TILLING-by-Sequencing+ to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis

Reverse genetic approaches have been widely applied to study gene function in crop species; however, these techniques, including gel-based TILLING, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members that share high homology in their DNA sequence. Chemical mutagenesis emerges as a genetically modified-free strategy to produce large-scale soybean mutants for economically important traits improvement. The current study uses an optimized high-throughput TILLING by target capture sequencing technology, or TILLING-by-Sequencing⁺ (TbyS⁺), coupled with universal bioinformatic tools to identify population-wide mutations in soybeans. Four ethyl methanesulfonate mutagenized populations (4032 mutant families) have been screened for the presence of induced mutations in targeted genes. The mutation types and effects have been characterized for a total of 138 soybean genes involved in soybean seed composition, disease resistance, and many other quality traits. To test the efficiency of TbyS⁺ in complex genomes, we used soybeans as a model with a focus on three desaturase gene families, GmSACPD, GmFAD2, and GmFAD3, that are involved in the soybean fatty acid biosynthesis pathway. We successfully isolated mutants from all the six gene family members. Unsurprisingly, most of the characterized mutants showed significant changes either in their stearic, oleic, or linolenic acids. By using TbyS⁺, we discovered novel sources of soybean oil traits, including high saturated and monosaturated fatty acids in addition to low polyunsaturated fatty acid contents. This technology provides an unprecedented platform for highly effective screening of polyploid mutant populations and functional gene analysis. The obtained soybean mutants from this study can be used in subsequent soybean breeding programs for improved oil composition traits.     

Assessment of new in-bin drying and storage technology for soybean seed

On-farm, in-bin drying and storage of soybean in environments with unconditioned air often result in repeated drying and rewetting of the grains which may have adverse effects on quality metrics; if done using natural air, as recommended for soybean destined to the seed market, the in-bin drying and storage method require operation at well-defined local weather-dependent strategies to maintain the seed quality. This study simulated in-bin drying and storage of soybeans. Different fan control options and drying strategies were used to assess performance in terms of drying duration to target final moisture content (MC), percent over drying, energy expenditure, and drying cost. Fan operation included running the fan continuously, only at night, only during the day, at a set window of equilibrium MC (EMC) of natural air, and set EMC window with supplemental heating of ambient air as an option (EMC-H). Drying and storage performance were tested for soybean at initial moisture content (IMC) (16–22%, wet basis), air flow rate (1.04–5.0?m³?min^?1 [air] t^?1 [soybean]), and harvesting start dates (August 15 to November 15). Simulation model was validated using a bench-scale pressure drop system filled with soybeans with IMC of 22% wet basis. The result shows that fan control strategies, air flow rates, harvest date, and initial MC of the soybeans significantly (P?χ² was 0.88.     

The comparative value of heated ground unextracted soybeans and heated dehulled soybean flakes as a source of soybean oil and energy for the chick

Experiments were conducted to evaluate heated unextracted soybean fractions as sources of soybean oil and protein for the growing chick. Heated dehulled unextracted soybean flakes produced growth rate and feed efficiency equal to that obtained with the combination of soybean oil meal and degummed soybean oil while heated ground unextracted soybeans were less satisfactory in this respect. The poorer results obtained with ground unextracted soybeans were shown to be related to a poorer absorbability of the oil in them. Flaking the soybeans markedly improved the absorbability of the oil by the chick, probably by causing a greater disruption of cellular structure than was obtained by the grinding of the soybeans. The metabolizable energy of ground unextracted soybeans was substantially less than that of unextracted soybean flakes. Most of the differences in metabolizable energy were accounted for by differences in absorbability of the oil. Soybean hulls at a level equivalent to that contained in soybeans were found to have no effect on growth rate and only a slight effect on feed efficiency. Autoclaving soybean oil did not lower its value for the chick. The relationship between the poorer growth obtained with ground unextracted soybeans and the low absorbability of the oil in them was discussed. To obtain maximum efficiency in the use of unextracted soybean products in chick rations, some such means as flaking must first be employed to increase the availability of the oil.     

Size determination of shriveled and wrinkled soybeans

Drought stress created shriveled and wrinkled (S/W) soybeans in the 1988 soybean crop. Seven lots of 1988 soybeans were examined to validate the Federal Grain Inspection Service (FGIS) definition of S/W. Lots were subdivided into sized fractions with both slotted and round-hole screens. Shriveled and wrinkled soybeans were found in all size fractions, whether those fractions were determined by a slotted or a round-hole screen. None of the size fractions adequately isolated or characterized S/W soybeans. The FGIS definition of shriveled and wrinkled does not sonsider larger wrinkled soybeans, but only shriveled soybeans passing through a 10/64″ by 3/4″ slotted screen. The most accurate determination of S/W soybeans can be made by examining the entire soybean sample, not a sized fraction.     

Preparation of Glycinin and β-Conglycinin from High-Sucrose/Low-Stachyose Soybeans

A new soybean line, known as high-sucrose/low-stachyose (HS/LS) soybeans, has been developed having elevated sucrose content and reduced content of flatus-causing oligosaccharides, especially stachyose. There is also increased interest in understanding the health benefits, functional properties and potential applications for the two major storage proteins of soybeans, glycinin and β-conglycinin. We evaluated the protein fractionation behavior of a HS/LS soybean line and compared it to normal soybeans when using the three-step Wu procedure, which employs SO₂, NaCl and precipitations at pH 6.4 and 4.8 and a new two-step Deak procedure, which employs SO₂, CaCl₂ and precipitations at pH 6.4 and 4.8. Both soybean variety and fractionation procedure significantly affected fraction yields, purities and functional properties. The Wu procedure gave glycinin- and β-conglycinin-rich fractions with 100% purities and high yields of solids (15.4%) and protein (31.7%) from HS/LS soy flour, which were significantly higher than the purities and yields achieved with normal soybeans. The Deak procedure was less efficient in fractionating proteins from HS/LS soybeans than from normal soybeans, producing protein fractions from HS/LS soybeans with purities ranging from 71 to 80%. The Deak procedures yielded products with unique solubilities, surface hydrophobicities, and emulsification and foaming properties.     

Estimating the processed value of soybeans

Interest in marketing soybeans on the basis of protein and oil content is increasing. Producers, breeders, handlers and buyers of soybeans need a method of evaluating soybean lots of different composition. A model is presented that predicts, given soybean composition and processing conditions, the yield of crude soybean oil and soybean meal from the processing of soybeans in a solvent extraction plant. From these yields, an estimated processed value (EPV) was calculated. For one set of price conditions, the EPV of typical soybeans had a range of $0.93 per bushel if premiums were paid for meal protein in excess of specifications and a range of $0.53 per bushel if meal protein premiums were not paid. Trading rules established by the National Oilseed Processors Association for domestic meal markets have a significant effect on the value and composition of soybean meal.     

Improving the quality of the soybean

Soybeans, once a relatively minor farm commodity, have become the world’s most abundant source of vegetable protein and oil. This growth in popularity, much of which has occurred in the last 20 years, has been made possible partly by genetic improvements that have successfully adapted soybeans to a wide variety of environmental conditions. To date, however, soybean breeders have concentrated their research efforts on increasing the quantity rather than the quality of soybeans. This paper summarizes genetic research currently underway to improve the quality of soybeans and/or soybean products. It also examines research efforts to improve the soybean’s fatty acid composition, change the amino acid profile and reduce antinutritional factors.     

A new approach to genetic alteration of soybean protein composition and quality

Although soybeans produce high-quality meal, modern animal and fish production systems often require synthetic essential amino acid supplements to fortify feed rations. However, biotechnology may enable development of soybeans with naturally adequate levels of certain essential amino acids for advanced feed formulations. One approach involves genetic manipulation of glycinin (11S) and β-conglycinin (7S) contents, the principal components of soybean storage proteins. Because 11S contains more cysteine and methionine than 7S protein, a higher 11S:7S ratio could lead to beneficial changes in the nutritional quality of soybean meal. Although genotypic variation for 11S:7S may be low among soybean [Glycine max (L.) Merr.] germplasm, ratios ranging from 1.7–4.9 were observed among accessions of the wild ancestor of cultivated soybean (Glycine soja Sieb, and Zucc.). Thus, wild soybean germplasm was evaluated as a potential source of genes that govern protein synthesis that may have been lost during the domestication of G. max. Change in the amount of 11S protein accounts for a significant portion of the genotypic variation in protein concentration and composition among wild soybeans. Strong positive correlation exists between the 11S:7S ratio and methionine or cysteine concentration of total protein. Moderate positive associations were found for threonine or tyrosine. A moderate negative correlation was found between lysine and 11S:7S. No association was found for leucine and phenylalanine or for total essential amino acid concentration. Based on these data, G. soja may contain a different complement of genes that influence expression of 11S and 7S proteins than G. max germplasm. Thus, through interspecific hybridization, wild soybeans may be a useful genetic resource for the further improvement of protein quality in cultivated soybeans.     

Measurement of fatty acids in whole soybeans with near infrared spectroscopy

Improvement of nutritional and/or functional properties of soybean oil by modification of soy fatty acid composition is one of the objectives of plant breeders. A major element of breeding is rapid identification and tracking of traits in seed samples. This discussion summarizes the progression of whole‐soybean fatty acid calibration developments at Iowa State University. Emphasis was placed on linolenic acid (18:3) and total saturates (16:0 + 18:0). Normal soybeans have 12–20% (of the oil) saturated fats; modified low saturate soybeans have 6–8% saturated fats. Normal soybeans have 6–12% linolenic acid; modified low linolenic soybeans have 1–3% linolenic acid. Infratec 122x/1241 and Bruins OmegaG NIRS units were calibrated to measure fatty acid levels as a percentage of total oil content, in whole soybeans. The first Infratec calibrations (in 1998) did not remain accurate as soybean genetics changed. Iterations of the calibration process yielded calibrations for total saturates and linolenic acid with standard errors of prediction (on 2005 crop samples not included in the calibration pool) of 1.0% percentage points and 0.8% points, respectively. These were sufficient to classify modified versus normal concentrations of the two fatty acids. The NIRS units could not determine the specific percentages within the classes of modified and normal soybeans.     

Photochemical production of conjugated linoleic acid from soybean oil

Conjugated linoleic acid (CLA), an anticarcinogenic compound with numerous other health benefits, is present mainly in dairy and beef lipids. The main CLA isomer present in dairy and beef lipids is cis 9, trans 11 CLA at a 0.5% concentration. The typical minimum human dietary intake of CLA is 10 times less than the 3 g/d suggested requirement that has been extrapolated from animal and cell-line studies. The objectives of this study were to produce CLA isomers from soybean oil by photoisomerization of soybean oil linoleic acid and to study the oxidation status of the oil. Refined, bleached, and deodorized soybean oil with added iodine concentrations of 0, 0.1, 0.25, and 0.5% was exposed to a 100-W mercury lamp for 0 to 120 h. An SP-2560 fused-silica capillary GC column with FID was used to analyze the esterified CLA isomers in the photoisomerized oil. The CLA content of the individual isomers was optimized by response surface methodology. Attenuated total reflectance (ATR)-FTIR spectra in the 3400 to 3600 cm⁻¹ range and ¹H NMR spectra in the 8 to 12 ppm range of the photoisomerized soybean oil were obtained to follow hydroperoxide formation. The largest amount of cis 9, trans 11 CLA isomer in soybean oil was 0.6%, obtained with 0.25% iodine and 84 h of photoisomerization. Lipid hydroperoxide peaks in the ATR-FTIR spectra and aldehyde peaks in the ¹H NMR spectra were not observed in the photoisomerized soybean oil, and the spectra were similar to that of fresh soybean oil. This study shows that CLA isomers can be produced simply and inexpensively from soybean oil by photoisomerization.     

Direct consumption of the soybean

Efforts in some Latin American countries directed toward the use of soybeans as a primary source of proteins for human nutrition have especially focused attention on simple home-level procedures such as the soaking and cooking of soybeans and the lime-cooking of corn-soybean mixtures. Data obtained with these two procedures indicate there is great potential in using soybeans directly in human feeding. Soaking soybeans in 0.25% NaHCO₃ for 8 hr and cooking for 20 min decreases trypsin inhibitor activity more than 80%, and 40 min of cooking gives chewiness indexes similar to those of common beans with acceptable texture (10–20). The protein efficiency ratio (PER) of a mixture that was 50% soybeans and 50% common beans was 60% higher than that of common beans alone. Considering acceptability and functional characteristics of “masa” (dough) and “tortilla,” an optimum soybean level within the lime-cooking procedure was found to be 16%. Green pods of soybean varieties adapted to the tropics, at 65 to 85 days of maturation, have the same nutrient content (dry basis) as mature soybeans, with a good quality protein and a good content of B complex vitamins.     

Soybean oil: Update on number one

The growth and present stature of soybeans and soybean oil production and utilization in the world and in the USA is presented. Compositions of soybeans and soybean oil are compared with other common vegetable oils. The current and optimal processing practices of extraction, degumming, neutralization (caustic and physical), hydrogenation, and deodorization are discussed. Where appropriate, new and innovative approaches are introduced. Utilization of soybean oil is covered, followed by a historical and present view on the subject of soybean oil flavor and present and future nutritional considerations of soybean oil.     

Effect of soybean pretreatment on the color quality of soybean oil

Color reversion in soybean oil can be prevented by reducting the enzyme activity of soybeans before cracking and flaking. Soybean oil extracted from steamed, intact soybeans (18% moisture) had lower Rm (max. red) values in RBD oil, higher amounts of γ-tocopherol, plus its isomers, in both crude and RBD oil, and also higher amounts of hydratable phosphatides in crude oil than those in the oils from the same beans without steam treatment. For soybean pretreatments, a toasting process is less effective than the steaming process for the inhibition of color reversion of soybean oil. To prevent the occurrence of color reversion in RBD soybean oil, the amount of γ-tocopherol and γ-TED (5-[tocopheryloxy]-γ-tocopherol) should be above 550 ppm in crude oil.     

Soybean protein food in China

This paper introduces the history of soybeans and soybean protein foods in China. For 4,000 years, soybeans have been one of the main crops cultivated in this country. The history of extracting protein to prepare a protein food (bean curd, tu-fu) is about 1,000 years old. Our ancestors had long been aware of the edible value of the soybean and had developed a technique for preparing many kinds of soybean foods. The traditional methods of preparing soybean protein foods such as bean curd (tu-fu), fermented bean curd (fu-ru) and dried bean milk cream (fu-tsu) are discussed.     

Bis-phosphatidic acid in developing soybeans and soybean suspension cultures

A phospholipid which rapidly accumulates radioactivity from [1-¹⁴C] acetate administered to slices of developing soybeans or to suspension cultures of soybean cells was isolated. Its structure was identified by comparison of its properties and degradation products with those of authentic lipid standards using infrared absorption spectrometry, thin layer chromatography, acetolysis, mild alkaline hydrolysis, and determination of molar ratios of phosphorus, glycerol, and acyl moieties. The structure of the phospholipid was found to bebis-phosphatidic acid, a phospholipid previously unreported in higher plants.     

Effect of phosphorus and potassium fertilizer on crop response and soil fertility in a long-term experiment

Data from 32 years of a rotation-fertility experiment were analyzed to determine the average P and K application rates required for maximum yield and for optimum yield. A four-year rotation of corn, soybean, wheat, hay was used for the first 10 years and then changed to corn-1, soybean, wheat, corn-2. Rates of P application per 4-year rotation ranged from 0 to 196 kg ha^–1 and for K from 0 to 558 kg ha^–1. Multiple regression equations were fitted to the mean yields per 4-year rotation for the response of each crop to P and K applications. The range in P application rates in kg of P per 4-year rotation required to get maximum yields of corn was 118 to 172, for soybeans was 134 to 150, and for wheat was 116 to 138. The range in K application rates in kg K per 4-year rotation to get maximum yields of corn was 378 to 411, for soybeans was 324 and 476, and for wheat was 11 to 323. For rates of application where P and K added exceeded crop removals, soil test P and K increased linearly with the cumulative positive balance of P and K. Where crop removal exceeded application rate, no relation was found between crop removal and soil test.Journal paper No. 10, 915 Purdue University Agric. Exp. Stn., West Lafayette, IN 47907. Contribution from the Department of Agronomy.     

Fate of Phytic Acid in Producing Soy Protein Ingredients

Phytic acid (myo-inositol hexaphosphate) is present in soybeans and soy protein products at 1–2% dry matter. Phytate causes poor absorption of essential electrolytes and minerals, and binds to proteins and co-precipitates with isoelectric soy protein isolates. We determined how phytic acid partitioned during different procedures to prepare soy protein ingredients. Procedure and soybean variety significantly affected phytic acid content and recovery. High-sucrose/low-stachyose (HS/LS) soybeans contained significantly (P < 0.05) less phytate than did a typical variety of commodity soybeans (IA2020). In addition, phytate was more readily extracted from the commodity soybeans than from HS/LS soybeans. Among all procedures studied, ethanol-washed soy protein concentrate had the highest phytate contents and yields in the protein products for both soybean varieties (~80 mg/g and 99%, respectively). When protein extraction was carried out at room temperature the protein products had significantly lower phytate yields (60–78%) than when extraction was at 60 °C (80–99%). The protein products obtained from normal soybeans had significantly higher phytate contents than the same products made from HS/LS soybeans. When fractionating soy proteins, the glycinin-rich fraction contained significantly less phytate than the β-conglycinin fraction except for the fractionation procedure performed at room temperature instead of 4 °C.     

Nutritional contribution of soy protein to food systems

From the nutritional point of view, soybeans can play a significant role in at least three aspects: as a source of supplementary and complementary protein, as a source of calories, and as a source of nitrogen. The protein role is probably the most important for food systems of developed and underdeveloped populations, while the role as a source of protein and calories applies more to food systems of developing populations. Soy protein efficiently supplements cereal grain protein, because it corrects the lysine deficiency of cereals. In some cases, for example with maize, it also corrects the tryptophan deficiency. On the other hand, the essential amino acid pattern of soybean protein complements that of other protein sources, for example cereal grains, cottonseed flour, and, in general, lysine deficient protein sources. This makes feasible the preparation of foods of optimum protein quality and of a high protein content. Because of its quality, soybean protein can replace animal protein without a significant decrease in nutritive value, for example as milk and meat extender; for diets low in quantity and quality of protein and deficient in calories, soybeans, as full-fat flour, provide both. Because of cultural eating habits, it is difficult to conceive the use of soybeans as complete substitutes of common beans; therefore, efforts should be made to use soy protein in combination with common foods used by populations to whom soybeans are foreign food. Examples of the nutritional benefits derived from the use of soybean protein as flour or protein concentrate or as full-fat soybean flour are given, particularly for foods consumed in Latin American countries. Besides the role soybeans play in human foods, they also play a significant role in the animal industry as a very important component of diets. The efficiency of the swine and poultry industry would be lower if it were not for the nutritional contribution of soybeans.     

Government role and participation in development and marketing of soy protein foods

Japan has a history of utilizing soybeans as human foods. Currently, a great quantity of defatted soybeans is used as animal feed in Japan, and governmental and commercial enterprises are anxious to turn the defatted soybeans directly into foods for humans. Therefore, they are putting great efforts into soybean research and development. Since the demands for better foods are rising and their resources are not abundant, I feel that soybean protein will play an important role by exerting its unique properties, not only to supplement other foods, but also to grow into new types of foods when their unknown properties are disclosed.     

Soybean drying in fluidized bed. effect on the hydratable and nonhydratable phosphatide concentration in crude and degummed crude oil

The content of nonhydratable phosphatides in soybean crude oil is increased by phospholipase D activity during the oil-making process. Enzyme inhibition would allow to minimize them. Recently harvested soybeans with high moisture levels require adequate drying to store safely. Simultaneous soybean drying and phospholipase D inactivation in a single operation when applying a thermal treatment by the fluidized-bed technique was evaluated. The process conditions for performing the drying and a complete enzyme inhibition on soybeans, with an initial moisture content between 7.4 and 20.6% wet basis, similar to that at the time of the harvest, were fluidizing and drying medium temperature between 110 and 140°C, and a drying time between 1 and 2 min. For the treated soybeans, the phosphorus content increased up to 223% in crude oil and decreased 17% in degummed crude oil with regard to the values of the control sample.