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a Dep. of Agronomy, Iowa State Univ., Ames, IA 50011-1010
* Corresponding author (wfehr{at}iastate.edu).
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ABSTRACT |
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Abbreviations: ACP, acyl carrier protein • DAF, days after flowering • EMS, ethyl methanesulfonate
Received for publication April 7, 2007.
a Dep. of Agronomy, Iowa State Univ., Ames, IA 50011-1010
* Corresponding author (wfehr{at}iastate.edu).
Genetic modification of the fatty acid composition of soybean [Glycine max (L.) Merr.] oil has been successful in better meeting the needs of end users than is possible with conventional oil. Three modified oils are or have been sold commercially. Oil in which the linolenic acid (18:3) content has been reduced from 8 to 1% makes it possible to reduce or eliminate the need for chemical hydrogenation to achieve the stability and shelf life necessary for some food applications. Elimination of chemical hydrogenation and the trans-fatty acids produced by the process is important for human health. Oil in which the oleic acid (18:1) has been increased from 25 to >80% also have increased its stability and shelf life. An oil with palmitic acid (16:0) reduced from 11 to <4% makes it possible to achieve a low content of saturated fatty acids, which is desirable for cardiovascular health. The genetic changes in soybean oil were achieved by conventional breeding and genetic engineering. Mutagenesis was the conventional breeding method used to develop the major genes for reduced palmitic and linolenic acids that are in the cultivars currently grown for commercial production. Genetic engineering was used to elevate oleic acid to >80%. The purpose of this paper is to review the methods that have been used to develop the fatty acid modifications, the inheritance of the modifications, the impact of the trait on agronomic and seed characteristics, the methods of phenotypic and genotypic selection, and the commercial status of the modified oils.
Abbreviations: ACP, acyl carrier protein • DAF, days after flowering • EMS, ethyl methanesulfonate
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INTRODUCTION |
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There are five steps in the development of a modified oil that can be sold commercially. (i) Genes for modified fatty acid composition are developed by conventional breeding or genetic engineering. (ii) The inheritance of the modification is determined. (iii) An assessment is made of the impact of the modification on agronomic and seed traits. (iv) Breeding is conducted to develop acceptable cultivars. (v) Markets for the novel oil are established. Each of these components will be discussed for the five major fatty acids in soybean oil.
The fatty acid data provided in this paper were determined by gas chromatography. The general method has been described by Hammond and Fehr (1984b) and Hammond (1991). The fatty acids in the oil are converted to fatty esters for measurement by gas chromatography. The data obtained from the gas chromatograph are converted to a percentage of the total fatty acids that are measured. The percentages used in this paper can be multiplied by 10 to obtain grams per kilogram.
The fatty acid modifications will be discussed in the order in which they have been commercialized. An oil with reduced linolenic acid was sold commercially from the crop grown in 1994, a low-saturate oil from the crop of 1996, and a high oleic acid oil from the crop of 2001. An oil with modified stearic acid content has not been commercialized.
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Linolenic Acid (Linolenate) (18:3) |
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Reduced Linolenate
Dutton et al.(1951) reported that there was circumstantial evidence that linolenate was a primary cause of the poor oxidative stability of soybean oil. Subsequent research demonstrated that the three double bonds in linolenate made it susceptible to oxidation, which resulted in the formation of undesirable off-flavors in food products. As a result, chemical hydrogenation was adopted to reduce the content of linolenate in the oil (Dutton, 1963; Okkerse et al., 1967).
Breeding was initiated by the USDA-ARS in 1952 to identify soybean germplasm with reduced linolenate as an alternative to chemical hydrogenation (White et al., 1961). Plant introductions with lower linolenate than conventional cultivars were identified, but none of them had <4% linolenate (Kleiman and Cavins, 1982). Our breeding research on fatty acid modification at Iowa State University began in 1968 as the result of a visit by E.G. Hammond to the Unilever Corporation in Vlaardingen, the Netherlands. The company was interested in reducing the linolenate content of soybean oil as a means of minimizing the need for chemical hydrogenation and the trans-fat produced by the process. With their financial support, we began our research by obtaining from A.H. Probst of the USDA-ARS at Purdue University the plant introductions with the lowest linolenate identified in the research he had been conducting (White et al., 1961). We initiated a recurrent selection program with the accessions, but were not able to identify any genotypes with substantially reduced linolenate. A recurrent selection program for increased oleate and a high [18:1/(18:2 + 18:3)] ratio conducted by the USDA-ARS and North Carolina State University was successful in decreasing linolenate below that of conventional soybeans, but the best lines they reported had 4.2% (Wilson et al., 1981).
Our second strategy was to use X-ray irradiation to induce genetic changes (Hammond and Fehr, 1975; Hammond et al., 1972). We found individual mutant plants that had interesting modifications in fatty acid composition, but none of them produced progeny with the same modification. The use of X-ray irradiation was replaced by chemical mutagenesis.
In 1978, we conducted our first seed treatments with ethyl methanesulfonate (EMS) using the lines with the lowest linolenate from our recurrent selection program (Hammond and Fehr, 1984a, 1984b). The treatment of FA9525 that traced to PI 80476 and PI 85671 obtained from A.H. Probst produced the line A5 that had about two percentage units lower linolenate than its parent (Hammond and Fehr, 1983a). At the same time we were conducting our mutagenesis program, Wilcox et al. (1984) treated ‘Century’ with EMS and selected the line C1640 that had 3.6% linolenate. The reduced linolenate in C1640 was controlled by allele fan1 (Wilcox et al., 1984; Wilcox and Cavins, 1985, 1987). When C1640 was crossed to A5, no transgressive segregation for linolenate was observed and the allele in A5 was designated fan1(A5) (Rennie and Tanner, 1991).
Additional alleles at the fan1 locus have been reported. An allele for reduced linolenate in PI 123440 was reported by Rennie and Tanner (1989). The allele was originally designated fan (PI 123440), but has been redesignated fan1-nc (Joseph W. Burton, personal communication, 2006). Stojsin et al. (1998b) developed the line RG10 with the fan1-b allele by treating seeds of C1640 with EMS. The lines M-5, IL-8, and KL-8 were developed by X-ray irradiation of ‘Bay’ (Rahman and Takagi, 1997; Rahman et al., 1995). M-5 and IL-8 had alleles at the fan1 locus that are designated herein as fan1(M-5) and fan1(IL-8). The allele in KL-8 is not at the same locus as fan1(M-5) (Rahman and Takagi, 1997).
The molecular basis of some of the alleles at the fan1 locus has been determined. Byrum et al. (1997) indicated that reduced linolenate in A5 was at least partially due to a full or partial deletion of a microsomal omega-3 desaturase gene. Bilyeu et al. (2003) reported that the soybean genome has at least three versions of the FAD3 omega-3 fatty acid desaturase gene that is responsible for conversion of linoleate to linolenate. They designated the three forms GmFAD3A, GmFAD3B, and GmFAD3C. They determined that the reduced-linolenate line A5 with the fan1(A5) allele had a deletion in GmFAD3A. They also found that the fan1 allele in C1640 has a mutation in GmFAD3A (Chapell and Bilyeu, 2006).
Molecular analysis of mutant lines developed in Japan has been reported by Anai et al. (2005). They considered four forms of the FAD3 gene that they designated GmFAD3-1a, GmFAD3-1b, GmFAD3-2a, and GmFAD3-2b. The GmFAD3-1a gene of Anai et al. (2005) is equivalent to the GmFAD3B designation of Bilyeu et al. (2003), GmFAD3-1b is equivalent to GmFAD3A, and GmFAD3-2a is equivalent to GmFAD3C (K. Bilyeu, personal communication, 2006). The mutant lines J18 and M5 with reduced linolenate had deletions at GmFAD3-1b (GmFAD3A) (Anai et al., 2005) Based on their molecular data, the allele in J18 is designated herein as fan1(J18).
Molecular analysis of germplasm with the fan1-nc allele indicated that it has a mutation in GmFAD3A gene (Ralph Dewey, personal communication, 2006). The molecular basis of the fan1-b allele in RG10 has not been determined (Stojsin et al., 1998b).
In 1982, we treated the line FA47437 with EMS and recovered a line with greater palmitate than its parent. The line was originally designated PA2 and later given its permanent designation A23 (Bubeck et al., 1989). We crossed A5 with A23 to determine if we could combine the reduced linolenate of A5 with elevated palmitate of A23. To our surprise, we found the lines A16 and A17 that had about 2.5% linolenate (Fehr and Hammond, 1998; Fehr et al., 1992). A subsequent inheritance study indicated that A23 had the mutant allele fan2 (Fehr et al., 1992). The fan2 allele in A16 and its descendant ‘IA3018’ has a single nucleotide mutation in GmFAD3C that results in the substitution of a tyrosine for a histidine (Bilyeu et al., 2006).
A second allele at the fan2 locus was identified in AX5152-44 by molecular analysis (Bilyeu et al. 2005). The allele will be referred to herein as fan2-b. It has a single nucleotide mutation in GmFAD3C that results in the substitution of a glutamic acid for a glycine.
We continued our mutagenesis program and found a reduced-linolenate line A26 by treating A89-144003 with EMS. A26 had the mutant allele fan3 (Fehr and Hammond, 2000). By combining the fan1(A5), fan2, and fan3 alleles, the line A29 was developed that had only 
1.0% linolenate (Fehr and Hammond, 2000; Ross et al., 2000). The fan3 allele has a mutation in the gene GmFAD3B (Bilyeu et al., 2006).
A second mutant allele at the fan3 locus has been identified in the mutant line M24 that was developed by X-ray mutation of Bay (Anai et al., 2005). It is the result of a deletion in the gene GmFAD3-1a (GmFAD3B). The allele has been designated fan3-b (T. Anai, personal communication, 2006).
An understanding of the DNA sequence of the major alleles for reduced linolenate has made it possible to develop direct molecular markers for selection. The markers have been used effectively to select for reduced linolenate in breeding populations (Beuselinck et al., 2006).
Modifying genes influence the linolenate content of lines with major fan alleles. Graef et al. (1988) evaluated the segregation from crosses of A5 with two conventional cultivars, ‘Weber’ and ‘Pella’. The continuous distribution for linolenate among F2:3 lines from both crosses indicated that modifying genes were important in the populations segregating for the fan1(A5) allele. Practical experience in breeding cultivars with reduced linolenate has confirmed that modifying genes are responsible for differences among cultivars that have the same major fan alleles.
Phenotypic selection for reduced linolenate is possible with single seeds, individual plants, individual plots, or replicated plots. Single seed can be evaluated by cutting or chipping a seed and analyzing by gas chromatography the part without the embryonic axis. We used this procedure in the early years of our backcrossing program to incorporate the fan alleles into elite conventional cultivars. The F2 seeds with the lowest linolenate were planted and backcrosses made with as many plants as possible. A five-seed bulk sample of selfed seed was analyzed from each plant used for crossing and the hybrid seed was advanced from those plants with the lowest linolenate.
We have worked with MTEC BioAnalytical, Inc., Ames, IA (www.mtecbioanalytics.com), for their development of calibrations for single-seed selection of linolenate by near-infrared transmission spectroscopy. In 2006, the company analyzed individual F2 seed from single-cross populations formed by crossing 1%-linolenate parents to elite conventional parents. After seeds were selected for <3% linolenate, we analyzed the fatty acid content of selected and unselected seeds to determine how many seeds with <3% linolenate had been correctly and incorrectly classified. There were 53 of 100 selected seeds that had <3.0% linolenate and only 4 of 100 unselected seeds that had <3% linolenate. As a result of the selection, we were able to reduce the amount time, space, and cost associated with planting subsequent generations.
The impact of reduced linolenate on agronomic and seed traits has been evaluated in several studies. Walker et al. (1998) compared lines that had 2.8% linolenate and normal lines with 7.6% linolenate from three single-cross populations. The reduced-linolenate lines were not significantly different in agronomic traits than the normal lines in two of the three populations. Ross et al. (2000) compared lines that had an average of 1.3% linolenate with lines that had an average of 2.5% linolenate from three single-cross populations. The lines with 1.3% linolenate were not significantly different in yield than those with 2.5% linolenate in two of the three populations. Both studies indicated that the difference in yield between lines that differ in linolenate content varies among populations. Therefore, it is important to evaluate progeny with reduced linolenate from as many matings between reduced-linolenate and conventional parents as possible. In both studies, lines with different levels of linolenate had similar performance for other agronomic and seed traits that were evaluated. McCord et al. (2004) evaluated the tocopherol (vitamin E) content of lines with 1 and 7% linolenate from three single-cross populations. The average total tocopherol content of the reduced-linolenate lines was 6.0% less than the normal-linolenate lines.
The performance of the newest cultivars with reduced linolenate is the same as that of conventional cultivars. Our cultivar ‘IA3024’ that has 1% linolenate was compared in 2005 with ‘IA3023’, the highest yielding conventional cultivar in maturity group III, at 10 locations in the Uniform Tests, Northern States and at 13 locations in the Quality Traits Test that are conducted by public soybean breeders in the United States. The two cultivars were the highest yielding entries in both tests. IA3024 had an average yield that was 54 kg ha–1 (0.8 bu acre–1) less than IA3023 in the Uniform Test and 40 kg ha–1 (0.6 bu acre–1) more that IA3023 in the Quality Traits Test.
The flavor quality and stability of oils with different levels of linolenate has been assessed. Mounts et al. (1988) compared the stability of soybean oils with 3.3, 4.2, 4.8, and 7.7% linolenate. The three oils with 3.3 to 4.8% had improved odor stability at high temperatures compared with the 7.7% linolenate oil. Liu and White (1992) evaluated the oxidative stability of oil from three lines that had 1.5 to 2.0% linolenate compared with oil from two conventional cultivars. The reduced-linolenate oils were more stable to peroxide development and had more acceptable flavor quality scores than the conventional soybean oil. Warner and Gupta (2003) fried potato chips with 2% (low) and 0.8% (ultra-low) linolenate soybean oils. They found that for some potato chip samples the decrease in linolenate from 2 to 0.8% improved the flavor quality and oxidative stability.
Our initial cultivar development for reduced linolenate was with the fan1(A5) allele. In 1989, sufficient seed of the lines was produced by farmers in Iowa to obtain oil for commercial testing. The oil with 3.4% linolenate was not considered stable enough to eliminate the need for hydrogenation and no commercial interest developed in the oil.
In cooperation with Pioneer Hi-Bred International, Inc. (Pioneer), we developed cultivars with 2.5% linolenate that were grown commercially for the first time in 1994 on about 8000 ha in Iowa. The oil was used for a few years in Japan in a blend with canola oil, but there was no commercial interest in the oil in the United States. Production of the low linolenate soybeans was discontinued when the oil was no longer used in Japan. Ironically, soybean oil with 2.5% linolenate is an important commercial product today.
Although there was some concern before 1970 about the possible negative health consequences of trans-fatty acids in the human diet, it was not until 9 July 2003 that the Food and Drug Administration (FDA) in the United States announced that it would require the labeling of all food products for their trans-fat content beginning 1 Jan. 2006. As a result of the new federal regulation, the food industry in the United States has been actively pursuing alternatives to hydrogenated oils so that their products can be labeled as containing 0 g of trans-fat. Soybean oil with reduced linolenate has been adopted as one of the alternatives to hydrogenated oil.
Since the FDA announcement, soybean oil with two levels of linolenate have entered the market: a low linolenate oil with 2.5 to <3.0% of the fatty acid and an ultra-low linolenate oil with 1.0%. In 2006, there were about 280,000 ha (700,000 acres) of the low- and ultra-low linolenate soybeans in the United States. Production is expected to grow in the future as the food industry decreases its use of hydrogenated oils. Most public and private soybean breeders in the United States are in the process of developing cultivars with low or ultra-low linolenate.
Elevated Linolenate
An oil with elevated linolenate may be useful as a drying oil for paint and other industrial products. Accessions of G. soja, the wild relative of the cultivated soybean, have been reported to contain up to 23% linolenate (Wilson, 2004). Pantalone et al. (1997) crossed three G. soja plant introductions with up to 14.7% linolenate to conventional cultivars and observed progeny with a range of linolenate from 5.0 to 13.1%. Although the linolenate in G. soja accessions is elevated, it is unlikely to be high enough to compete with alternative sources of drying oils.
Cahoon (2003) indicated that soybean seeds with >50% linolenate have been generated by increasing expression of the FAD3 gene that controls the conversion of linoleate to linolenate. He suggested that genetic engineering may be useful for increasing linolenate high enough for use as a drying oil. No commercial cultivars with elevated linolenate have been developed by either conventional breeding or genetic engineering.
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Palmitic Acid (Palmitate) (16:0) |
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Reduced Palmitate
In the United States, all food products must be labeled for their total content of saturated fatty acids because they raise low-density lipoprotein cholesterol that is undesirable for cardiovascular health (Hu et al., 1997). The American Heart Association recommends that the intake of saturated fat be limited to 7 to 10% or less of total calories consumed each day (www.americanheart.org). A food product cannot be advertised as "low in saturated fat" unless it contains 1 g or less of saturated fat per serving. For a liquid soybean oil, 1 g in a 14 g serving is equivalent to 7.1%. When rounding is considered, a product can be labeled as containing 1 g per serving if its content of saturated fatty acids is 1.25 g or less (8.9%).
Soybean oil has about 4.0% stearate (18:0) and about 1.0% of other saturated fatty acids in addition to palmitate. It is too time consuming for evaluating thousands of individuals in a breeding program to measure all of the saturated fatty acids by gas chromatography; therefore, only palmitate and stearate are measured in most breeding programs. In our breeding program at Iowa State University, the contents of the two fatty acids are added together for selection in a breeding program. To meet the requirements for a low-saturate oil, only individuals with <7.5% saturates (palmitate + stearate) are considered acceptable. The preferred level is <7.0% palmitate + stearate to account for environment effects on the content of the saturated fatty acids and unintended co-mingling with conventional soybeans during commercial production of the oil.
Low-saturate soybean oil is possible due to major alleles that have been developed that reduce the palmitate content. The first major allele reported for reduced palmitate was developed through treatment of seeds of Century with EMS by Erickson et al. (1988). The mutant line C1726 had 8.6% palmitate that was controlled by the mutant allele fap1 allele. We treated seeds of Asgrow ‘A1937’ with N-nitroso-N-methylurea (NMU) and identified a mutant line A1937NMU-173 that had 6.8% palmitate (Fehr et al., 1991a). The line, later given the permanent designation A22, had a single recessive allele fap3 (Schnebly et al., 1994). When A22 was crossed to C1726, transgressive segregates were observed that had only 4.4% palmitate (Fehr et al., 1991a). This reduced level of palmitate made it possible to initiate a cultivar development program to produce a soybean oil that could be labeled as low in saturated fatty acids.
Additional lines with alleles at the fap3 locus have been identified. The fap3-nc allele is a natural mutation in the line N79–2077 derived from the fifth cycle of recurrent selection for elevated oleate (Burton et al., 1994). The allele has a deletion in the GmFATB1a gene encoding 16:0-ACP thioesterase activity (Cardinal et al., 2006). The cross of C1726 to N79-2077-12 resulted in individuals with 4.0% palmitate (Wilcox et al., 1994). The fap3-ug allele, originally designated fap*, in the line ELLP2 was developed by EMS treatment of ‘Elgin 87’ (Stojsin et al., 1998a). The line RG3 with 4.0% palmitate was selected from the cross C1726 x ELLP2 (Primomo et al., 2002a; 2002b). Takagi et al. (1995) treated Bay with X-ray irradiation and recovered a line J3 that had reduced palmitate. The allele in J3 was designated sop1(J3). Kinoshita et al. (1998a) crossed J3 to C1726 with fap1 and C1727 with fap2 and determined that sop1(J3) was not at either of those loci. They obtained a genotype from the cross J3 x C1726 with 3.5% palmitate.
Modifying genes are an important consideration in breeding low-saturate cultivars with major alleles at the fap1 and fap3 loci (Horesji et al., 1994; Rebetzke et al., 1998). The first cultivar grown commercially for the purpose of producing a low-saturate oil was XB37ZA (Fehr and Hammond, 1996). It was developed jointly by Iowa State University and Pioneer. In 1994, the cultivar was grown and processed by Rose Acres Farms, Seymour, IN. The saturate content of the oil from the crop exceeded 1 g per serving and had to be labeled as a "reduced saturate" oil. It was sold in 1995 as the brand New Horizons and the label read: "30% less saturated fat than regular soybean oil" and "Only 1.5 g of saturated fat per serving". The oil was not successful commercially and there was no production of XB37ZA after 1994.
XB37ZA taught us important lessons about the breeding of low-saturate cultivars. (i) To achieve the desired saturate content in progeny from a single cross between a low-saturate parent and a high-yielding conventional parent, it is preferable that the saturate content of the low-saturate parent not exceed 7.0% to maximize the frequency of acceptable individuals in the population. This may exclude as parents high yielding cultivars with >7% saturates that are grown commercially. (ii) As many possible crosses of low-saturate and conventional parents should be made because the frequency of acceptable individuals for saturate content and yield varies among crosses due to segregation of modifying genes for palmitate and stearate (Scherder et al., 2006). In our breeding program, the average frequency of acceptable individuals from a single cross of a low-saturate and a normal parent is 5%.
The relationship of modified palmitate content in the seed to its content in vegetative tissues was evaluated by Schnebly et al. (1996). The palmitate content of root, stem, and leaf tissue at vegetative and reproductive stages was assessed for Elgin 87 with normal palmitate (11.6%), A18 with reduced palmitate (4.5%), and A19 with elevated palmitate (25.5%) in the seed. The palmitate content in the three tissues was the least for A18 and the greatest for A19. The consistent ranking among the three lines for palmitate content in the seed and vegetative tissues indicated that the fap1 and fap3 alleles in A18 and the fap2-b and fap4 alleles in A19 were constitutively expressed, which could influence the agronomic and seed traits of cultivars with reduced and elevated palmitate.
The impact of reduced palmitate on agronomic and seed traits has been evaluated in several studies and the results for some characteristics have not been consistent. Ndzana et al. (1994) crossed reduced-palmitate parents with the fap1 and fap3 alleles to normal-palmitate parents. They found that the mean yield of reduced-palmitate lines from the populations was 7.2% less than normal lines. The oil content of the reduced-palmitate lines was consistently less than the normal lines in all the populations by an average of 1.4% units, but the protein content was not consistently different. Horesji et al. (1994) backcrossed the fap1 and fap3 alleles into ‘Kenwood’ and ‘Marcus’. Lines with reduced palmitate were recovered that had higher yield than the recurrent parents. Rebetzke et al. (1998) crossed a reduced-palmitate line with the fap1 and fap3-nc alleles to Kenwood and Pioneer ‘P9273’. They found that the mean yield of the reduced-palmitate lines was 10% lower than for the normal lines. Protein and oil contents were not consistently different for the two types of lines. Cherrak et al. (2003) evaluated random lines from a single-cross population ranging in palmitate from 4.1 to 12.9% and linolenate from 4.1 to 11.3%. They reported that the phenotypic correlation of 0.12 between yield and palmitate and of 0.00 between yield and linolenate indicated that reduced palmitate and linolenate should not negatively impact yield. Scherder et al. (2006) crossed reduced-palmitate lines with the fap1 and fap3 alleles to normal parents. They found that the mean yield of reduced-palmitate lines was 5.5% less than the normal lines; however, the highest yielding line in one population had reduced palmitate. The total tocopherol (vitamin E) content of oil from their reduced-palmitate lines was 15% greater than for the normal lines. They indicated that it was important to mate as many reduced- and normal-palmitate parents as possible to increase the likelihood of obtaining reduced-palmitate lines that yield as much as normal lines. In all of the aforementioned studies, the frequency distributions of reduced- and normal- palmitate lines for other traits have overlapped, which indicated that it should be possible to select reduced-palmitate cultivars that are comparable to conventional cultivars for maturity, height, lodging, protein content, oil content, seed size, and the content of the other major fatty acids. These results have been confirmed by our practical experience in breeding low-saturate cultivars that are in commercial production.
The performance of the newest cultivars with low saturates is the same as conventional cultivars. Our cultivar IA1020 of maturity group I and IA2075 of maturity group II were compared with the highest yielding conventional cultivars in 10 tests in Iowa during 2005. The two cultivars had higher average yields than conventional cultivars in the tests.
Phenotypic selection for reduced palmitate in a breeding program can be done on single seeds, individual plants, or plots. The most reliable method for determining the palmitate content of a seed for breeding purposes is by splitting the seed and analyzing it by gas chromatography. We have assisted MTEC BioAnalytics, Inc. with the development of a nondestructive method of single-seed selection for palmitate based on near-infrared transmission spectroscopy (www.mtecbioanalytics.com). Our goal was to select 50% of the seeds with the lowest palmitate from populations derived by crossing a reduced-palmitate parent with a conventional one. In 2006, the instrument was calibrated to select F2 seeds with <6.0% palmitate. We analyzed 100 selected and 100 unselected seeds by gas chromatography. There were 76 out of 100 selected seeds with <6.0% palmitate and only 2 out of 100 unselected seeds with <6.0% palmitate. Single-seed selection reduced the number of individuals that had to be grown each generation and that had to be analyzed for saturate content to select plants with <7.5% saturates for the initial yield evaluation.
Palmitate content can be evaluated effectively in a range of environments (Hawkins et al., 1983a, 1983b). Horesji et al. (1994) found no significant genotype x environment interaction for palmitate content across three Iowa locations in 1 yr. Primomo et al. (2002b) evaluated reduced and elevated palmitate lines during 3 yr at four locations in Canada. The genotype x year interaction for palmitate was significant, but not the genotype x location or the genotype x year x location.
In our breeding program, individual plants from a breeding population are analyzed with a five-seed bulk sample and those with <7.5% palmitate + stearate are evaluated for yield and saturate content in one replication at each of two locations. Lines with acceptable yield and <7.5% saturates are evaluated the next season in one replication at five locations. By the end of the second year of testing, the genetic potential of a line for saturate content is well defined.
The first low-saturate cultivar that was grown commercially in 1996 was developed jointly by our program and Pioneer. The low-saturate oil from the crop was sold for the first time in the fall of 1997. It was sold in grocery stores in the midwestern United States and distributed to schools in the United States through the USDA National School Lunch Program and School Breakfast Program as a means of lowering the saturated fat content of the meals served to young people (Mahnken et al., 2001). The oil has not been widely adopted for home use because its saturated fat content is not superior to canola and its cost is higher than canola because it has to be kept separate from conventional soybeans during production of the grain and processing of the oil. Less than 12,000 ha of low-saturate soybeans were grown in the United States during 2006 from cultivars developed by Pioneer or our program.
Elevated Palmitate
Oxidative stability of an oil determines how long it can be used before it develops an unacceptable level of off-flavors. Soybean oil with elevated saturated fatty acids has better oxidative stability than conventional oil (Shen et al., 1997).
Major alleles at five loci have been developed that cause an increase in palmitate and additional mutants have been reported for which allelism tests have not been conducted (Palmer et al., 2004). The fap2 allele in C1727 was developed by EMS treatment of seeds of Century (Erickson et al., 1988). The elevated palmitate was associated with a single base pair substitution in the GmKAS IIA gene that converted a tryptophan codon into a premature stop codon (Aghoram et al., 2006). The fap2-b allele in A19 was developed by NMU treatment of Asgrow ‘A1937’ (Fehr et al., 1991b), the fap4 allele in A24 by EMS treatment of ‘Elgin’ (Schnebly et al., 1994), the fap5 allele in A27 by EMS treatment of Kenwood (Stoltzfus et al., 2000b), the fap6 allele in A25 by EMS treatment of Kenwood (Narvel et al., 2000), and the fap7 allele in A30 by NMU treatment of A89-144026 (Stoltzfus et al., 2000c). Takagi et al. (1995) developed the line J10 by X-ray irradiation of Bay. Rahman et al. (1996a) designated the allele for elevated palmitate as sop2 (J10) because they had not determined its relationship to other fap alleles. By combining fap alleles for elevated palmitate, it has been possible to develop a range of palmitate from normal to >40% of the fatty acid (Stoltzfus et al., 2000a).
Phenotypic selection for elevated palmitate has been conducted on single seeds by gas chromatography (Bravo et al., 1999). It is likely that single-seed selection could be done by near-infrared transmission spectroscopy, but such selection has not been evaluated.
Elevated palmitate can have a negative impact on agronomic and seed traits. Stoltzfus et al. (2000a) evaluated the seed traits of 92 soybean cultivars and lines ranging in palmitate content from 10.3 to 42.7%. They found significant positive phenotypic correlations between palmitate and protein (0.25), stearate (0.58), and linolenate (0.86) contents. Palmitate was negatively correlated with oil (–0.84), oleate (–0.94), and linoleate (–0.96). Hartmann et al. (1996) compared lines with normal palmitate content of 11.0% and lines that had 25.0% palmitate due to the fap2-b and fap4 alleles. The lines with elevated palmitate from two populations averaged 6.3% lower seed yield, 14 g kg–1 lower protein, and 35 g kg–1 lower oil than the normal lines. Bravo et al. (1999) evaluated lines with the fap2-b and fap4 alleles from four single-cross populations and did not find any significant phenotypic correlation of palmitate with seed yield. However, palmitate had a average phenotypic correlation across the populations of –0.47 with oil content, –0.61 for oleate, and –0.59 for linoleate. To evaluate the effects of even higher levels of palmitate, Hayes et al. (2002) compared lines with 26.0% palmitate and lines with 40.0% palmitate that were derived from three populations. The lines with 40% palmitate had an average of 23% less seedling emergence, 814 kg ha–1 lower seed yield, 9 cm less plant height, 15 mg seed–1 smaller seed, 9 g kg–1 higher protein, 16 g kg–1 lower oil, 3.9% units lower oleate, and 8.0% units lower linoleate compared with the lines containing 26% palmitate. These studies indicated that developing elevated-palmitate cultivars comparable to normal cultivars for some agronomic and seed traits would be progressively more difficult as palmitate contents increased.
There is no commercial production of soybean oil with elevated palmitate, even though the oil has greater oxidative stability than conventional soybean oil (Shen et al., 1997). This may be due to the availability of other oils with high saturated fat content, particularly palm oil.
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Oleic Acid (Oleate) (18:1) |
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Conventional Breeding
The highest content of oleate that has been achieved by conventional breeding is >70% (Alt et al., 2005b). It was obtained by intercrossing three lines with 50% oleate: FA22, M23, and N98-4445A. We found FA22 in a population while breeding for reduced linolenate. M23 was developed by treatment of Bay with X-ray irradiation (Rahman et al., 1994, 1996b; Takagi and Rahman, 1996). N98-4445A was selected in a breeding program for modified oil quality and has a complex pedigree (Burton et al., 2006). M23 has the ol allele, which corresponds to a deletion in Fad2-1a, one of the major genes that control the desaturation of oleate to linoleate (Alt et al., 2005a; Kinoshita et al., 1998b). The same deletion does not occur in FA22 and N98-4445A, neither of which are known to have any major genes for elevated oleate content (Alt et al., 2005a; Ralph Dewey, personal communication, 2006).
Although there is a major allele for elevated oleate in M23, individuals in a segregating population that are homozygous for the ol allele have a wide range of oleate content due to modifying genes. Alt et al. (2005a) compared single plants and lines with the olol, Olol, and OlOl genotypes at three environments. Averaged across environments, the olol lines had a mean oleate of 39.4% and a range of 35.0 to 45.1%, the Olol lines had a mean of 31.7% and a range of 25.2 to 42.4%, and the OlOl lines had a mean of 28.9% and a range of 22.0 to 35.7%.
It is our experience that phenotypic selection for oleate content on a single-seed basis is not as reliable as it is for palmitate and linolenate. Alt et al. (2005a) reported that narrow-sense heritability estimates were 0.33 on a single-seed basis in a population segregating for the ol allele. Our results indicate that selection of individual F2 seeds with the intent of using those with the highest oleate for crossing would not be as successful as it is for reduced palmitate or linolenate in capturing the individuals with the best genetic potential for the trait (Curtis Scherder, personal communication, 2006). A sample of 152 individual F2 seeds were analyzed from populations developed from lines with >50% oleate derived from M23 that had been crossed to lines with normal oleate. A five-seed bulk sample was analyzed from each of the 152 F2 plants that were grown from the seeds. Of the 34 F2 seeds with >50% oleate, only 13 of the F2 plants had >50% oleate. Of the 118 F2 seeds with <50% oleate, 21 of the F2 plants had >50% oleate. Selection can be improved by the combination of phenotypic and molecular analysis (Curtis Scherder, personal communication, 2006). In populations segregating for the deletion in M23, individual seeds or a bulk of seeds from plants or lines can be evaluated by gas chromatography and those with the highest oleate can be analyzed for the deletion or the two steps can be reversed. Individuals with elevated oleate that are homozygous for the deletion have a high likelihood of producing elevated oleate in subsequent generations.
Significant genotype x environment interactions have been reported for oleate content (Alt et al., 2005a). Nevertheless, lines with the highest oleate commonly are among the best lines across environments. When Alt et al. (2005a) evaluated lines segregating for the ol allele in two replications at Ames, IA, and Portageville, MO, four of the five olol lines with the highest oleate at Ames were in the top five of the olol lines at Portageville, despite a significant genotype x environment interaction for the trait. The genetic potential of a line for oleate can be reliably estimated by testing in a relatively few environments.
The goal of our breeding program has been to develop a cultivar that would consistently produce an oil with greater than 50% oleate in Iowa and have low saturates, 1% linolenate, or both. Rahman et al. (2001) crossed M23 that has 50% oleate to M5 that has 4.9% linolenate and recovered a line with 54.4% oleate and 4.2% linolenate. They crossed that line to one with 33.9% oleate and 2.9% linolenate and recovered a line with 56.3% oleate and 2.8% linolenate. Later, they crossed a line LPKKC-3 with the fap1 and sop1 alleles for reduced palmitate to a line DHL that has the ol allele for elevated oleate and the fan1 and fanxa alleles for reduced linolenate (Rahman et al., 2004). They recovered a line LPDHL with 4.0% palmitate, 51.0% oleate, and 2.9% linolenate.
We began our research in 2002 with the three sources of mid-oleate. FA22 of maturity group I matured at Ames, but did not consistently have 50% oleate across environments. When we grew M23 of maturity group V and N98-4445A of maturity group IV at Ames, M23 was killed by frost before it matured and N98-4445A matured later than commercial cultivars. As a result, their oleate content was <50% (Alt et al., 2005b). Each of the mid-oleate lines was crossed to the most elite lines with the other fatty acid modifications. Individual F2 plants with the highest oleate and the best content of the other fatty acids were selected, the selected F2:3 lines were used for crossing to elite lines with the other fatty acid modifications, selfed seed from the lines was analyzed, and the hybrid seed was advanced from those lines with the best fatty acid profile. Subsequent breeding cycles were conducted with the same procedure.
After two breeding cycles, we were not able to identify lines with >50% oleate at Ames that were derived from FA22 and N98-4445A, but did find such lines that were derived from M23. Subsequent breeding cycles have only been continued with the M23-derived lines. N98-4445A may be more useful in the southern United States where temperatures are higher than in Iowa during seed filling (Burton et al., 2006; Oliva et al., 2006).
We have been able to combine the mid-oleate trait from M23 with low saturates, 1% linolenate, or with low saturates and 1% linolenate. In 2005, we planted a seed increase at Ames of F2–derived lines with >50% oleate and 1% linolenate from the third breeding cycle. All of the lines were maturity group III or earlier. One line was increased in Argentina during the winter of 2005–2006 to obtain additional seed for processing. A total of 1636 kg of seed from Ames and the same quantity from Argentina was blended and processed into refined oil by the POS Pilot Plant Corporation in Sasketoon, SK, during June 2006. The fatty acid composition of the refined oil was 9.3% palmitate, 5.4% stearate, 53.3% oleate, 31.1% linoleate, and 1.0% linolenate. The oil was sent to more than 30 food companies to evaluate its characteristics in comparison with a normal oleate/1%-linolenate oil that is produced commercially from the cultivars we have developed. The purpose of the comparison was to determine if the increase in oleate adequately improves the performance of the 1%-linolenate oil to justify breeding cultivars with the two traits. The oxidative stability was about 15 h for the mid-oleate/1%-linolenate oil and 9 h for the normal oleate/1%-linolenate oil. Based on the interest in the mid-oleate/1% linolenate by food companies, large-scale production of the oil will begin in 2007 with 12 soybean cultivars from our breeding program that have >50% oleate/1% linolenate. The varieties were released in November 2006 through the Iowa State University Research Foundation.
The stability of oleate content across environments has been a major consideration. Alt et al. (2005b) compared FA22, M23, and N98-4445A at four environments. FA22 had oleate contents that ranged from 50.1 to 60.4%, M23 ranged from 35.4 to 66.2%, and N98-4445A ranged from 32.8 to 63.7%. The low values for M23 and N98-4445A occurred when the lines were killed by frost at Ames before they matured. Oliva et al. (2006) evaluated M23 and N98-4445A in nine environments in the southern United States. The range in oleate for M23 was from 39.4 to 48.9% and the range for N98-4445A was from 39.6 to 67.1%. The oleate content of our lines derived from M23 that have low saturates, 1% linolenate, or both are undergoing evaluation for environmental stability. In 2005, the lines were tested at multiple locations and planting dates in Iowa and the oleate content of the lines was >50% for all the plantings (Curtis Scherder, personal communication, 2006).
The next phase of our breeding program will be to determine the influence of mid-oleate from M23 on agronomic and seed traits and to attempt to increase oleate content further by use of the transgressive segregates from the M23 x N98-4445A cross. In the long term, the development of cultivars with elevated oleate in combination with either low saturates or 1% linolenate likely will be a two-step process because the traits are independently inherited and multiple genes are involved. A cross of a mid-oleate/low saturate or a mid-oleate/1%-linolenate line to a high-yielding conventional cultivar will produce very few, if any, progeny that are acceptable for the two traits. As a result, it will be necessary to develop elite lines with either low saturates or 1% linolenate and to cross those lines to the most elite mid-oleate/low-saturate or mid-oleate/1%-linolenate lines. It is likely that the necessity of the two-step process will result in a yield differential between cultivars with low saturates or 1% linolenate alone and those with mid-oleate/low saturates and mid-oleate/1% linolenate.
Breeding for the combination of low saturates/mid-oleate/1% linolenate likely will be a three-step process in which the third step will be crossing elite mid-oleate/low-saturate or mid-oleate/1%-linolenate lines to the best lines with low saturates/mid-oleate/1% linolenate. The three-step process likely will result in a yield differential between low-saturate/mid-oleate/1%-linolenate cultivars and those with mid-oleate/low saturates or mid-oleate/1% linolenate.
Genetic Engineering
A soybean oil with >80% oleate was developed by the DuPont Company through genetic engineering (Kinney and Knowlton, 1998). Detailed descriptions of its development have been provided by Kinney (1996) and by Parrott and Clemente (2004). In brief, the soybean naturally has one copy of the gene GmFad2-1 that controls the conversion of oleate to linoleate in the seed. DuPont inserted a second copy of the FAD2-1 gene that silenced the gene that naturally occurs in the plant resulting in suppression of linoleate formation. The event used for cultivar development was designated 260-05.
The contents of the five major fatty acids in the high oleate soybean as a percentage of all the fatty acids were 6.3% palmitate, 3.7% stearate, 84.6% oleate, 0.9% linoleate, and 2.4% linolenate (Kinney and Knowlton, 1998). Oleate contents >90% have been reported by Buhr et al. (2002). The high oleate oil had exceptional oxidative stability that may be desirable for food and industrial applications (Warner and Gupta, 2005).
According to Kinney and Knowlton (1998), the high oleate trait did not have any negative effects on yield or other agronomic traits. The high-oleate soybean received regulatory approval in the Australia, Canada, Japan, New Zealand, and the United States. Regulatory approval was not granted by the European Union and the application was withdrawn (S. Knowlton, personal communication, 2002). About 1.4 million kg (3 million lb) of high oleate oil was sold for the first time in 2001 for industrial use. It was grown under strict identity preservation for several years, but the lack of regulatory approval by the European Union limited its production. In 2006, there was no production of the high-oleate soybean oil. (Dennis Byron, personal communication, 2006).
The event 260-05 involved the use of the bla gene for ampicillin resistance as a selectable marker (Kinney and Knowlton, 1998). Although the likelihood of transferring the gene from the high-oleate soybean to a microorganism in the environment is extremely small, DuPont chose to replace event 260-05 with an event that does not involve the use of antibiotic resistance as a selectable marker (S. Knowlton, personal communication, 2006). The development of cultivars with the new event for high oleate is underway. DuPont anticipates submitting the new event for regulatory approvals worldwide during the coming year and commercialization following regulatory approval in 2009.
Monsanto also is conducting genetic engineering research on a high oleate soybean oil (M.S. Hawbaker, personal communication, 2006). The relationship of their research to that of DuPont is not available.
Although a high-oleate soybean oil has excellent oxidative stability, one potential weakness it may have for some products is a different flavor than the same products prepared with low-linolenate or conventional soybean oil. Warner and Gupta (2005) evaluated the quality of potato chips fried in a high-oleate, a low-linolenate, and a 1:1 blend of high-oleate/low-linolenate soybean oils. Potato chips fried in the high-oleate oil had a significantly higher intensity of an undesirable fishy flavor that the other oils. Chips fried in the blended oil had improved flavor compared with the high-oleate oil and improved oil life and oxidative stability compared with the low-linolenate oil.
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Linoleic Acid (Linoleate) (18:2) |
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Stearic Acid (Stearate) (18:0)
Stearate is one of the two major saturated fatty acids in soybean oil. Its content in conventional soybean oil is about 4%. The range of the fatty acid in soybean lines is from 3% to >35%.
Reduced Stearate
Reducing the stearate content of soybean oil would be desirable for achieving a low-saturate oil. The stearate content of conventional soybean oil is 4%. No major alleles for reduced stearate have been reported. There is a limited amount of genetic variation among conventional soybean lines that can influence the breeding of cultivars with reduced or elevated saturated fatty acids (Hawkins et al., 1983a). In our breeding program for low-saturate cultivars, selection for reduced stearate is integrated with the selection for reduced palmitate by considering palmitate + stearate as a single trait. Only lines with <7.5% palmitate + stearate are considered acceptable for a low-saturate oil.
Elevated Stearate
Elevated stearate may be useful to eliminate the need for chemical hydrogenation of soybean oil in the production of trans-free margarines and shortenings (List et al., 1997; Knowlton, 2001; Kok et al., 1999). Multiple alleles for elevated stearate have been identified in soybean. The fas1-a allele in A6 developed by treatment of FA8077 with sodium azide (NaN3), the fas1-b allele in A10 developed by EMS treatment of ‘Coles’, and the fas1 allele in A9 developed by EMS treatment of A81-606085 are at the same locus (Hammond and Fehr, 1983b; Graef et al., 1985a). Additional alleles for elevated stearate at that locus include fas1-(Ames 1) developed by EMS treatment of FA26625, fas1-(Ames 2) developed by EMS treatment of FA47473, and fas1-(Ames 3) developed by EMS treatment of FA47445 (Bubeck et al., 1989). An allele developed by EMS treatment of FA47394 seemed to be at a different locus than those at the fas1 locus (Bubeck et al., 1989). Rahman et al. (1997) used X-ray irradiation of Bay to develop the st1 allele in KK-2 and the st2 allele in M25 that are at different loci. The relationship of st1 and st2 to the fas alleles has not been determined. The fas1-nc allele was identified as a natural mutation and found to be allelic to fas1-a (Pantalone et al., 2002).
The molecular makeup of the fas alleles has not been determined. Three molecular markers have been identified that are associated with the Fas locus (Spencer et al., 2003).
Modifying genes can cause significant variation among lines that are homozygous for the major alleles for elevated stearate. Lundeen et al. (1987) crossed A6 (fas1-a), A9 (fas1), and A10 (fas1-b) to a conventional cultivar and did not recover lines that were as high in stearate as the original parents.
The influence of elevated stearate on fatty acid development in the seed was evaluated by comparison of the fatty acid content of seeds of A6 (fas1-a) and its parent FA8077 from 15 d after flowering (DAF) to maturity (Graef et al., 1985b). The stearate content of FA8077 was 22.6% at 15 DAF and declined steadily until maturity when it was 3.9%. The stearate content of A6 was 17.1% at 15 DAF, increased steadily to 45.4% at 25 DAF, and gradually decreased to 32.6% at maturity. Only oleate was significantly affected by the increase in stearate content. FA8077 had 45.0% oleate at maturity and A6 had 17.8%. Stearate is located primarily at the sn-1 position of phospholipids and changes in stearate content were associated primarily with this position (Wang et al., 1997).
Environmental conditions can significantly alter the stearate content of lines. The stearate content of A6 (fas1-a) ranged from 26.2 to 35.2% during 3 yr at Ames, IA (Schnebly and Fehr, 1993). The stearate content varied among four planting dates, but the effect was not consistent among years. The earliest planting date had the highest stearate in 1988, the earliest date had the lowest stearate in 1989, and there was no difference between the earliest and latest date in 1990.
The impact of the elevated stearate on agronomic and seed traits varies among the alleles, which is related, at least in part, to the level of stearate that they generate. High stearate progeny from a population segregating for the fas1-a allele in A6 had 7.7% lower seed yield than the average of the low stearate progeny, but there was no significant difference in yield between high and low stearate progeny with either the fas1-b allele from A9 or the fas1 allele from A10 (Lundeen et al., 1987). Hartmann et al. (1997) evaluated the relationship between stearate content and seed yield in the progeny from two crosses segregating for the fas1-a allele in A6. There was an average yield decrease of 3.4 kg ha–1 for each percentage unit increase in stearate. They suggested that it may be possible to develop acceptable cultivars with the fas1-a allele that have moderately elevated stearate content. Rahman et al. (1997) combined the st1 and st2 alleles and recovered seeds with >30% stearate that failed to produce plants after germination. They suggested that tissue culture would be needed to perpetuate the genotype.
We conducted a breeding program with the fas1-a allele to develop cultivars with >25% stearate. One of the problems we encountered was inconsistent seedling emergence of high stearate lines among environments. Under controlled temperature conditions, Wang et al. (2001b) found that there was a negative correlation between elevated lipid saturates and germination, soybean growth rate, and electrolyte retention. Wang et al. (2001a) indicated that increased concentrations of saturated fatty acids in the soybean seed may cause elevated melting transition temperatures of triacylglycerols and phospholipids that reduce germination and seedling growth rates.
There is no commercial production of a soybean oil with elevated stearate, even though it has been used experimentally to produce trans-free margarine. List et al. (1997) found that it was possible to manufacture a soft-tube margarine by interesterification of a soybean oil containing 20 to 33% stearate. Kok et al. (1999) produced a trans-fat free margarine by interesterification of oil obtained from our soybean lines with 23.3% palmitate and 20.0% stearate. Despite the possibility of using an elevated stearate oil to produce trans-free products, it is not clear if the cost of developing such cultivars is justified given the other methods that are being used to produce such products. For example, Archer Daniels Midland is producing trans-free margarine and shortening by enzymatic interesterification of liquid soybean oil and fully hydrogenated soybean oil (Lee, 2005; Kodali and List, 2005)
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Future Oil Modifications |
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Booth et al. (2005) combined the gene for >80% oleate in a line D2T that was developed by genetic engineering with major genes for other fatty acid modifications that were developed by mutagenesis. The cross of D2T to the high stearate line A6 with the fas1-a allele resulted in progeny with 26% stearate and 61% oleate. Knowlton (2001) indicated that an oil with elevated oleate and stearate could be used to produce oil for use in confectionary applications and other products requiring a high level of stability. From the cross of D2T to lines containing the fap2 allele for elevated palmitate and the fas1-a allele for elevated stearate, Booth et al. (2005) recovered lines with 12% palmitate, 21% stearate, and 58% oleate. They indicated that the combination of elevated saturates and oleate may be useful for production of margarine and other spread products. The combination of the D2T gene and the fan1 allele provided an oil with 87% oleate and 1.7% linolenate and the D2T gene with the fap1 and fap3 alleles for reduced palmitate produced an oil with 89% oleate and 6.7% palmitate + stearate.
The challenge in breeding for these and other combinations of modified fatty acids that are controlled by multiple major and minor genes will be developing cultivars that are acceptable in seed yield and other important agronomic and seed traits.
Genetic Engineering of Fatty Acids from Other Species
In addition to modifications in the five major fatty acids, there is genetic engineering research underway to modify soybean oil for human and industrial uses. Cahoon (2003) provided a description of some of the targets of genetic engineering of soybean for oil modification. These include an oil with >50% linolenate for use as a drying oil in paints, inks, and varnishes and >20% calendic acid for use as a drying oil. DuPont is utilizing genetic engineering to try and produce the omega-3 fatty acids found in fish oil, including eicosapentaenoic acid (20:5) and docoshexaenoic acid (22:6) (William D. Hitz, personal communication, 2006). If these genetic engineered traits become a reality, they will present new and interesting challenges for the development of cultivars with acceptable agronomic and seed traits for commercial production.
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Summary of General Breeding Principles |
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Received for publication April 7, 2007.
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REFERENCES |
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-linolenic acid mutants and elucidation of their contribution to fatty acid biosynthesis in soybean seed. Plant Sci. 168:1615–1623.
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ABSTRACT
INTRODUCTION