Crop Science 43:527-531 (2003)
© 2003 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
A Novel Soybean Germplasm with Elevated Saturated Fatty Acids
Shaikh M. Rahman*,a,
Toyoaki Anaib,
Takehito Kinoshitab and
Yutaka Takagib
a Laboratory of Plant Biotechnology, National Agricultural Research Center for Western Region, 6-12-1 Nishifukatsu, Fukuyama, Hiroshima 721-8514, Japan
b Laboratory of Plant Breeding, Fac. of Agric., Saga Univ., Saga 840-0085, Japan
* Corresponding author (mizanshaikh{at}yahoo.com)
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ABSTRACT
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Two soybean [Glycine max (L.) Merr.] germplasm lines have been identified for unique fatty acid content. The very high content of palmitic acid in HPKKJ10 is controlled by the fap2 and fapx loci, and the very high content of stearic acid in M25 is controlled by the st2 locus. If the fap2 and fapx loci are independently inherited from the st2 locus, soybean germplasm could be developed with more unique and useful combinations of these fatty acids. The objectives of this study were to combine the loci of high palmitic and stearic acids, and determine the effects of altered contents of palmitic and stearic acids on other fatty acids. HPKKJ10 was reciprocally crossed to M25. The data from F2 seed indicated that the fap2 and fapx loci controlling high palmitic acid were independently inherited from the st2 locus controlling high stearic acid. Thus, the germplasm (HPS) with the very high palmitic acid trait from HPKKJ10, and very high stearic acid trait from M25, was easily developed. The increases in palmitic acid due to the fap2 and fapx loci in HPKKJ10, and the increases in stearic acid due to the st2 locus in M25 were individually associated with changes in oleic acid. The combined increases in these two fatty acids in HPS were associated with decreases in oleic and linoleic acids, and increases in linolenic acid. The development of HPS with high content of saturated fatty acids could open new markets for soybean oil.
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INTRODUCTION
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INDUSTRIAL PROPERTIES and potential food application of an oil are largely determined by the ratio and amount of saturated and unsaturated fatty acids. Commercial oil crops cannot satisfy all requirements of the food industry. Therefore, interest in producing modifications in the oil composition of oilseed crops is increasing. The food industry wants specially tailored oils for specific purposes (Rattray, 1991).
The ratio and amount of saturated and unsaturated fatty acids in a seed oil can be changed by different methods (Robbelen et al., 1989). Although natural variability for these traits has been observed in most of the oilseed crops (Knowles, 1989; Stefansson et al., 1961), natural genetic variability for fatty acid composition in commercial soybean seed oil is limited (Rattray, 1991). Therefore, additional variability must be created by mutagenesis, the recombination of different mutant genes, or genetic engineering (Ohlrogge et al., 1991).
The saturated fatty acid content in natural soybean cultivars is approximately 150 g kg-1 in which palmitic acid predominates with 110 g kg-1, and a range from 100 to 120 g kg-1 (Hawkins et al., 1983; Cherry et al., 1985), and stearic acid contributes 40 g kg-1, with a range from 22 to 72 g kg-1 (Hymowitz et al., 1972). Development of cultivars with elevated contents of these fatty acids could increase the utility of the oil for specific edible purposes. Germplasm with elevated palmitic acid (Fehr et al., 1991; Takagi et al., 1995) or stearic acid (Graef et al., 1985; Bubeck et al., 1989; Rahman et al., 1995; Takagi and Rahman, 1995) have been developed by treatment with X-rays or chemical mutagens and further hybridization of mutants.
Genetic studies on elevated palmitic acid showed that this fatty acid was controlled by either a single locus or two loci. The single locus fap2 was found in C1727 (Erickson et al., 1988) and J10 (Rahman et al., 1996; Rahman et al., 1999). Fap4 was found in A24 (Schnebly et al., 1994) and fapx in KK7 (Rahman et al., 1999). Evidences of two loci were found in A19 (fap2fap4, Schnebly et al., 1994) and HPKKJ10 and HPKKC-7 (fap2fapx, Rahman et al., 1999). These germplasm contain 220 to 280 g kg-1 palmitic acid, which is the highest reported in soybean oil. On the other hand, limited genetic studies have been conducted on stearic acid. Graef et al. (1985) found that elevated stearic acid content in different mutants was controlled by different multiple recessive alleles at a single locus, and these were designated as fasa for A6, fasb for FA41545 and fas for A81-606085. Bubeck et al. (1989) crossed the mutants ST1, ST2, ST3, and ST4 with A6 and found that the alleles in ST1, ST3, and ST4 occurred at the same locus as the fasa allele in A6, whereas the allele in ST2 may occur at a different locus. Rahman et al. (1997) first observed that the elevated stearic acid contents in the mutants KK-2 and M25 are controlled by different major loci, designated as st1 for KK-2 and st2 for M25. The stearic acid content (>300 g kg-1) found in the combination of st1 and st2 is the highest reported in soybean, but it was not possible to develop the line with this genotype because the irregular seeds failed to grow into plants after germination.
The genetic relationship of the loci for elevated palmitic and stearic acids in soybean is still unknown, although studies on relationships of reduced linolenic acid with reduced and elevated palmitic acid (Nickell et al., 1991) and elevated oleic acid (Rahman et al., 2001) in soybean and with elevated palmitic acid in flax (Linum usitatissimum L.) (Ntiamoah et al., 1995) and of reduced palmitic acid with reduced stearic acid in sunflower (Helianthus annuus L.) (Miller and Vick, 1999) were conducted and soybean, flax, and sunflower cultivars were successfully developed. If the loci for elevated palmitic and stearic acids are independently inherited, a soybean cultivar with the desired content of saturated fatty acids can easily be developed, and this oil will have a significant market value. Therefore, this investigation was undertaken to combine the loci of elevated palmitic and stearic acids and determine if changes in contents of saturated fatty acids would affect contents of unsaturated fatty acids in these genetic systems.
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MATERIALS AND METHODS
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The recombinant line HPKKJ10 and mutant line M25 were used for the present study. The line HPKKJ10 is a very high palmitic acid selection from the cross J10 x KK7 (Table 1). M25 is a very high stearic acid selection from the M4 generation of the irradiated cultivar Bay.
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Table 1. Average (±SE) fatty acid composition and genotype of the original cultivar (Bay) and the soybean lines selected for high palmitic or high stearic acids.
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The lines HPKKJ10 and M25 were reciprocally crossed in the glasshouse at Saga University. Seed of parents and reciprocal F1 seed was planted in the field at Saga University in July 2000. Seed of J10 and KK7 was also planted. Each of the HPKKJ10, M25, KK7, J10, and F1 plants was harvested individually. To reduce the environmental effects on fatty acid composition, seed of HPKKJ10, M25, KK7, J10, and F2 seed of each F1 plant were collected from pods of similar maturity on the fifth to seventh nodes of the main stem. Fatty acid composition was determined from 136 F2 seeds of each reciprocal cross and 20 seeds of HPKKJ10, M25, KK7, and J10. The F2 seed was cut into two parts with a razor blade. The portion containing the embryonic axis was stored, and the portion containing a section of the cotyledon was used for fatty acid analysis. The identity of all F2 seeds was maintained during fatty acid analysis.
The range of stearic acid content of the parents grown in the same field conditions with the F1 plants was used to classify the F2 seed. An F2 seed was considered similar to the parent when its stearic acid content was within the range exhibited by that parental seed. Thus, the F2 seed was classified as = HPKKJ10, >HPKKJ10 to <M25, and = M25. But during the classification of F2 seed for palmitic acid, the range of this fatty acid content of the KK7 and J10, and HPKKJ10 was used, since fapx (KK7) and fap2 (J10) loci with additive effects are present in HPKKJ10 (Rahman et al., 1999) which made easy to divide the F2 seeds into three phenotypic classes. Thus, the F2 seed was classified as <KK7 and J10, = KK7 and J10, and = HPKKJ10. The same classification was also used to evaluate segregation of palmitic and stearic acids.
Seven F2 seeds, with palmitic acid similar to HPKKJ10 and stearic acid similar to M25, were identified. Four seed from these F2 seeds and four seeds from each of HPKKJ10, M25, and Bay were planted in the field in 2001, and the plants were harvested individually. Ten individual F3 seeds from each F2 plant, and from four plants in each of HPKKJ10, M25, and Bay, were analyzed for fatty acid composition.
Fatty acid composition was determined by gas chromatography, as described by Takagi et al. (1989). Chi-square analyses were calculated to test the best fit of data to expected genetic ratios. A single gene model for stearic acid and a two-gene model for palmitic acid were used to evaluate the segregation ratio in F2 seed, while a three-gene model was applied for the evaluation of these two fatty acids combined.
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RESULTS AND DISCUSSION
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Data from reciprocal F2 seed of the HPKKJ10 x M25 cross were analyzed for palmitic and stearic acids individually to determine if the loci for these two fatty acids are independently inherited. Data from reciprocal F2 seed were combined because their 
2 values were homogeneous for both palmitic and stearic acid content. Trimodal frequency distribution patterns for both palmitic and stearic acids were evident from the combined data (Fig. 1 and Fig. 2). The ranges of palmitic acid content in KK7 and J10, and HPKKJ10 were used to evaluate the segregation of F2 seeds (Fig. 1). The F2 seeds were classified as <KK7 and J10 (<152 g kg-1), = KK7 and J10 (152–185 g kg-1), and = HPKKJ10 (200–234 g kg-1). Results indicated a satisfactory fit to the 9:6:1 ratio (Table 2). This double homozygous recessive gene inheritance of the palmitic acid trait in HPKKJ10 is supported by the results previously described (Rahman et al., 1999). On the other hand, the ranges of stearic acid content in HPKKJ10 and M25 were used to evaluate the segregation of F2 seeds (Fig. 2). The F2 seeds were classified as = HPKKJ10 (29–42 g kg-1), > HPKKJ10 to <M25 (>42 to <156 g kg-1), and = M25 (156–216 g kg-1). The combined data for stearic acid produced a ratio of 68:133:71 (Fig. 2), with a 2 value of 0.20 (P > 0.90; Table 2) indicating a best fit to the expected 1:2:1 ratio, which is consistent with previous results (Rahman et al., 1997). The results from the individual analyses indicate that the loci for both high palmitic and stearic acids were independently inherited.
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Fig. 1. Distribution of palmitic acid content in soybean seeds of Bay, KK7, J10, HPKKJ10, and F2 of HPKKJ10 x M25 cross.
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Fig. 2. Distribution of stearic acid content in soybean seeds of M25, HPKKJ10, and F2 of HPKKJ10 x M25 cross.
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Table 2. Chi-square (2) analyses of palmitic and stearic acid contents individually and combined in F2 soybean seeds of HPKJ10 x M25 cross.
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For the two fatty acids combined, two loci (fap2 and fapx) for the palmitic acid trait and one locus (st2) for the stearic acid trait are expected to segregate in the HPKKJ10 x M25 cross. Because the loci fap2 and fapx in HPKKJ10 and the locus st2 in M25 segregate respectively to the 9:6:1 and 1:2:1 ratio, segregation for these three individual loci should result in a ratio of 9:6:1:18:12:2:9:6:1 for contents of the two fatty acids. Nine phenotypic classes were distinctly observed (Fig. 3), and the combined data for these fatty acids gave a consistent good fit to the expected ratio (2 = 6.39, P > 0.50; Table 2).
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Fig. 3. Scatter graph of palmitic acid vs. stearic acid in oil from F2 soybean seeds of HPKKJ10 x M25 cross.
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There were seven F2 seeds with the genotype fap2fap2fapxfapxst2st2. However, four from these seeds were grown to produce F3 seeds. The mean palmitic acid content in 40 F3 seeds of four F2 plants (HPS) was 214 g kg-1, compared with 212 g kg-1 for HPKKJ10, 93 g kg-1 for M25, and 108 g kg-1 for Bay (Table 3). The mean stearic acid content in HPS was 171 g kg-1, compared with 35 g kg-1 for HPKKJ10, 181 g kg-1 for M25, and 34 g kg-1 for Bay. These results also support a three-locus model in which fap2 and fapx are responsible for 214 g kg-1 palmitic acid (Rahman et al., 1999), and st2 is responsible for 171 g kg-1 stearic acid (Rahman et al., 1997) in the segregant HPS.
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Table 3. Average (±SE) fatty acid composition of the original cultivar (Bay) and the soybean lines developed for high palmitic or high stearic acids.
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The elevation of palmitic acid contents from 108 to 212 and 214 g kg-1 in HPKKJ10 and HPS was associated with a reduction in oleic and linoleic acid contents (Table 3). The elevation of stearic acid contents from 34 to 181 and 171 g kg-1 in M25 and HPS was also associated with a change in oleic and linoleic acid contents. The reduction of 76 g kg-1 oleic acid from 280 to 204 g kg-1 and of 33 g kg-1 linoleic acid from 501 to 468 g kg-1 in HPKKJ10 was directly associated with 104 g kg-1 increase in palmitic acid. The reduction of 172 g kg-1 oleic acid from 280 to 108 g kg-1 and of 90 g kg-1 linoleic acid from 501 to 411 g kg-1 in HPS cannot be solely associated with the elevation of 106 g kg-1 palmitic acid from 108 to 214 g kg-1, because the locus st2 for high stearic acid is present in HPS that elevated 137 g kg-1 stearic acid from 34 to 171 g kg-1. However, effects of combined high palmitic and stearic acids on the other fatty acids showed that the 243 g kg-1 elevation of these two fatty acids in HPS was associated with a 262 g kg-1 decrease in oleic and linoleic acids, and 19 g kg-1 increase in linolenic acid.
Because the very high palmitic acid trait of HPKKJ10 and the very high stearic acid trait of M25 are under simple Mendelian inheritance, the germplasm HPS was easily developed. The saturated fatty acid content (>380 g kg-1) in the oil of HPS is the highest known to date. This will increase the utility and improve the quality of soybean oil for specific edible purposes. First, this type of oil requires no chemical transformation such as hydrogenation or transesterification to obtain solid or semisolid fats. Such oil transformations have been related to cardiovascular diseases (Kritchevsky et al., 1995; Ascherio and Willett, 1997). Second, the oil from this germplasm will show higher stability than the oil from currently available cultivars with elevated palmitic or stearic acids (Schnebly et al., 1994; Rahman et al., 1995, 1999), and will find many applications in the field of solid and semisolid fats, such as the manufacture of margarine, shortenings for baking, or fats for deep frying. In our previous work, we attempted to develop the recombinant soybean with drastically high stearic acid (>300 g kg-1; Rahman et al., 1997), but the seeds with this content were irregular and failed to grow into plants after germination. The seeds of HPS are completely normal and grow into normal plants. Investigations into the effects of fap2, fapx, and st2 alleles on yield and other agronomic traits in HPS are under way.
Received for publication March 14, 2002.
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REFERENCES
|
|---|
- Ascherio, A., and W.C. Willett. 1997. Health effects of trans fatty acids. Am. J. Clin. Nutr. 66(Suppl.):1006S–1101S.[Abstract/Free Full Text]
- Bubeck, D.M., W.R. Fehr, and E.G. Hammond. 1989. Inheritance of palmitic and stearic acid mutants of soybean. Crop Sci. 29:652–656.[Abstract/Free Full Text]
- Cherry, J.H., L. Bishop, P.M. Hasegawa, and H.R. Leffler. 1985. Differences in the fatty acid composition of soybean seed produced in northern and southern areas of the U.S.A. Phytochemistry 24:237–241.[ISI]
- Erickson, E.A., J.R. Wilcox, and J.F. Cavin. 1988. Inheritance of altered palmitic acid percentage in two soybean mutants. J. Hered. 79:465–468.[Abstract/Free Full Text]
- Fehr, W.R., G.A. Welke, E.G. Hammond, D.N. Duvick, and S.R. Cianzio. 1991. Inheritance of elevated palmitic acid content in soybean seed oil. Crop Sci. 31:1522–1524.[Abstract/Free Full Text]
- Graef, G.L., W.R. Fehr, and E.G. Hammond. 1985. Inheritance of three stearic acid mutants of soybean. Crop Sci. 25:1076–1079.[Abstract/Free Full Text]
- Hawkins, S.E., W.R. Fehr, and E.G. Hammond. 1983. Resource allocation in breeding for fatty acid composition of soybean oil. Crop Sci. 23:900–904.[Abstract/Free Full Text]
- Hymowitz, T., R.G. Palmer, and H.H. Hadley. 1972. Seed weight, protein, oil, and fatty acid relationships within the genus Glycine. Trop. Agri. (Guildford, UK) 49:245–250.
- Knowles, P.E. 1989. Safflower. p. 363–374. In G. Robbelen et al. (ed.) Oil crops of the world, their breeding and utilization. Mcgraw-Hill, New York.
- Kritchevsky, D., S.A. Tepper, A. Kuksis, K. Eghtedary, and D.M. Kluefeld. 1995. Influence of triglyceride structure on experimental atherosclesosis. FASEB J. 9:A320.
- Miller, J.F., and B.A. Vick. 1999. Inheritance of reduced stearic and palmitic acid content in sunflower seed oil. Crop Sci. 39:364–367.[Abstract/Free Full Text]
- Nickell, A.D., J.R. Wilcox, and J.F. Cavins. 1991. Genetic relationships between loci controlling palmitic and linolenic acids in soybean. Crop Sci. 31:1169–1171.[Abstract/Free Full Text]
- Ntiamoah, G., G.G. Rowland, and D.C. Taylor. 1995. Inheritance of elevated palmitic acid in flax and its relationship to the low linolenic acid. Crop Sci. 35:148–152.[Abstract/Free Full Text]
- Ohlrogge, J.B., J. Browse, and C. Somerville. 1991. The genetics of plant lipids. Biochem. Biophys. Acta 1082:1–26.[Medline]
- Rahman, S.M., T. Kinoshita, T. Anai, and Y. Takagi. 1999. Genetic relationships between loci for palmitate contents in soybean mutants. J. Hered. 90:423–428.[Abstract/Free Full Text]
- Rahman, S.M., T. Kinoshita, T. Anai, and Y. Takagi. 2001. Combining ability in loci for high oleic and low linolenic acids in soybean. Crop Sci. 41:26–29.[Abstract/Free Full Text]
- Rahman, S.M., Y. Takagi, K. Miyamoto, and T. Kawakita. 1995. High stearic acid soybean mutant induced by X-ray irradiation. Biosci. Biotech. Biochem. 59:922–923.
- Rahman, S.M., Y. Takagi, and T. Kinoshita. 1996. Genetic analysis of palmitic acid contents using two soybean mutants, J3 and J10. Breed Sci. 46:343–347.
- Rahman, S.M., Y. Takagi, and T. Kinoshita. 1997. Gentic control of high stearic acid content in seed oil of two soybean mutants. Theor. Appl. Genet. 95:772–776.[ISI]
- Rattray, J. 1991. Plant biotechnology and oils and fats industry. p. 1–15. In J. Rattray (ed.) Biotechnology of plant fats and oils. Am. Oil Chem. Soc., Champaign, IL.
- Robbelen, G., R.K. Downey, and A. Ashri (ed.) 1989. Oil crops of the world, their breeding and utilization. McGraw-Hill, New York.
- Schnebly, S.R., W.R. Fehr, G.A. Welke, E.G. Hammond, and D.N. Duvick. 1994. Inheritance of reduced and elevated palmitate in mutant lines of soybean. Crop Sci. 34:829–833.[Abstract/Free Full Text]
- Stefansson, B.R., F.W. Hougen, and R.K. Downey. 1961. Note on the isolation of rape plants with seed oil free from erucic acid. Can. J. Plant Sci. 41:218–219.
- Takagi, Y., A.B.M.M. Hossain, T. Yanagita, and S. Kusaba. 1989. High linolenic acid mutant in soybean induced by X-ray irradiation. Jpn. J. Breed. 39:403–409.
- Takagi, Y., and S.M. Rahman. 1995. Variation of different fatty acids in mutants in comparison with natural soybean varieties. Bull. Fac. Agric. (Saga Univ.) 79:23–27.
- Takagi, Y., S.M. Rahman, H. Joo, and T. Kawakita. 1995. Reduced and elevated palmitic acid mutants in soybean developed by X-ray irradiation. Biosci. Biotech. Biochem. 59:1778–1779.
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ABSTRACT
INTRODUCTION
