30 to 50% reduction in trypsin inhibitor (TI) activity compared with ‘Amsoy 71’ that has the SBTI-A2 band.
Scientists at Pioneer Hi-Bred International, Inc. observed that the introduction into soybean of a gene from the Brazil nut tree [Bertholletia excelsa (Castanheira) Humb. & Bonpl.] encoding higher levels of methionine-rich 2S seed storage protein resulted in a reduction in TI and CI (Beach et al., 1995). One objective of this study was to determine if TI and CI activity could be further reduced by combining the Brazil nut protein (BNP) gene and the ti allele for KTI. The second objective was to evaluate the effect of the BNP gene and the ti allele on agronomic traits of soybean.
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MATERIALS AND METHODS |
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The single cross of BX4P9341B6 and Kunitz was made at Johnston, IA, in July 1993. F1 seeds were planted in Salinas, PR, in November 1993. In February 1994, F2 plants were grown in Puerto Rico under lights to extend the day length to obtain enough seeds for testing. A total of 400 F2 plants were harvested and threshed individually in May 1994. In May 1994, 35 seeds from each of the 400 F2:3 lines, 9341, BX4P9341B6, and Kunitz were planted in single-row plots 1.5 m long and spaced 77 cm apart at Johnston, IA. At maturity, each plot was harvested separately with a plot combine.
Experimental Procedures
An indirect enzyme-linked immunosorbent assay (ELISA) was used to assess accumulation of BNP in seeds and segregation of the BNP gene in 400 F2-derived lines. Eleven individual whole seeds from each line were placed in separate wells of 48-well Falcon microplates (BD Immunocytometry Systems, San Jose, CA). Samples of positive (BX6P9341B6) and negative (9341) control seeds also were present in each plate. The seeds in each well were crushed with a handmade 48-position pestle and plate support apparatus. An aliquot of 250 mL of assay buffer (5 mM Tris-HCL; 5 mM Trizma base; 0.5 M NaCl, 0.5% [v/v] Tween-20) was added to each well, and the plates were incubated at room temperature for 1 h to permit binding of soybean proteins to the wells. Soybean particulate matter and buffer were removed by inverting and rapidly shaking each plate. Plates were washed three times with assay buffer by filling each well for each rinse and removing buffer by inversion and shaking. This procedure was followed for all washes noted below. Subsequent nonspecific protein binding to wells was blocked by filling each well with assay buffer and incubating plates at room temperature for 1 h. The plates were washed. An aliquot of 250 mL of assay buffer containing polyclonal rabbit anti-BNP antibody at 7.2 mg mL-1 was added to each well and plates were incubated for an additional 30 min at room temperature, after which the plates were washed. The polyclonal rabbit anti-BNP antibody is specific to BNP protein and does not cross-react with any soybean seed protein. An aliquot of 250 mL of assay buffer containing alkaline phosphate-conjugated goat anti-rabbit antibody was added to each well. The plates were incubated at room temperature for 30 min, after which plates were washed. For the assay, 1 mg mL-1 p-nitrophenyl phosphate in 10% (v/v) diethanolamine: 0.5 mM MgCl2 was added to each well, and the plates were incubated at room temperature for at least 30 min. A yellow color indicated the presence of BNP and a clear color indicated absence of BNP.
For determination of segregation among F4 seeds from the 400 F2-derived lines, 11 seeds from each line were analyzed and results were scored. By analyzing 11 seeds, there was a 95% probability that lines would be accurately classified as homogeneous BNP+ BNP+ or BNP- BNP- (Sedcole, 1977). Lines with 10 to 11 BNP+ seeds were scored as homogeneous BNP+ BNP+, lines with 2 to 9 BNP+ seeds were scored as heterogeneous for BNP+ BNP-, and lines with 0 to 1 BNP+ seeds were scored as homogeneous for BNP- BNP-.
On the basis of the results of the BNP test, homogeneous lines for BNP+ or BNP- were evaluated to identify those that were homogeneous for the Ti and ti alleles. Eleven individual whole seeds of each line were evaluated individually by the rapid test for KTI activity in soybean seeds described by Hildebrand and Hymowitz (1980). For the lab conditions used, the amount of trypsin added to each culture tube was reduced from 100 to 75 mL for lines testing normal for BNP and to 20 mL for lines testing positive for BNP. Individual lines also were evaluated by polyacrylamide disc gel electrophoresis to verify the results of the rapid test.
There were 42 lines selected for field evaluation during the summer of 1995. Six lines were homogeneous for presence of the BNP gene and the Ti allele (BNP+, Ti), 18 lines homogeneous for presence of the BNP gene and the ti allele (BNP+, ti), six lines homogeneous for absence of the BNP gene and presence of the Ti allele (BNP-, Ti), and 12 lines homogeneous for absence of the BNP gene and presence of the ti allele (BNP-, ti). The 42 F2:5 lines, 9341, Kunitz, and BX4P9341B6 were planted in two replications of a randomized complete-block design at three locations: Johnston and Sheldahl, IA, and Salinas, PR. The soil type at Johnston is a Waukegan loam (fine-loamy over sandy or sandy skeletal, mixed, superactive, mesic Typic Hapludoll), at Sheldahl is a Nicollet loam (fine-loamy, mixed, superactive, mesic Aquic Hapludoll), and at Salinas is a Guamani silty clay loam (fine-loamy over sandy or sandy skeletal, mixed, isohyperthermic, Flurentic Ustropepts). The plots were single rows 1.5 m long with 77 cm between rows. The planting rate was 28 seeds m-1 of row.
Seed yield, lodging score, maturity date, and plant height were determined for each plot at Johnston and Sheldahl. Seed yield expressed in kilograms per hectare was measured as the weight of the harvested sample that had been dried artificially at 40°C for 2 d before weighing. Maturity was measured as days after 31 August when 95% of the pods had reached their mature color. Plant height was the distance in centimeters from the soil surface to the terminal node with a pod on the main stem. Lodging score was rated on a scale of 1 to 9, with 9 representing all plants erect and 1 all plants prostrate. Agronomic traits were not measured in Puerto Rico because the lines were not adapted to the location; however, each plot was harvested to evaluate protein inhibitor content of the seed. A 50-seed sample from all the plots was ground in a coffee grinder, and the meal was analyzed for TI and CI activity by the procedure outlined by Kollipara and Hymowitz (1992). TI and CI activity were reported as the number of units inhibited per gram of tissue.
Statistical Analyses
Analyses of variance were performed by the general linear model procedure of the SAS software package (release 6.11) (SAS Institute, 1992). Environments and replications within environments were considered random effects and entries were considered fixed effects. The variation among lines and the comparisons among the means of the four genotypic classes were evaluated by F tests. The entries mean squares were evaluated with their appropriate entries x environments interaction mean squares. Phenotypic correlation coefficients were computed for all traits on the entry means basis across locations with the procedure (CORR) of SAS (SAS Institute, 1992).
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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There was a significant reduction in the mean chymotrypsin inhibitor units (CIU) in seeds expressing the BNP gene (Table 1). The mean of BNP+ lines was 31 CIU less than BNP- lines when the Ti allele was present and 43 CIU less when the ti allele was present. The average reduction of 37 CIU associated with the BNP gene was 52.9% of the mean CIU of the lines with the BNP-, Ti genotype of conventional soybean. The ti allele was not associated with any reduction in CIU when the BNP gene was absent, but there was a significant reduction of 12 CIU when the BNP gene was present. The combination of the BNP gene and ti allele had a mean reduction of 43 CIU or 61.4% compared with conventional soybean.
The significant phenotypic correlation coefficient between TIU and CIU of 0.92 indicated the development of both the TI and CI was influenced by expression of BNP (Table 2). The positive correlation would be desirable for developing cultivars with a reduction in total protease inhibitors. The protease inhibitors are rich in sulfur containing amino acids and expression of BNP may act to shunt the amino acids from both TI and CI to BNP. This mechanism of reducing the TIU and CIU would likely be effective with high levels of expression of other high methionine proteins.
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The mean seed yield, maturity, lodging, and protein content of the four genotypic classes were not significantly different (Table 1). There were significant differences among the genotypic classes for height, but the range was only 6 cm. The mean oil content of the BNP+ lines was significantly greater than for the BNP- lines by 
10 g kg-1. The phenotypic correlations of TIU and CIU with the agronomic traits were not significant, but there was a significant negative correlation with oil content (Table 2). The results suggested that the BNP gene and ti allele did not have a negative impact on the agronomic and seed traits and that it should be possible to develop high-yielding cultivars that have reduced TIU and CIU.
Research is needed to determine if this level of reduction in the protease inhibitors would reduce or eliminate the need for heat treatment of soybean for use in animal feed. The increase in methionine with the BNP gene and the reduction or elimination of heat treatment would be desirable improvements in soybean. There will not be any commercial cultivars containing BNP because the protein is likely to be an allergen (Nordlee et al., 1996). Cultivars expressing other proteins high in sulfur-containing amino acids are likely to have both increased methionine content and a reduced or eliminated need for heat treatment of the meal.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication May 24, 2000.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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