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CROP BREEDING, GENETICS & CYTOLOGY

Influence of Genotype and Environment on Isoflavone Contents of Soybean

^a Dep. of Agronomy, Iowa State Univ., Ames, IA USA
^b Dep. of Food Science and Human Nutrition, Iowa State Univ., Ames, IA 50011 USA

wfehr{at}iastate.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Isoflavones in soybean [Glycine max (L.) Merr.] may have positive impacts on human health. The objective of this study was to determine the role of the genotype, environment, and genotype x environment interactions on isoflavone contents of soybean. Nine isoflavones were measured in six cultivars grown at eight locations during 2 yr. The mean contents of total isoflavones and six of nine individual isoflavones were significantly higher in 1996 than 1995. There were significant differences among locations in one or more years for total and individual isoflavone contents. The year x location interactions were significant due to changes in rank and magnitude among the locations during the 2 yr. The genotype, genotype x year, genotype x location, and genotype x year x location interactions were significant for total and individual isoflavone contents. Despite the significant genotype x environment interactions, the differences between the cultivars with the highest and lowest total and individual isoflavone contents were relatively consistent among the 16 environments. It should be possible to breed for isoflavone content as a quantitative trait in a cultivar development program.

Abbreviations: HPLC, high performance liquid chromatography

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
ISOFLAVONES are a group of plant compounds that have potential anticarcinogenic properties (Adlercreutz et al., 1986). Isoflavones may act as antiestrogens (Adlercreutz et al., 1986), antioxidants (Naim et al., 1976), and tyrosine protein kinase inhibitors (Akiyama et al., 1987).

Significant environmental effects on isoflavone concentration in soybean have been reported. Tsukamoto et al. (1995) showed that isoflavone content was significantly lower in seeds that developed in high temperatures during seed fill than in seeds exposed to low temperatures. They reported that 80 to 90% of the total isoflavones were located in the cotyledon and the remainder in the hypocotyl. Isoflavone content of cotyledons exhibited a large response to temperature during seed fill, but the isoflavone content of the hypocotyls remained relatively constant across different temperature regimes (Tsukamoto et al., 1995).

Eldridge and Kwolek (1983) reported that total isoflavone content varied from 1160 to 3090 µg g^-1 among four soybean cultivars grown in the same environment and from 460 to 1950 µg g^-1 among four locations. Wang and Murphy (1994) found that total isoflavone content of a single cultivar ranged from 1176 to 3309 µg g^-1 among years and from 1176 to 1749 µg g^-1 among locations within the same year.

None of the previous studies on isoflavone contents have evaluated the same soybean cultivars at the same locations during different years. The objective of this study was to evaluate the relative importance of genotypes, years, locations, and their interactions on isoflavone contents by testing multiple cultivars at the same locations during different years.

	Materials and methods

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The six cultivars used in this experiment were grown by the Iowa State University soybean breeding project in 1995 and 1996 at eight Iowa locations. Kenwood 94 was a high-yielding commodity-type cultivar, Vinton 81 a large-seeded and high-protein cultivar, IA2011 a large-seeded and high-protein cultivar that lacked the lipoxygenase-2 enzyme, and IA2012, IA2013, and IA2016 were large-seeded cultivars. The cultivars were grown in three replications of a randomized complete-block design at Ames, Fairfield, Greene, Grand Junction, Kanawha, Keystone, Pocahontas, and Tingley, IA. The soil types were a Nicollet loam (fine-loamy, mixed, superactive, mesic Aquic Hapludoll) at Ames and Grand Junction, a Haig silty clay loam (fine, smectitic, mesic Vertic Argiaquoll) at Fairfield and Tingley, a Kenyon silty clay loam (fine-loamy, mixed, mesic Typic Hapludoll) at Greene, a Webster silty clay loam (fine-loamy, superactive, mesic Typic Endoaquoll) at Kanawha, a Muscatine silty clay loam (fine-silty, mixed, mesic Aquic Hapludoll) at Keystone, and an Okoboji (fine, smectitic, mesic Cumulic Vertic Endoaquoll) at Pocahontas. The plots consisted of four rows 3.7 m long spaced 69 cm apart and planted at a seeding rate of 33 seed m^-1. The center two rows were harvested with a self-propelled combine.

The seed from each plot was analyzed for isoflavone contents. Two samples from each plot were analyzed consecutively and the average of the samples was used for data analysis. The isoflavones measured were daidzin, daidzein, 6''-O-acteyldaidzin, 6''-O-malonyldaidzin, genistin, genistein, 6''-O-acetylgenistin, 6''-O-malonylgenistin, glycitin, glycitein, 6''-O-acetylglycitin, and 6''-O-malonylglycitin. Total daidzein, total genistein, total glycitein, and total isoflavones were calculated by adjusting for the molecular weights of the different isoflavones using the following formulas: total daidzein = 254.23(daizin/416.36 + 6''-O-malonyldaidzin/502.41 +6''-O-acetyldaidzin/458.4 + daidzein/254.23), total genistein = 270.23(genistin/432.37 + 6''-O-malonylgenistin/518.41 + 6''-O-acetylgenistin/474.4 + genistein/270.23), total glycitein = 284(glycitin/446 + 6''-O-malonylglycitin/532 + 6''-O-acetylglycitin/488 + glycitein/284), and total isoflavones = total daidzein + total genistein + total glycitein (Murphy et al., 1997).

The concentrations of the isoflavones were determined by C₁₈ reverse-phase high-performance liquid chromatography (HPLC) (Murphy et al., 1997). For each sample, 2 g of whole soybean seed were ground, mixed with 2 mL of 0.1 M HCl and 10 mL of acetonitrile (ACN) in a 125-mL screw-top flask, and stirred for 2 h at room temperature. After the samples were stirred, 7 mL of distilled water was added. The solution was filtered through Whatman no. 42 filter paper and the filter was rinsed three times with ACN. The filtrate was transferred to a 100-mL round-bottomed flask and rotary evaporated to dryness at 30°C. The dried material was redissolved in 10 mL of 80% (v/v) methanol and transferred to a 10-mL volumetric flask. The redissolved material was filtered through a 0.45-µm filter and 1 mL of the filtrate was transferred to sample vials. A 20-µl aliquot of the filtrate was tested in the HPLC analysis.

For the analysis of variance of all traits, cultivars were considered fixed effects, and replications, years, and locations were considered random effects. The analysis of variance was performed with the general linear models procedure (GLM) of the SAS software package (release 6.11) (SAS Institute, 1992). The significance of years, locations, and genotypes mean squares were calculated by deriving the expected mean squares and determining the appropriate F tests.

	Results and discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
The mean contents of total isoflavones and six of nine individual isoflavones were significantly greater in 1996 than 1995 (Tables 1 and 2) . The three isoflavones that did not have significant differences between years had mean contents of <20 µg g^-1. Eldridge and Kwolek (1983) found significant differences among years for the content of total isoflavones and six individual isoflavones of `Clark' soybean grown at Urbana, IL. They suggested that unknown climatic and environmental conditions contributed to the differences among years.

Significant differences among genotypes were observed for the content of total and individual isoflavones in both 1995 and 1996 (Tables 1 and 2). Genotype differences had been reported previously in other studies (Eldridge and Kwolek, 1983; Wang and Murphy, 1994). It was possible in our study to evaluate the interaction of genotypes with years and locations. The genotype x year and genotype x year x location interactions in the combined analyses of variance across years as well as the genotype x location interactions in at least one of the 2 yr were significant for total and individual isoflavone contents (Table 1). The significant interactions were associated primarily with changes in rank and magnitude of differences among genotypes with intermediate isoflavone concentrations. Despite the significant interactions, the performance of the two genotypes with the highest and lowest mean total isoflavone concentration was relatively consistent among the 16 environments (Table 3). Kenwood 94 had the highest mean total isoflavone content, the highest rank among the genotypes in nine of the 16 environments, and a lower content than Vinton 81 in only one environment. Vinton 81 had the lowest mean content of total isoflavones and the lowest rank in nine environments. Previous studies have evaluated total and individual isoflavone contents of genotypes at different locations. Eldridge and Kwolek (1983) found that `Corsoy 79' had greater isoflavone contents than `Hardin' at each of four locations in Illinois during 1980. Tsukamoto et al. (1995) evaluated cultivars ranging from Maturity Group I to VI at two locations in Japan for two or more planting dates. At Tsukuba, the ranking of four cultivars for total isoflavone content was identical for two planting dates; however, the cultivar with the greatest content at Tsukuba ranked only third at Kyushu at two planting dates. In contrast, the cultivar with the lowest total isoflavone content at two planting dates at Tsukuba had the lowest content at two planting dates at Kyushu. The consistency of the ranking among genotypes for the contents of total and individual isoflavones seems to depend on the magnitude of the differences in their inherent genetic potential for the traits.

The cause of the fluctuations in isoflavone concentrations among environments observed in our study could not be determined because stages of development were not recorded. Tsukamoto et al. (1995) conducted the only study that has been reported in which the plants were grown in a growth chamber to control the temperature. They found that isoflavone concentrations were significantly greater at low temperature during seed development than at high temperature. In the field, they observed that seed that matured at low temperatures had a greater isoflavone content than seed that matured at high temperatures.

The genotype differences for total and individual isoflavone contents indicated that it would be possible to select for the traits in a cultivar development program. In designing a breeding strategy, total and individual isoflavone contents can be considered quantitative traits because of the continuous variation that has been observed among genotypes and the significant role of environment on the contents that have been obtained. Only a limited number of cultivars have been included in the studies that have been reported. A more extensive evaluation of elite and plant- introduction germplasm would be desirable to determine the range of contents that are available for a breeding program. It would be critical for such evaluations to plant the test genotypes on the same date in the same location and to take into consideration their time of maturity (Tsukamoto et al., 1995). To compare genotypes grown in different tests, multiple check genotypes should be included that are common to the tests. A limited number of environments could be used for the initial evaluation to identify genotypes that differ appreciably for isoflavone contents. More extensive tests across years and locations would be desirable to select among an elite group of genotypes.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Journal Paper no. 18417 of the Iowa Agric. and Home Econ. Exp. Stn., Ames, IA; Projects no. 3353 and 3107, and supported by the Hatch Act, State of Iowa, and Iowa Soybean Promotion Board.

Received for publication May 24, 1999.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 

• Adlercreutz H., Fotsis T., Bannwart C. Determination of urinary lignans and phytoestrogen metabolites, potential antiestrogens and anticarcinogens, in urine of women on various habitual diets. J. Steroid Biochem. 1986;25:791-797.[ISI][Medline]
• Akiyama T., Ishida J., Nakagawa S., Ogawara H., Watanabe S., Itoh N., Shiuya M., Fukami Y. Genistein, a specific inhibitor of tyrosine protein kinases. J. Biol. Chem. 1987;262:5592-5595.[Abstract/Free Full Text]
• Eldridge A., Kwolek W. Soybean isoflavones: Effect of the environment and variety on composition. J. Agric. Food Chem. 1983;31:394-396.[ISI][Medline]
• Murphy P., Song T., Buseman G., Barua K. Isoflavone in soybased-infant formula. J. Agric. Food Chem. 1997;45:4635-4638.
• Naim M., Gestetner B., Bondi A., Birk Y. Antioxidative and antihemolytic activity of soybean isoflavones. J. Agric. Food Chem. 1976;22:806-811.
• SAS Institute. SAS guide for personal computers, 6th ed Cary, NC: SAS Inst, 1992.
• Tsukamoto C., Shimada S., Igita K., Kudou S., Kokubun M., Okubo K., Kitamura K. Factors affecting isoflavone content in soybean seeds: Changes in isoflavones, saponins, and composition of fatty acids at different temperatures during seed development. J. Agric. Food Chem. 1995;43:1184-1192.
• Wang H., Murphy P. Isoflavone composition of American and Japanese soybeans in Iowa: Effects of variety, crop year, and location. J. Agric. Food Chem. 1994;42:1674-1677.

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