Published in Crop Sci. 44:772-776 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
CROP BREEDING, GENETICS & CYTOLOGY
Tocopherol Content of Soybean Lines with Reduced Linolenate in the Seed Oil
Kristen L. McCorda,
Walter R. Fehr*,a,
Tong Wanga,
Grace A. Welkea,
Silvia R. Cianzioa and
Steven R. Schneblyb
a Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011
b Pioneer Hi-Bred International, P.O. Box 177, Johnston, IA 50131-0177
* Corresponding author (wfehr{at}iastate.edu).
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ABSTRACT
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Soybean [Glycine max (L.) Merr.] oil is an important source of the tocopherols (vitamin E) used as a dietary supplement and as an antioxidant in foods. Previous research studies have indicated that genotypes with lower linolenate than that of conventional soybean genotypes have less tocopherols and that the relative amounts of 
- and 
-tocopherol may change as linolenate content is reduced by genetic modification. Those studies have utilized a limited number of genotypes with diverse genetic backgrounds and with greater linolenate content than that of currently available genotypes. The purpose of our study was to evaluate the tocopherol content of 20 lines with reduced linolenate of about 10 g kg–1 and 20 lines with normal linolenate of about 70 g kg–1 derived from each of three single-cross populations segregating for the trait. The lines were grown in replicated tests at Isabela, PR, and at two locations near Ames, IA, during 2002. The mean total tocopherol content of the lines with reduced linolenate was 6.0% less than for the normal-linolenate lines averaged across populations and environments. There was significant variation for total tocopherol among the lines of each type in each population, and some reduced-linolenate lines were not significantly different from normal-linolenate lines for the trait. Lower total tocopherol in reduced-linolenate lines was due to a proportionate decrease in , , and 
tocopherol, and the mean percentages of the three tocopherols in the total tocopherol were not significantly different in the reduced- and normal-linolenate lines. It should be possible to develop cultivars with about 10 g kg–1 linolenate that have an acceptable content of total tocopherol and with the same proportions of , , and tocopherol as conventional soybean cultivars.
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INTRODUCTION
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THE LINOLENATE CONTENT of soybean oil has been reduced from about 70 g kg–1 in conventional cultivars to about 10 g kg–1 in a cultivar that was released in 2003 for commercial production. The reduction in linolenate was achieved by combining three independent mutations for reduced linolenate: fan1(A5), fan2, and fan3 (Fehr and Hammond, 1998; Ross et al., 2000).
Soybean oil is a primary source of tocopherols (Vitamin E), which are important for human health (Stone and Papas, 2003). Previous research has indicated that soybean genotypes with reduced linolenate have lower total tocopherol. Almonor et al. (1998) evaluated the tocopherol content of one conventional cultivar with normal linolenate and one plant introduction and two breeding lines with 31 to 42 g kg–1 when grown at 27.5°C. They reported that lower linolenate content was associated with lower total tocopherol, a lower percentage of tocopherol, and a greater percentage of tocopherol than conventional soybean oil. Dolde et al. (1999) studied soybean lines and cultivars with diverse genetic backgrounds that ranged in linolenate from 26 to 112 g kg–1. They indicated that linolenate content was positively correlated with total, , and tocopherol. All the previous studies used a limited number of genotypes with diverse genetic backgrounds. This makes it difficult to determine if the observed relationships between linolenate and tocopherol were associated with linolenate content or with the genetic backgrounds of the genotypes that were studied. The linolenate contents of the genotypes they evaluated were not as low as the linolenate content of genotypes currently available. The objective of our study was to evaluate the tocopherol content of soybean lines with reduced linolenate of about 10 g kg–1 and normal linolenate of about 70 g kg–1 derived from each of three populations.
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MATERIALS AND METHODS
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Three soybean populations developed by Pioneer Hi-Bred International, Inc. (Pioneer) were used for the study. Population (Pop.) 1 was from the cross of A97-553017 with CVL 1, Pop. 2 from the cross of A97-553010 with CVL 2, and Pop. 3 from the cross of A97-553018 with CVL 3. CVL 1, CVL 2, and CVL 3 were high-yielding lines of maturity group III from Pioneer with normal-linolenate content. A97-553017, A97-553010, and A97-553018 are lines of maturity group II with about 10 g kg–1 linolenate developed cooperatively by Pioneer and the soybean breeding project at Iowa State University.
The F1 seeds of the three crosses were produced at Johnston, IA, by Pioneer during the summer of 2000. The F1 seeds of the populations were planted during October 2000 in the Pioneer nursery at Salinas, PR, and F2 seeds were harvested in bulk. The F2 seeds for each population were planted during January 2001 at Salinas. Several pods were harvested from each plant and the pods from all the plants of a population were threshed together in bulk. The F3 seeds for each population were planted during May 2001 at the Pioneer nursery at Johnston and individual plants were harvested and threshed separately. A five-seed bulk from each of 693 F3 plants in Pop. 1, 682 plants in Pop. 2, and 653 plants in Pop. 3 was analyzed by gas chromatography as described by Hammond (1991). The 25 plants in each population with the highest linolenate and the 25 with the lowest linolenate were selected for the study.
The 50 F3–derived lines of each population were grown as separate experiments. Each experiment was grown as a randomized complete-block design with two replications at three environments during 2002. One environment was planted during January 2002 under natural daylength conditions at the Iowa State University-University of Puerto Rico soybean nursery at Isabela, PR. The soil type is a Coto clay (very-fine, kaolinitic, isohyperthermic Typic Eutrustox). The single-row plots were 0.3 m long with a 1-m spacing between rows. The seeding rate was eight seeds in a plot. The plants in each plot were harvested in bulk with a stationary plot thresher. Seeds harvested in Puerto Rico were used to plant the experiments near Ames, IA, at the Agronomy Farm and Burkey Farm of Iowa State University. The soil type at both locations is a Nicollet loam (fine-loamy, mixed, superactive, mesic Aquic Hapludoll). Single-row plots 0.76 m long with a row spacing of 1.02 m were planted with 20 seeds at each location. The plots were harvested with a self-propelled plot combine.
The resources available for tocopherol analysis by high performance liquid chromatography (HPLC) were not adequate to obtain data from the 50 lines in each population. To reduce the number of lines, a five-seed bulk from each plot was analyzed for fatty ester content of palmitate, stearate, oleate, linoleate, and linolenate. The 20 lines with the highest and the lowest linolenate of each population were chosen for further analysis.
The lines from each replication of an experiment were analyzed for tocopherol and fatty ester content in the same random order as the field plots. Thirty random seeds of each plot were used for analysis. Six of the 30 seeds were placed in each of five wells of a metal plate and subjected to 2.76 x 105 kPa pressure with a hydraulic press. After the seeds were crushed, 2 mL of hexane were added to each well and allowed to soak for 6 h. A 0.3-mL sample of the hexane-oil mixture was removed from each well and the samples from the five wells of the same plot were pooled in a 1.5-mL glass vial that had been weighed. The samples were placed under a chemical hood at ambient temperature for 8 to 12 h and transferred to a vacuum oven (National Appliance Company, Portland, OR) equipped with a model 1400 Welch Duo-Seal vacuum pump (Sargent Welch Scientific Co., Skokie, IL) for solvent evaporation for 2 h at ambient temperature. The weight of oil in each vial was determined. The samples were redissolved in HPLC-grade hexane (Fisher Scientific, Fair Lawn, NJ) to a total volume that was level with the vial neck, averaging 1.8109 mL of hexane-oil mixture for each sample. A 15- or 20-µL aliquot of each sample was injected. Each replication within a population was analyzed in the same injection volume. External standards with known quantities of , , and tocopherol (Sigma-Aldrich, Inc., St. Louis, MO) as determined by spectrophotometry were used with each replication with the same injection volume as the analyzed samples. The results for the standards were used to calculate the content of individual tocopherols on the basis of the AOCS Official Method Ce 8-89 (1993). Samples were eluted with 1% (v/v) isopropanol in HPLC-grade hexane at a 0.3 mL min–1 flow rate with a Beckman Coulter System Gold module 126 solvent delivery system, 508 autosampler, and 168 UV detector (Beckman Coulter, Inc., Fullerton, CA) at a wavelength of 292 nm as specified by the AOCS Official Method Ce 8-89 (1993). The column was a 250 mm by 2.1 mm Alltech Solvent Miser Silica (Alltech Associates, Inc., Deerfield, IL) with 5-µm particle size. The integration of ß tocopherol, a minor constituent of total tocopherol, was included in the -tocopherol component because of insufficient separation in the analysis. The tocopherol data were expressed in mg kg–1. The contents of -, -, and -tocopherol also were determined as a percentage of total tocopherol.
Fatty ester content of each sample was determined by gas chromatography from a subsample of the oil used for HPLC analysis. The amount of each fatty ester was determined as a normalized percentage of the five major fatty esters. The percentages were converted to g kg–1 by multiplying times 10.
The fatty ester and tocopherol data were analyzed as a randomized complete-block design with the general linear model procedure of SAS version 8.01 (SAS Institute, 2001). Environments and replications were considered random effects. The reduced- and normal-linolenate types were considered a fixed effect with one degree of freedom in the analysis of variance. Lines within the reduced- and normal-linolenate types were considered random effects because there was no prior knowledge of the tocopherol content of the lines. The sums of squares for lines were partitioned into among reduced- and among normal-linolenate lines. The environment x type and environment x lines within type interactions were used to evaluate the significance of the appropriate main effects in the F-tests. Line mean correlations among traits were computed based on means averaged across environments, and correlations among environments were computed based on line means for each environment. The correlations were computed by the correlation procedure of SAS version 8.01 (SAS Institute, 2001). Broad-sense heritabilities on a plot and entry-mean basis were computed based on the appropriate variance components derived from the combined analysis of variance across environments, as described by Hallauer and Miranda (1981).
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RESULTS AND DISCUSSION
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The mean linolenate contents of the reduced-linolenate lines (reduced lines) and the normal-linolenate lines (normal lines) were significantly different in the three populations (Table 1). There were significant differences among lines within each type, except for the reduced lines in Pop. 1 (Table 1). Ross et al. (2000) observed similar variation among reduced-linolenate lines in three soybean populations and attributed it to modifying genes that influence the trait.
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Table 1. Mean and range for tocopherol and fatty ester composition of 20 reduced- and 20 normal-linolenate lines from three populations averaged across three environments in 2002.
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The reduced lines had a lower total tocopherol content than the normal lines of 72 mg kg–1 in Pop. 1, 74 mg kg–1 in Pop. 2, and 90 mg kg–1 in Pop. 3 (Table 1). The lower total tocopherol of the reduced lines was significant for every population at each of the three individual environments. In the combined analysis across environments, the difference between types was not significant in Pop. 3. In that population, the total tocopherol of the reduced lines was lower by 60 mg kg–1 at Isabela, 153 mg kg–1 at the Agronomy Farm, and 57 mg kg–1 at the Burkey Farm. This variation among the environments resulted in an environment x type interaction mean square that was sufficiently large to make the mean difference between the two types not significant. The same interaction was not significant for total tocopherol in the other two populations due to more consistent differences between the reduced and normal lines among environments.
Although the reduced lines had a lower mean total tocopherol than the normal lines, there was overlap in the distributions among lines for the two types (Table 1). This overlap indicated that it should be possible to select reduced lines with similar total tocopherol to some conventional cultivars. Dolde et al. (1999) evaluated the tocopherol content of three soybean lines with linolenate contents of <30 g kg–1 and 14 conventional cultivars. One of the three reduced-linolenate lines had greater total tocopherol than three of the conventional cultivars.
The lower mean total tocopherol content of the reduced lines compared to the normal lines was consistent with the lower total tocopherol reported by Almonor et al. (1998) and Dolde et al. (1999) in lines with reduced linolenate. Although Dolde et al. (1999) observed a positive association between linolenate and total tocopherol for genotypes ranging from 26 to 112 g kg–1 linolenate, they did not have an explanation for the positive correlation between linolenate and total tocopherol obtained for their conventional soybean cultivars that ranged in linolenate from 67 to 95 g kg–1. They suggested that the relationship between the two traits should be further investigated using lines with similar genetic backgrounds. The positive association between linolenate and total tocopherol they observed among the conventional cultivars of diverse genetic backgrounds was not confirmed in our study with normal lines from the same population (Table 2). The line mean correlations between the two traits among the 20 normal lines in the three populations were all negative and not significant. The range in linolenate among lines in our study was less than that evaluated by Dolde et al. (1999), which may partially account for the difference in results of the two studies.
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Table 2. Line mean correlations between linolenate and tocopherol contents of 20 reduced-linolenate, 20 normal-linolenate, and all lines in each of three soybean populations.
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The reduction in total tocopherol of the reduced lines was associated with a decrease in , , and tocopherol (Table 1). For each of the tocopherols, the significance of the main effect of types in the combined analysis across environments was dependent on the significance of the environment x type interaction. The main effect of types was not significant whenever the interaction was significant because of variation in the magnitude of the differences between the two types across environments.
The mean percentages of , , and tocopherol in the total tocopherol were not significantly different between the reduced and normal lines, which indicated that the changes in total tocopherol content were due to proportional changes in the three tocopherols (Table 1). The , , , and total tocopherol of the 40 lines in each population were positively correlated with each other (Table 2). Our results did not support the conclusion of Almonor et al. (1998) that cultivars with reduced linolenate should be expected to have proportionately more tocopherol and less tocopherol than conventional cultivars. Their results were based on the evaluation of only four soybean genotypes that ranged in linolenate from 31 g kg–1 to 82 g kg–1 when grown at 27.5°C. In our study, there were significant differences among the lines within each type for the percentages of , , and tocopherol in the total tocopherol (Table 1). By evaluating only four genotypes, Almonor et al. (1998) did not account for this variation and could not adequately evaluate the average changes in , , and tocopherol for lines with different linolenate contents.
The significant variation in tocopherol among lines within the reduced and normal types should make it possible to select for the trait in a cultivar development program (Table 1). The genotype x environment interactions for , , , and total tocopherol were only significant for tocopherol in Pop. 2 and total tocopherol in Pop. 3. The consistency of performance of lines in different environments should make it possible to limit the number of environments used for tocopherol evaluation (Table 3). The correlation coefficients between Isabela, PR, and the two Iowa locations generally were as large as the correlations between the two Iowa locations. It should be possible to evaluate lines for tocopherol with seed produced in the subtropical environment. The broad-sense heritability estimates for , , , and total tocopherol indicated that selection for the trait should be effective, particularly when lines are selected based on entry means from tests conducted in multiple replications, environments, or both (Table 4).
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Table 3. Line mean correlations among three environments for tocopherol contents of 20 reduced-linolenate lines, 20 normal-linolenate lines, and all lines from each of three soybean populations.
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Selection for increased total tocopherol in a breeding program will increase the contents of , , and tocopherol. There may be limited opportunity to select for the proportion of , , or tocopherol among lines in a population. The range among lines in the three populations for the percentages of the three tocopherols in the total tocopherol was a maximum of 5.4% units for tocopherol, 8.4% units for tocopherol, and 8.5% units for tocopherol (Table 1). The limited range is due to the positive correlations among the three traits (Table 2). Selection for one of the tocopherols generally increases the contents of the other two tocopherols.
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NOTES
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Project No. 3732 and supported by the Hatch Act, State of Iowa, Raymond F. Baker Center for Plant Breeding, Iowa Soybean Promotion Board, United Soybean Board, and Pioneer Hi-Bred International.
Received for publication August 4, 2003.
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REFERENCES
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- Almonor, G.O., G.P. Fenner, and R.F. Wilson. 1998. Temperature effects on tocopherol composition in soybeans with genetically improved oil quality. J. Am. Oil Chem. Soc. 75:591–596.
- American Oil Chemists' Society. 1993. Determination of tocopherols and tocotrienols in vegetable oils and fats by HPLC. Uniform Methods Committee. AOCS Official Method Ce 8–89. AOCS, Champaign, IL.
- Dolde, D., C. Vlahakis, and J. Hazebroek. 1999. Tocopherols in breeding lines and effects of planting location, fatty acid composition, and temperature during development. J. Am. Oil Chem. Soc. 76:349–355.
- Fehr, W.R., and E.G. Hammond. 1998. Reduced linolenic acid production in soybeans. U.S. Patent 5850030. Date issued: 15 Dec.
- Hallauer, A.R., and J.B. Miranda. FO. 1981. Quantitative genetics in maize breeding. p. 45–113. Iowa State University Press, Ames, IA.
- Hammond, E.G. 1991. Organization of rapid analysis of lipids in many individual plants. p. 321–330. In H.F. Linskens and J.F. Jackson (ed.) Modern methods of plant analysis. Vol. 12. Springer-Verlag, Berlin.
- Ross, A., W.R. Fehr, G.A. Welke, and S.R. Cianzio. 2000. Agronomic and seed traits of 1%-linolenate soybean genotypes. Crop Sci. 40:383–386.[Abstract/Free Full Text]
- SAS Institute. 2001. The SAS system for Windows. Release 8.01. SAS Inst., Cary, NC.
- Stone, W.L., and A. Papas. 2003. Tocopherols, tocotrienols, and vitamin E. p. 53–72. In F.D. Gunstone (ed.) Lipids for functional foods and nutraceuticals. PJ Barnes & Associates, Bridgwater, England.
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ABSTRACT
INTRODUCTION
