Published online 8 September 2006
Published in Crop Sci 46:2069-2075 (2006)
© 2006 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
CROP BREEDING & GENETICS
Stability of Fatty Acid Profile in Soybean Genotypes with Modified Seed Oil Composition
M. L. Olivaa,
J. G. Shannonb,*,
D. A. Sleperc,
M. R. Ellersieckd,
A. J. Cardinale,
R. L. Parisf and
J. D. Leeb
a Nidera S.A., Ruta 8 Km 376, 2600, Venado Tuerto, Argentina
b Univ. of Missouri-Delta Center, P.O. Box 160, Portageville, MO 63873
c Division of Plant Sciences, 271-F Life Sciences Center, Univ. of Missouri-Columbia, Columbia, MO 65211
d Dep. of Statistics, 105 Math Science, Univ. of Missouri-Columbia, Columbia, MO 65211
e Dep. of Crop Science, 840 Method Rd. Unit 3, Box 7629, North Carolina State Univ., Raleigh, NC 27695
f USDA-ARS-MSA, P.O. Box 345, Stoneville, MS 38776
* Corresponding author (shannong{at}missouri.edu)
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ABSTRACT
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Genetic effects and temperature during the reproductive period for unsaturated fatty acids in soybean [Glycine max (L.) Merr.] seed oil affect oil composition. Increasing oleic and reducing linolenic acids are desirable to improve oil for food and other uses. The objective of this study was to access the environmental effect on fatty acids of seed oil for seventeen soybean genotypes with normal and modified fatty acid profiles. Stability coefficients (b values) were calculated from the regression of fatty acid level on average temperature over the final 30 d of the reproductive period across 10 environments. Mid-oleic acid genotypes were generally less stable for oleic acid content than genotypes with reduced oleic acid. Significant differences, however, were found for oleic acid stability among mid-oleic acid genotypes. Mid-oleic acid lines N98–4445A and N97–3363–4 were the most unstable among the 17 genotypes with stability coefficients of 3.28 and 2.53, respectively. However, the higher oleic acid line M23 was relatively stable in oleic acid with a stability coefficient of 0.13 over environments. IA 3017 at 10 g kg–1 was the most stable in linolenic acid content across environments while progressively higher linolenic acid genotypes were less stable. Soybean lines similar to M23 and IA 3017 will be essential to develop increased oleic acid and reduced linolenic acid cultivars to ensure consistent production of soybean oil with the desired fatty acid levels.
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INTRODUCTION
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MODIFYING seed oil composition has become a major goal in soybean breeding programs. The oil portion of the soybean seed is made up of five major fatty acids, which include palmitic, stearic, oleic, linoleic, and linolenic acids. Changing the proportions of these fatty acids in soybean seed to reduce palmitic acid, increase oleic acid, and reduce linolenic acid will enhance food, fuel, and other applications of the oil (Wilson, 2004). Several soybean genotypes that produce novel fatty acid profiles have been developed through genetic recombination, mutation breeding, or transgenic approaches. It is important to study the effect of the environment on the oil composition of novel fatty acid genotypes to determine their stability over different growing conditions and their potential utility in plant breeding programs (Primomo et al., 2002).
Environmental influence on the fatty acid profile of soybean oil from typical cultivars has been addressed in several studies. Seed development at higher temperatures resulted in significant decreases in linoleic acid and linolenic acid contents, and a significant increase in oleic acid content (Howell and Collins, 1957; Wolf et al., 1982; Dornbos and Mullen, 1992). Palmitic acid and stearic acid contents generally were unaffected by changes in temperature. Wilcox and Cavins (1992) studied the response of fatty acid composition to planting dates for the cultivar Century and two low linolenic acid soybean lines (C1640 and 9509) in Indiana across years. Planting date had a significant effect on fatty acid profile in 3 of 5 yr of the study. Levels of palmitic acid decreased slightly and levels of stearic acid increased slightly with successively later planting dates. Linolenic acid content showed a consistent increase in all genotypes with later planting dates. Stability of linolenic acid content across planting dates was greater in low linolenic acid soybean lines than in the cultivar Century. Regression coefficients of linolenic acid on mean daily maximum air temperature during the 0 to 20 d before maturity were –4.9 g kg–1 °C–1 for Century and –3.0 g kg–1 °C–1 for C1640. Temperature during the final 20 d before maturity showed the highest correlation with linolenic acid content among the different plant reproductive periods evaluated in the study.
Primomo et al. (2002) studied the effect of environment at four locations in Ontario, Canada over 3 yr on the fatty acid profiles of three soybean cultivars and 14 lines with modified fatty acid composition. The stability of the fatty acid profile across environments for each genotype was evaluated by using the b-values from regression as the stability parameter according to Finlay and Wilkinson (1963). They found that lines with modified fatty acid levels, except for lines with elevated stearic acid content and high oleic acid content, had more stable fatty acid profiles over years and locations than normal cultivars.
Genotypes with elevated oleic acid content and reduced linolenic acid content are desirable to improve functionality of soybean oil by increasing oil utility at higher temperatures. Also, higher oleic acid, lower linolenic acid oil will reduce the need for hydrogenation which produces undesirable trans-fats in foods, which causes increased cholesterol and heart disease in humans (Wilson, 2004). Evaluation of stability of oleic acid and linolenic acid contents of genotypes with modified fatty acid profiles is necessary to determine their utility in plant breeding programs to develop soybean cultivars with enhanced oil quality and adaptation to a wide array of environments (Primomo et al., 2002). The objective of this study was to determine the stability of fatty acid composition across environments for 17 soybean genotypes with normal and modified fatty acid profiles.
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MATERIALS AND METHODS
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Four soybean cultivars and 13 soybean genotypes from maturity group III to V with modified fatty acid levels in seed oil were used in this study. The cultivars and modified fatty acid genotypes represent seven fatty acid profiles as follows: (i) reduced linolenic acid, (ii) reduced palmitic acid, (iii) elevated palmitic acid, (iv) mid-oleic acid, (v) mid-oleic acid and reduced linolenic acid, (vi) reduced palmitic acid and reduced linolenic acid, and (vii) common cultivars with typical fatty acid profiles (Table 1).
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Table 1. Maturity Group (MG), phenotype and corresponding average fatty acid profile over ten environments for each of 17 soybean genotypes evaluated, 2004.
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The experiment was conducted in 2004 at five locations (Columbia, MO; Portageville loam soil, MO; Portageville clay soil, MO; Sandhills, NC; and Stoneville, MS) which represent relatively different seasonal growing conditions. Locations, latitude for each location, planting dates, soil types, and herbicides used are listed in Table 2. The experimental design was a randomized complete block design with four to six replications at each location. Six replications were planted at Columbia, Portageville loam soil, Sandhills (NC), and Stoneville (MS) and four replications were planted on Portageville clay soil. The plots consisted of a factorial arrangement of planting dates and genotypes. Two planting dates were used at each location (early and late) separated by about 30 d (Table 2). Plots consisted of three rows, 61 cm long, with 76 cm between rows, except for Stoneville and Sandhills, where row width was 66 cm and 96 cm, respectively. Planting was done by hand with a seeding rate of 60 seeds per plot (about 33 seed m–1 of row).
Due to emergence problems, the first block at Stoneville had too many missing plots and therefore was not included in the statistical analysis. Also, in the second planting date at Stoneville some maturity group IV and group V genotypes had problems with green stems and delayed maturity, producing only a few shrunken seeds that were not representative of seed to analyze for fatty acid profile. Therefore, data from some genotypes at Stoneville was excluded from the analyses.
Concentrations of palmitic, stearic, oleic, linoleic, and linolenic acids as a percentage of the total fatty acids in the seed oil were determined for each plot by randomly selecting four plants from the center row and picking two pods each from the seventh and eighth node down from the top of the plant. The 16 pods were threshed by hand and the seeds represented a bulked-plot sample. Ten seeds were randomly selected from each sample for fatty acid analysis. Each 10 seed sample was placed in a paper envelope, manually crushed with a hammer and put in separate test tubes for fatty acid extraction. Crushed seeds were extracted in 5mL chloroform:hexane:methanol (8:5:2, v/v/v) overnight. Derivatization was done by transferring 100 µL of extract to vial and adding 75 µL of methylating reagent (0.25 M methanolic sodium methoxide:petroleum ether:ethyl ether, (1:5:2, v/v/v). Hexane was added to dilute samples to approximately 1 mL. An Agilent (Palo Alto, CA) series 6890 capillary gas chromatograph fitted with a flame ionization detector (275°C) was used with an AT-Silar capillary column (Alltech Associates, Deerfield, IL). Standard fatty acid mixtures (Animal and Vegetable Oil Reference Mixture 6, AOACS) were used as calibration reference standards.
The 10 growing environments from the combination of five locations, each with two planting dates were used to obtain stability parameters by regression analysis. Mean fatty acid content of each of the 17 genotypes was regressed on mean temperature during the last 30 d of the reproductive period in each environment. A modified approach used by Finlay and Wilkinson (1963) was used to obtain a quantitative measure of the environment. Wilcox and Cavins, (1992) reported that temperature during the final 20 d before maturity showed the highest correlation with linolenic acid content among the different plant reproductive periods for lines evaluated over several planting dates over 5 yr. In this study the correlation of temperature during the last 20, 30, or 40 d of the reproductive period with linolenic acid and oleic acid content was evaluated (data not shown). Because average temperature during the last 30 d before maturity showed the highest correlation among the three periods studied, it was used in this study as the quantitative measure of the environment. The use of average temperature provided an environmental measure that was independent of the genotypes used in the study. Genotypes having stability regression coefficients (b-values) closer to zero are more stable while those that deviate significantly from zero (either positive or negative) are considered less stable to changes in the environment. Since genotypes ranged from maturity group III to V and varied widely for maturity, the average temperature was calculated separately for each genotype by averaging the mean daily temperature for the final 30 d of seed filling to physiological maturity. Average temperature during the final 30 d of the reproductive period is shown in Table 3.
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Table 3. Average temperature in °C over the final 30 d of the reproductive period for 17 soybean genotypes at each of two planting dates at each of five environments, 2004.
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Stearic acid and palmitic acid were not significantly affected across growing environments and were not analyzed for stability. This reaction of stearic acid and palmitic acid is in agreement with other studies (Wolf et al., 1982; Primomo et al., 2002). The stability analysis was performed for the unsaturated fatty acids oleic acid, linoleic acid, and linolenic acid, which are significantly influenced by temperature in various growing environments.
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RESULTS AND DISCUSSION
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Mean oleic, linoleic, and linolenic acid contents of seed of 17 genotypes for two planting dates at each of the five locations with corresponding LSD's and ranges are shown in Tables 4, 5, and 6, respectively. Oleic acid, linoleic acid, and linolenic acid stability coefficients (b values) with corresponding coefficients of determination from regression (r2) and significance levels (p) for each genotype are shown in Table 7. Stability coefficients for each genotype varied for the unsaturated fatty acids showing differences in their response to changes in seed-filling temperature.
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Table 4. Mean linolenic acid content (g kg–1) of 17 soybean genotypes for two planting dates at each of five locations, 2004.
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Table 5. Mean linoleic acid content (g kg–1) of 17 soybean genotypes for two planting dates at each of five locations, 2004.
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Table 6. Mean linolenic acid content (g kg–1) of 17 soybean genotypes for two planting dates at each of five locations, 2004.
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Table 7. Stability coefficients (b values), coefficients of determination (r2) and probabilities for significance (p) for 17 soybean genotypes calculated from the regression of mean oleic acid, linoleic acid and linolenic acid contents on mean temperature during the final 30 d of the reproductive period over 10 seed-filling temperature environments, 2004.
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Oleic acid and linoleic acid contents showed a strong negative correlation (r = –0.93, p < 0.0001). Thus, results for oleic acid content were opposite of linoleic acid content (Tables 4 and 5). Increasing oleic acid levels and decreasing linolenic acid levels is important in improving functionality of the soybean oil. Therefore, the discussion of the stability analysis will focus primarily on stability of these two unsaturated fatty acids. In general, oleic acid stability coefficients were positive (Table 7), which shows that oleic acid content increased with an increase in temperature. Oleic acid content for 16 of the 17 genotypes evaluated increased 0.5 to 3.28% or 5 to 32.8 g kg–1 per °C increase in average temperature. The coefficient of determination provides a measure of the proportion of variability in fatty acid content accounted for by temperature changes. The most unstable genotypes for oleic acid were mid-oleic acid lines N97–3363–4 or N98–4445A which had high b values (2.53 and 3.28) and high and statistically significant (p < 0.01) r2 values. This indicated a great proportion of the variability for oleic acid content in these genotypes can be attributed to temperature changes. Range values (Table 4) of 336 and 275 g kg–1, respectively, also showed that these genotypes fluctuated widely in oleic acid content under the various growing conditions. In the most stable genotypes, such as the cultivar AG 4902, the range value in mean oleic acid levels across locations was only 79 g kg–1 (Table 4), and mean temperature (Table 7) was not a significant factor affecting oleic acid content (r2 = 0.04, p = 0.58). Warmer environments would enhance expression of high oleic acid content in mid-oleic acid genotypes with low stability. The genotypes Holl and M23 with relatively high mean oleic acid content (400–500 g kg–1) were more stable across environments with stability coefficients of 0.73 and 0.13, respectively (Table 7), and range values (Table 4) of 152 and 95 g kg–1, respectively, compared to the high oleic acid genotypes N98–4445A and N97–3363–4 mentioned above. Figure 1A
shows linear regressions of oleic acid content on mean temperature across environments for two increased oleic acid genotypes and the cultivar AG4902. Genotypes M23 and N97–3363–4 exhibited a crossover interaction in their response of oleic acid content to temperature. The low and nonsignificant (p = 0.70) regression coefficient (.13) shown in Table 7, and flat slope of the regression line of oleic acid content on temperature in Fig. 1A, shows M23 had much greater stability across temperature regimes than N97–3363–4. The high stability coefficient (3.28, p = 0.0002) and steep slope of the N97–3363–4 for regression of oleic acid content on temperature indicates oleic acid content for this genotype fluctuates significantly with changes in temperature. Therefore, factors other than mean temperature were responsible for variability in oleic acid content in M23 and AG4902 across environments (Table 4). A similar crossover interaction was found among mid-oleic genotypes N98–4445A and Holl (Fig. 1B). The elevated oleic acid genotype Holl and the cultivar Manokin had nonsignificant stability coefficients of 0.73 (p = 0.076) and 0.27 (p = 0.10), respectively, as compared to the high and significant stability coefficient of 2.53 (p = 0.005) for N98–4445A. The lower stability coefficient of the increased oleic acid genotype Holl shows it is significantly more stable than N98–4445A. Holl is derived from M23, which was relatively stable in oleic acid across growing environments in this study. Both lines likely share the same single gene for high oleic acid content (Rahman et al., 2001). N98–4445A and N97–3363–4 also are genetically related (J. W. Burton, Personal Communication, 2005) and share several minor genes for high oleic acid. N98–4445A and N97–3363–4 have high stability coefficients and low environmental stability, whereas M23 and Holl have low stability coefficients and high stability across growing environments. These differences in stability may be due to genotypes with several minor genes being more affected by the environment than genotypes with single genes for higher oleic acid content (Primomo et al., 2002). It is not known whether these different high oleic acid germplasm sources affect the same or different enzymes in the fatty acid pathway. It is possible that they affect different enzymes and that these enzymes are affected differentially by temperature. It is also possible that genes in these genotypes affect the same enzyme or enzymes, while different genes within each genotype at other loci may regulate the activity of these enzymes.
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Fig. 1. Regression of oleic acid content on mean temperature during the final 30 d of the reproductive period for genotypes N97–3363–4, M23 and AG 4902 (1A) and N98–4445A, Holl and Manokin (1B) calculated over 10 seed-filling temperature environments, 2004.
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M23, Holl, or other mid-oleic acid genotypes like the genotype RG9 reported by Primomo et al. (2002), with genetic mechanisms for greater stability across environments would be favored in breeding programs to develop increased oleic acid genotypes which are less influenced by temperature. Selection for stable oleic acid content in a breeding program would be useful to develop lines that maintain high oleic acid content when grown under a wide array of environments. It would be especially important to have the desired oleic acid contents in soybean seed grown under cooler growing conditions.
Genotypes differed in the fluctuation of linolenic acid content across environments and showed differences in stability (Table 7). Linolenic acid stability coefficients are negative since linolenic acid content decreased as temperature increased. The stability coefficients for the 17 soybean genotypes ranged from 0.02 to 0.53% or 0.2 to 5.3 g kg–1 decrease in linolenic acid content per °C increase in temperature. Most genotypes showed high and statistically significant coefficients of determination in regressions of linolenic acid content on mean temperature. Stability coefficients agreed with the range in linolenic acid content across environments within genotypes. Genotypes C1943, M23, and S01–9267 had high b values (Table 7), high range values (Table 6), and were the least stable for linolenic acid level. Therefore, mean temperature accounted for a high proportion of variability in linolenic acid content in these genotypes. On the other hand IA3017, IA3018, and S01–9370 had low b values, low ranges, and were the most stable for linolenic acid level, showing mean temperature had little effect on linolenic acid in these genotypes.
Genotypes with reduced linolenic acid had more stable expression than did genotypes with normal linolenic acid content (Fig. 2
). This confirms similar results reported by Primomo et al. (2002). Linolenic acid in lowest linolenic acid lines was influenced less by changes in temperature, as compared to normal linolenic acid or other genotypes with altered palmitic acid or oleic acid content in these studies. Stability regressions for linolenic acid of cultivar DKB 38–52 and three reduced linolenic acid genotypes, ‘MD 00–6605’, ‘IA 3018’, and ‘IA 3017’, are compared in Fig. 2A. IA 3017 with about 10 g kg–1 mean linolenic acid content was the most stable genotype with a stability coefficient of –0.02 and a range value of 2 g kg–1 (Tables 6 and 7). Figure 2B shows the increased stability for linolenic acid of two reduced linolenic acid genotypes S01–9370 and CR03–529 compared to the cultivar Manokin. These results show that breeding to decrease linolenic acid content would enhance stability of low linolenic acid levels over different growing conditions.
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Fig. 2. Regression of linolenic acid content on mean temperature during the final 30 d of the reproductive period for genotypes DKB38–52, MD00–6605, IA3017 and IA3018 (2A) and Manokin, S01–9370, and CR03–529 (1B) calculated over 10 seed-filling temperature environments, 2004.
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CONCLUSIONS
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Stability analysis showed the contents of oleic acid, linoleic acid and linolenic acid were strongly correlated with average temperature during the final 30 d of the reproductive period. Even though temperature affected unsaturated fatty acid content on all genotypes, the magnitude of the temperature effect varied widely among genotypes. Mid-oleic acid genotypes N97–3363–4 and N98–4445A were less stable for oleic acid content across environments than genotypes with normal oleic acid content. Mid-oleic acid genotypes M23 and Holl were relatively stable across environments and therefore represent good germplasm lines to breed mid-oleic acid lines adapted to cooler environments, or for adaptability to a wider range of growing conditions. Breeding programs for increased oleic acid content in soybean should evaluate genotypes in several environments to characterize stability of mean oleic acid content under different growing conditions.
Reduced linolenic acid genotypes are desirable and were more stable across environments than progressively higher linolenic acid genotypes. Therefore, one or a few environments may be adequate to obtain a good estimate of the mean linolenic acid content and its stability across locations for a genotype.
Soybean cultivars with novel fatty acid profiles, like reduced linolenic acid are currently available for commercial production. Cultivars with other modified fatty acid traits, such as mid-oleic acid, will likely be introduced for production in the near future. Knowing the best growing conditions for these novel cultivars with regard to adaptation to different locations, planting dates, and other agronomic practices will ensure that desired fatty acid profile of seed oil is consistently met.
Received for publication December 15, 2005.
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REFERENCES
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- Finlay, K.W., and G.N. Wilkinson. 1963. The analysis of adaptation in a plant breeding program. Aust. J. Agric. Res. 14:742–754.[CrossRef][ISI]
- Howell, R.W., and F.I. Collins. 1957. Factors affecting linolenic acid and linoleic acid content of soybean oil. Agron. J. 49:593–597.
- Primomo, V.S., D.E. Falk, G.R. Ablett, J.W. Tanner, and I. Rajcan. 2002. Genotype x environment interactions, stability, and agronomic performance of soybean with altered fatty acid profiles. Crop Sci. 42:37–44.[Abstract/Free Full Text]
- Rahman, S.M., T. Kinoshita, T. Anai, and Y. Takagi. 2001. Combining ability in loci for high oleic acid and low linolenic acid in soybean. Crop Sci. 41:26–29.[Abstract/Free Full Text]
- Wilcox, J.R., and J.F. Cavins. 1992. Normal and low linolenic acid soybean strains: Response to planting date. Crop Sci. 32:1248–1251.[Abstract/Free Full Text]
- Wilson, R.F. 2004. Seed Composition. p. 621–677. In H.R. Boerma and J.E. Specht (ed.) Soybeans: Improvement, Production, and Uses. 3rd ed. ASA, CSSA, and SSSA, Madison, WI.
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ABSTRACT
INTRODUCTION
