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ANALYSIS OF SOYBEAN SEED LIPOXYGENASES.

SOYBEAN SEEDS contain three lipoxygenase isozymes designated L-1, L-2, and L-3 that are controlled by three separate genes, Lx1, Lx2, and Lx3 (Axelrod et al., 1981). The isozymes play a role in the development of grassy and beany off-flavors in foods containing soy protein by oxidation of polyunsaturated fatty acids (Wilson, 1996). The absence of each isozyme is controlled by the null alleles lx1, lx2, and lx3 (Hildebrand and Hymowitz, 1981; Kitamura et al., 1983; Davies and Nielsen, 1986). There was a tight repulsion-phase linkage between the alleles at the Lx1 and Lx2 loci (lx1Lx2/ Lx1lx2), and the Lx3 locus was independent of Lx1 and Lx2 (Hildebrand, 1996). The tight repulsion-phase linkage was broken by mutagenesis resulting in a tight coupling-phase linkage between the alleles at the Lx1 and Lx2 loci (lx1lx2/Lx1Lx2), which made it possible to obtain the triple-null genotype lx1lx1lx2lx2lx3lx3 (Hajika et al., 1991; Kitamura, 1991). For a breeding program, the three null alleles can be considered a two-gene system because of the extremely tight linkage between lx1 and lx2.

The triple-null genotype leads to no detectable level of the lipoxygenase proteins in mature soybean seed with a resultant absence of lipoxygenase activity. The absence of the three seed lipoxygenase isozymes does not influence the performance of soybean for agronomic traits and seed composition (Narvel et al., 1998). The use of lipoxygenase-free soybean has been shown to improve the flavor of tofu and soymilk, but no marked improvement in the flavor or quality of oil has been observed (King et al., 1998; Torres-Penaranda et al., 1998).

The development of triple-null soybean cultivars requires testing procedures to select for the appropriate genotype. Suda et al. (1995) developed three colorimetric assays to select for the triple-null genotype by detecting an absence in activity of each isozyme. Their procedure utilized homogenized soy flour from seed samples to screen for the lipoxygenases with a dye-substrate (test) solution. Test specificity for the L-1, L-2 or L-3 test is based on the pH of the test solution for which the corresponding isozyme has optimum activity. The substrate for each test is linoleic acid, and the dye is used to detect its oxidation by one of the lipoxygenase isozymes. The dye for the L-1 and L-2 tests is methylene blue and for the L-3 test is [Beta]-carotene. L-3 has stronger activity towards the oxidation of both linoleic acid and [Beta]-carotene than do L-1 or L-2 (Suda et al., 1995). This enables separate detection of L-2 and L-3 that otherwise have similar pH ranges for activity. Each test is based on the principle that if the isozyme is present, linoleic acid is oxidized, the dye is reduced for the L-1 and L2 tests or oxidized for the L-3 test, and the test solution is bleached clear. If the isozyme is absent due to a null genotype, no reactions occur, and the test solution remains colored.

Although the assays developed by Suda et al. (1995) are effective, the length of time required and materials needed to prepare homogenized soy flour would be a limiting factor for analyzing a large number of samples in a breeding program. An alternative procedure was tested that consisted of splitting the seed with a razor blade or breaking it with a hammer to expose the cut or broken surface to the test solution. The objective of this study was to report on the modifications that have been made to the original assays to facilitate large-scale analysis.

Materials and Methods

Method of Analysis

Detailed descriptions of the assays are provided to facilitate their use by other researchers. Individual seeds were tested for the triple-null genotype by splitting each seed with a razor blade into two portions, with about one-third of the seed lacking the embryonic axis and two-thirds containing it. The portion containing the embryonic axis was kept for planting and the other portion was split again into at least two pieces, one piece each for the L-1 and L-3 tests. When the L-2 test was conducted, the portion of the seed that lacked the embryonic axis was split into three pieces. To identify triple-null plants in a population segregating for the alleles, four individual seeds were tested from each plant to be 99% certain that the loci were homozygous (Sedcole, 1977; Fehr, 1987). Each of the four seeds were placed into separate envelopes and broken with a hammer to produce several seed chips. The size of the seed chip needed for each test was as small as 10 mg.

Before adding water or test solution to the test samples, control samples were analyzed to make sure that the solutions were properly prepared. A few seeds from a triple-null and a normal cultivar were used as the control samples. Two blanks consisting of 0.5 mL of water and 2.0 mL of test solution were also used for color comparison during analysis.

A single chip from an individual seed or a single chip from each of four seeds from an individual plant was placed into a 12- X 75-mm test tube, and 0.5 mL of distilled water was added. Samples were left undisturbed for at least 10 min for the L-1 or the L-2 test or at least 30 min for the L-3 test. After the soaking period, 2 mL of the appropriate test solution was added.

The samples were scored after 15 min following the addition of the L-1 test solution. Samples were scored as lx1lx1 when the test solution remained blue-colored or as Lx1_ when the solution turned clear. Only lx1lx1 seeds or plants were tested for L-3. Samples were scored as lx3lx3 when the test solution remained yellow-colored or as Lx3_ when the solution turned clear.

Preparation of Solutions

The minimum amount of test solution that was prepared was for 20 samples to ensure pipetting accuracy. To avoid running out of test solution during the analyses, enough test solution was prepared for the equivalent of an additional five samples when 100 samples were tested or an additional 10 samples when more than 100 samples were tested. The test solutions were prepared at the time of the analysis and were stored in the refrigerator when not in use.

The L-1 test solution was prepared by adding 1.25 mL of L-1 buffer, 0.25 mL of methylene blue, 0.25 mL of distilled water, and 0.25 mL of substrate solution for each sample. The L-2 test solution was prepared by adding 1.25 mL of L-2 buffer, 0.25 mL of methylene blue, 0.25 mL of acetone, 7.8 mg of dithiothreitol, and 0.25 mL of substrate solution for each sample. The amount of dithiothreitol needed was 3.9 mg [mL.sup.-1] of the total volume of test solution desired. The L-3 test solution was prepared by adding 1.25 mL of L-3 buffer, 0.25 mL of dye solution, and 0.5 mL of substrate solution for each sample. The test solutions were gently mixed by hand. The substrate solution was made daily to avoid auto-oxidation of the substrate. Suda et al. (1995) reported that the substrate solution could be stored for several weeks at -20 [degrees] C if oxygen is removed from the sample bottle by blowing it out and replacing it with an inert gas, such as nitrogen or argon. The minimum amount of substrate solution prepared was for 20 samples to ensure pipetting accuracy. The total volume of substrate solution needed for the L-1 or L-2 test was 0.25 mL per sample and for the L-3 test was 0.50 mL per sample.

The substrate solution was prepared in an Erlenmeyer flask by adding a volume of autoclaved distilled water equal to one-fourth of the total volume of substrate solution desired. An aliquot of 1.2 [micro]L of polyoxyethylenesorbitan mono-laurate (Tween 20) per sample for the L-1 or L-2 test or 2.4 [micro]L per sample for the L-3 test was added by using a pipette tip with the tip cut off. An aliquot of 0.98 [micro]L of linoleic acid per sample for the L-1 or L-2 test or 1.96 [micro]L per sample for the L-3 test was added and gently mixed to avoid the formation of air bubbles until the solution turned milky white. An aliquot of 8.75 [micro]L of 0.5 M sodium hydroxide per sample for the L-1 or L-2 test or 17.5 [micro]L per sample for the L-3 test was added and gently mixed until the solution turned clear. The substrate solution was brought to its total volume by adding autoclaved distilled water. The substrate solution was covered with Para-film (American Can Co., Greenwich, CT) and was kept in the refrigerator until used.

The dye for the L-1 or L-2 tests (200 [micro]M methylene blue) was prepared by adding 0.075 g of methylene blue to 1.0 L of distilled water in a screw-top bottle. The bottle was capped and shaken vigorously for about 10 s. This was enough dye to test about 4000 samples. The dye was stored at room temperature for up to 6 mo. The minimum amount of dye made for the L-3 test was for 20 samples to ensure pipetting accuracy. The dye was prepared by adding 0.125 mg of [Beta]-carotene to 0.125 mL of acetone on a per sample basis. The solution was mixed by hand and filtered with a Buchner funnel using Whatman No. I filter paper. The filtrate was transferred to a clean flask and 0.125 mL of acetone was added on a per sample basis to bring the solution to 50% saturation. The solution was mixed by hand and filtered again using new filter paper. The filtrate was transferred to a clean flask, covered with Parafilm, and stored in a refrigerator until needed. The dye was kept in a brown-colored glass container if stored for longer than 1 h. The dye may be prepared by the centrifugation method described by Suda et al. (1995).

The L-1 buffer (200 mM sodium borate; pH 9.0) was prepared by adding 11.1 g of sodium chloride (NaCl) and 3.8 g of borax ([Na.sub.2][B.sub.4][O.sub.7] [multiplied by] 10 [H.sub.2]O) to 1.0 L of distilled water. The L-2 buffer (200 mM sodium phosphate; pH 6.0) was prepared by adding 8.8 g of NaCl, 6.0 g of sodium phosphate monobasic (Na[H.sub.2][PO.sub.4] [multiplied by] [H.sub.2]O), and 0.9 g of sodium phosphate dibasic ([Na.sub.2][HPO.sub.4]) to 1.0 L of distilled water. The L-3 buffer (200 mM sodium phosphate; pH 6.6) was prepared by adding 8.8 g of NaCl, 5.2 g of sodium phosphate monobasic, and 1.8 g of sodium phosphate dibasic to 1.0 L of distilled water. The buffers were adjusted to their desired pH with an appropriate strong acid or base.

Results and Discussion

The information presented is based on the analysis of thousands of samples over several years for development of lipoxygenase-free soybean cultivars. The average reaction time for the L-1 test was [approximately equals] 3 min for the analysis of individual seed chips or a bulk of chips from four seeds from an individual plant. Suda et al. (1995) reported a similar reaction time for the analysis of homogenized soy flour for L-1. There was some variation in reaction time among different seed samples. To account for the variability, the samples were scored 15 min after the addition of the test solution. Additional time before scoring did not influence the results because the L-1 test was found to be very stable and the test solution remained blue for more than 1 d with lx1lx1 samples. One variation that was encountered in the analysis of the bulk samples was a pale blue color of the test solution that often made it difficult to differentiate between lx1lx1 nulls and the Lx1_ genotype. To circumvent this problem, the concentration of methylene blue dye was increased to 200 [micro]M from the 100 [micro]M concentration used by Suda et al. (1995), which was found to be superior for color separation of individual or bulk samples.

The average reaction time for the L-2 test was [approximately equals] 5 min for individual seed chips, similar to the time reported by Suda et al. (1995). The L-2 test was scored 15 min after the addition of the test solution to account for the variation in reaction time that was observed among seed samples. The samples were scored within 1 h because the solution occasionally turned clear whether or not L-2 was present. This was likely due to nonspecific dye bleaching by dithiothreitol. Evaluation of a bulk of seed chips did not provide satisfactory results for the L-2 test. Bleaching of the dye did not consistently occur when at least one normal seed chip was mixed with lx2lx2 null chips.

The L-3 test was the slowest and the least stable of the three tests. The average reaction time for the analysis of individual seed chips was [approximately equals] 15 min, but varied considerably. The primary difficulty with the test was that the bleaching of [Beta]-carotene often was incomplete in the presence of L-3 when conducted under conditions similar to the L-1 and L-2 tests. Suda et al. (1995) used an L-2 isozyme extract instead of water for soaking the samples because they found that L-2 had slight activity towards [Beta]-carotene bleaching. In our evaluation of the procedure, the L-2 isozyme extract had no significant effect. To enhance the bleaching process, twice the concentration of linoleate substrate was used in the test solution. Because the bleaching is dependent both on the presence of L-3 and on the oxidation of linoleate, doubling the linoleate concentration enhanced the bleaching of [Beta]-carotene. Several other modifications were employed to further increase the accuracy of the L-3 test. The amount of time the seed chips were allowed to soak in water before the addition of the test solution was three times greater than the time allowed for the L-1 or L-2 test. This presumably increased the amount of L-3 that was extracted. To account for the wide variation in bleaching time that was observed among seed samples, the samples were scored 1.5 h after the addition of the test solution. The samples were scored within 2 h because the solution often turned clear whether or not L-3 was present. This was likely due to light-activated bleaching of [Beta]-carotene. The use of blank and control samples also greatly improved the accuracy in scoring the test. It should be noted that triple-null seeds be used as controls for the L-3 test and not seeds that are null for lx3lx3 because of the potential for endogenous L-2 to contribute to the bleaching process.

Evaluation of a bulk of seed chips did not provide satisfactory results for the L-3 test. Bleaching of the dye did not consistently occur when at least one normal seed chip was mixed with lx3lx3 null chips. It also was difficult to score the results due to turbidity that developed in the test sample.

The use of individual seed chips for the three tests and the use of a bulk sample of seed chips for the L-1 test proved successful. Although the allotted time before scoring was greater for the modified procedures compared with the procedures reported by Suda et al. (1995), the time saved in sample preparation more than compensated for the delay in scoring. In an 8-h day, two people could prepare and evaluate at least 300 seed samples for a single assay. Preparing homogenized soy flour for analysis would limit the number of samples that could be evaluated to only a fraction of this amount.

One of the most attractive features of the assays is their low cost. The cost of the test solutions was approximately $0.01 per sample for the L-1 test, $0.09 for the L-2 test, and $0.02 for the L-3 test. Because of the tight coupling-phase linkage between lx1 and lx2, the use of the L-2 test is optional because a seed or plant that is lx1lx1 should be lx2lx2, if it was derived from a triple-null X normal cross. For procedural efficiency, the L-1 test was conducted first because it was the most rapid, stable, and least expensive. In testing thousands of samples in our breeding program, only a few putative recombinants have been detected. The L-2 test has been used primarily during backcrossing to ensure the transfer of the triple-null alleles or during the purification stages of cultivar development.

References

Axelrod, B., T.M. Cheesborough, and S. Laasko. 1981. Lipoxygenase from soybean. Meth. Enzymol. 71:441-451.

Davies, C.S., and S.S. Nielsen. 1986. Genetic analysis of a null-allele for lipoxygenase-2 in soybean. Crop Sci. 26:460-462.

Fehr, W.R. 1987. Backcross method, p. 366-369. In Principles of cultivar development. Vol. 1. Theory and technique. Macmillian Publ., New York.

Hajika, M., K. Igita, and K. Kitamura. 1991. A line lacking all three seed lipoxygenase isozymes in soybean induced by gamma-ray irradiation. Jpn. J. Breeding. 41:507-509.

Hildebrand, D.F. 1996. Genetics of soybean lipoxygenases, p. 33-38. In G. Piazza (ed.) Lipoxygenase and lipoxygenase pathway enzymes. Am. Oil Chem. Soc. Press, Champaign, IL.

Hildebrand, D.F., and T. Hymowitz. 1981. Soybeans lacking lipoxygenase-1. J. Am. Oil Chem. Soc. 58:583-586.

King, J.M., L.K. Svendsen, W.R. Fehr, J.M. Narvel, and P.J. White. 1998. Oxidative and flavor stability of oil from lipoxygenase-free soybeans. J. Am. Oil Chem. Soc. 75:1121-1126.

Kitamura, K. 1991. Spontaneous and induced mutations of seed storage proteins in soybean [Glycine max (L.) Merr.]. Gamma Field Symposia 30:61-69.

Kitamura, K., C.S. Davies, N. Kaizuma, and N.C. Nielsen. 1983. Genetic analysis of a null-allele for lipoxygenase-3 in soybean seeds. Crop Sci. 58:583-586.

Narvel, J.M., W.R. Fehr, and G.A. Welke. 1998. Agronomic and seed traits of soybean lines lacking seed lipoxygenases. Crop Sci. 38: 926-928.

Sedcole, J.R. 1977. Number of plants necessary to recover a trait. Crop Sci. 17:667-668.

Suda, I., M. Hajiki, Y. Nishibi, S. Fururta, and I. Kazunori. 1995. Simple and rapid method for the selective detection of individual lipoxygenase isozymes in soybean seeds. J. Agric. Food Chem. 43:742-747.

Torres-Penaranda, A.V., C.A. Reitmeier, L.A. Wilson, W.R. Fehr, and J.M. Narvel. 1998. Sensory characteristics of soymilk and tofu made from lipoxygenase-free and normal soybeans. J. Food Sci. 63(6):1084-1087.

Wilson, L.A. 1996. Comparison of lipoxygenase-null and lipoxygenase containing soybeans for foods, p. 209-222. In G. Piazza (ed.) Lipoxygenase and lipoxygenase pathway enzymes. Am. Oil Chem. Soc. Press, Champaign, IL.

JAMES M. NARVEL, WALTER R. FEHR,(*) AND LINDA C. WELDON

Dep. of Agronomy, Iowa State Univ., Ames, IA 50011. Journal Paper No. J-18494 of the Iowa Agric. and Home Econ. Exp. Stn., Ames. Project No. 3107 and supported by the Hatch Act, State of Iowa, and Iowa Soybean Promotion Board. Received 9 July 1999. (*) Corresponding author (wfehr@iatate.edu).
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Author:NARVEL, JAMES M.; FEHR, WALTER R.; WELDON, LINDA C.
Publication:Crop Science
Date:May 1, 2000
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