CRISPR/Cas9-Mediated Knockout of GmFATB1 Significantly Reduced the Amount of Saturated Fatty Acids in Soybean Seeds

Soybean (Glycine max) oil is one of the most widely used vegetable oils across the world. Breeding of soybean to reduce the saturated fatty acid (FA) content, which is linked to cardiovascular disease, would be of great significance for nutritional improvement. Acyl-acyl carrier protein thioesterases (FATs) can release free FAs and acyl-ACP, which ultimately affects the FA profile. In this study, we identified a pair of soybean FATB coding genes, GmFATB1a and GmFATB1b. Mutants that knock out either or both of the GmFATB1 genes were obtained via CRISPR/Cas9. Single mutants, fatb1a and fatb1b, showed a decrease in leaf palmitic and stearic acid contents, ranging from 11% to 21%. The double mutant, fatb1a:1b, had a 42% and 35% decrease in palmitic and stearic acid content, displayed growth defects, and were male sterility. Analysis of the seed oil profile revealed that fatb1a and fatb1b had significant lower palmitic and stearic acid contents, 39–53% and 17–37%, respectively, while that of the unsaturated FAs were the same. The relative content of the beneficial FA, linoleic acid, was increased by 1.3–3.6%. The oil profile changes in these mutants were confirmed for four generations. Overall, our data illustrate that GmFATB1 knockout mutants have great potential in improving the soybean oil quality for human health.     

Evaluation of Agronomic and Seed Characteristics in Elevated Oleic Acid Soybean Lines in the South-Eastern US

Increasing oleic acid, a monounsaturated fatty acid, is reported to strike the best balance between cold flow properties and oxidative stability in soybean seed oil to enhance biodiesel and produce a better burning fuel. In addition, it is important that elevated oleic acid soybeans have the agronomic traits of local cultivars and maintain oleic acid stability across environments. Research was conducted in 2007–2008 to evaluate six Roundup Ready^® soybean recombinant inbred lines exhibiting enhanced levels of oleic acid. The six elevated oleic lines averaged a 55% increase in oleic acid and a 43% decrease in linolenic acid over the two commercial cultivars (AG3906 and AG4103). Some elevated oleic acid genotypes fulfilled the linear regression definition of a stable genotype. TN03-93RR was the best genotype because of its oleic acid content (397 g kg^?1) and desirable regression estimates for stability. Iodine value (IV), peroxide value (PV), and induction period (IP) were used to evaluate the fuel properties of the two lines with the highest oleic acid content and the two commercial cultivars. The elevated oleic acid lines had significantly better IP, PV and IV for better biodiesel properties and oxidative stability than the two commercial cultivars.     

Combination of the Elevated Stearic Acid Trait with Other Fatty Acid Traits in Soybean

Stearic acid is one of five major fatty acids found in soybean oil. It is a fully saturated lipid and is known for neutral or positive effects on LDL cholesterol when consumed by humans. Unfortunately, stearic acid only accounts for about 4% of the total seed oil produced in commodity soybean. Previous work has shown that stearic acid can reach levels as high as 28% of the total oil fraction when the SACPD-C gene, encoding the delta-9-stearoyl-acyl carrier protein desaturase responsible for most of the stearic acid variation in soybean seed, is ablated in combination with other loci. In order to increase stearic acid content and create soybeans with improved utility based on fatty acid composition, we combined mutations in SACPD-C with other mutations in the fatty acid biosynthetic pathway. Soybean plants carrying mutant alleles of both SACPD-C and FAD2-1A produce seed with stearic acid levels from 14% to 21%, and with elevated levels of oleic acid. Soybeans carrying mutations in both SACPD-C and FAD3A or FAD3C have both statistically significantly elevated levels of stearic acid (from 15–21%) and statistically reduced linolenic acid levels. Neither mutant combination appears to affect other agronomic properties such as plant morphology or seed protein levels making this a potentially viable trait.     

Genetic Variability Studies in Seed Biochemical Traits of Pongamia pinnata (L.) Pierre Accessions

A genetic variability study of oil content, fatty acid composition and karanjin content of seeds was carried out in candidate plus trees of Pongamia pinnata from selected agro-ecological zones of southern peninsular India. Significant zonal variation (P < 0.01) for biochemical traits was recorded. Significant positive correlation (P < 0.05) was recorded for latitude with oleic acid. Longitude showed significant negative correlation with palmitic acid and oleic acid. Altitude positively correlated with oleic acid and linolenic acid; negatively correlated with linoleic acid and seed oil content. Phenotypic co-efficient of variation was higher than the genotypic co-efficient of variation for all the biochemical traits. The oleic acid positively correlated with seed oil content (P < 0.05) and negatively correlated with linoleic acid and linolenic acid (P < 0.01). The zone 1, zone 2, zone 3 and zone 4 showed high heritability and genetic advance for oleic acid, linoleic acid and oleic to linoleic acid ratio. Based on Ward's method, the pongamia accessions were grouped into three major clusters and four sub-groups depending on their geographical locations. Further, the results obtained in this study could be used as background information for biofuel programs in India and other tropical countries.     

Fatty acid composition of oil from soybean leaves grown at extreme temperatures

Leaves from soybean (Glycine max (L.) Merr.) plants were assayed to determine if the relationship between temperature and relative fatty acid composition observed in the seed oil also existed for the triglycerides in the leaf oil. Leaf samples were harvested from eight soybean lines (A5, A6, C1640, Century, Maple Arrow, N78-2245, PI 123440 and PI 361088B) grown at 40/30,28/22 and 15/ 12°C day/night. At 40/30 and 28/22°C, seven fatty acids were observed at a level greater than 1.0%. These included the five major fatty acids found in the seed oil: palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) acid; plus two fatty acids that had retention times the same as palmitoleic (16:1) and γ-linolenic (18:3 g) acid. In addition, an eighth fatty acid that had a retention time the same as behenic (22:0) acid was found in the leaves of all lines at 15/12°C. Palmitic, palmitoleic and stearic acid content did not differ significantly over temperatures. The oleic and linoleic acid content were each highest at 15/12°C, while the γ-linolenic and the linolenic acid content were each highest at 40/30°C. The fatty acid composition of the triglyceride portion of the leaf oil did not display the same pattern over temperatures as that observed for seed oil.     

The Interaction of the Soybean Seed High Oleic Acid Oil Trait With Other Fatty Acid Modifications

Oil value is determined by the functional qualities imparted from the fatty acid profile. Soybean oil historically had excellent use in foods and industry; the need to increase the stability of the oil without negative health consequences has led to a decline in soybean oil use. One solution to make the oil stable is to have high oleic acid (>70%) and lower linolenic acid content in the oil. Other fatty acid profile changes are intended to target market needs: low‐saturated fatty acid and high stearic acid content in the oil. The objective of this study is to determine the interaction of the high oleic acid oil trait with other alleles controlling fatty acid profiles. Soybean lines containing high oleic acid allele combinations plus other fatty acid modifying alleles were produced, and the seed was produced in multiple field environments over 2 years. Stable high oleic acid with low linolenic acid (<3.0%) was achieved with a 4‐allele combination. The target of >20% stearic acid in the seed oil was not achieved. Reducing total saturated fatty acids below 7% in a high oleic acid background was possible with mutant alleles of both an acyl‐ACP thioesterase B and a β‐ketoacyl‐[acyl‐carrier‐protein] synthase III gene. The results identified allele combinations that met the target fatty acid profile thresholds and were most stable across environments.     

Restriction fragment length polymorphism analysis of soybean fatty acid content

The quality of soybean [Glycine max (L.) Merr.] oil depends greatly upon its fatty acid composition. The objective of this study was to map quantitative trait loci affecting the relative content of the fatty acids composing soybean seed oil. The mapping was done in a population formed from a cross between aG. max experimental line and aG. soja plant introduction. Sixty F2-derived lines from this population were genotyped with 243 restriction fragment length polymorphism, five isozyme, one storage protein and three morphological markers. The genotyped lines were grown in a replicated trial, and the seed harvested was analyzed for palmitic, stearic, oleic, linoleic and linolenic fatty acids. Markers significantly (P>0.005) associated with each fatty acid were found. Individual markers were associated with up to 38% of the total variance in specific fatty acids among the F2 lines. The greatest associations between markers and fatty acid content were observed with markers on two major linkage groups. Joint contribution of North Central Region, USDA-ARS and Journal Paper no. J-14849 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011; Project 2974.     

In Silico Analysis of Fatty Acid Desaturases Structures in Camelina sativa,and Functional Evaluation of Csafad7 and Csafad8 on Seed Oil Formation and Seed Morphology

Fatty acid desaturases add a second bond into a single bond of carbon atoms in fatty acid chains, resulting in an unsaturated bond between the two carbons. They are classified into soluble and membrane-bound desaturases, according to their structure, subcellular location, and function. The orthologous genes in Camelina sativa were identified and analyzed, and a total of 62 desaturase genes were identified. It was revealed that they had the common fatty acid desaturase domain, which has evolved separately, and the proteins of the same family also originated from the same ancestry. A mix of conserved, gained, or lost intron structure was obvious. Besides, conserved histidine motifs were found in each family, and transmembrane domains were exclusively revealed in the membrane-bound desaturases. The expression profile analysis of C. sativa desaturases revealed an increase in young leaves, seeds, and flowers. C. sativa ω3-fatty acid desaturases CsaFAD7 and CsaDAF8 were cloned and the subcellular localization analysis showed their location in the chloroplast. They were transferred into Arabidopsis thaliana to obtain transgenic lines. It was revealed that the ω3-fatty acid desaturase could increase the C18:3 level at the expense of C18:2, but decreases in oil content and seed weight, and wrinkled phenotypes were observed in transgenic CsaFAD7 lines, while no significant change was observed in transgenic CsaFAD8 lines in comparison to the wild-type. These findings gave insights into the characteristics of desaturase genes, which could provide an excellent basis for further investigation for C. sativa improvement, and overexpression of ω3-fatty acid desaturases in seeds could be useful in genetic engineering strategies, which are aimed at modifying the fatty acid composition of seed oil.     

Mutation of GmIPK1 Gene Using CRISPR/Cas9 Reduced Phytic Acid Content in Soybean Seeds

Phytic acid (PA) acts as an antinutrient substance in cereal grains, disturbing the bioavailability of micronutrients, such as iron and zinc, in humans, causing malnutrition. GmIPK1 encodes the inositol 1,3,4,5,6-pentakisphosphate 2-kinase enzyme, which converts myo-inopsitol-1,3,4,5,6-pentakisphosphate (IP₅) to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP₆) in soybean (Glycine max L.). In this study, for developing soybean with low PA levels, we attempted to edit the GmIPK1 gene using the CRISPR/Cas9 system to introduce mutations into the GmIPK1 gene with guide RNAs in soybean (cv. Kwangankong). The GmIPK1 gene was disrupted using the CRISPR/Cas9 system, with sgRNA-1 and sgRNA-4 targeting the second and third exon, respectively. Several soybean Gmipk1 gene-edited lines were obtained in the T₀ generation at editing frequencies of 0.1–84.3%. Sequencing analysis revealed various indel patterns with the deletion of 1–9 nucleotides and insertions of 1 nucleotide in several soybean lines (T₀). Finally, we confirmed two sgRNA-4 Gmipk1 gene-edited homozygote soybean T₁ plants (line #21-2: 5 bp deletion; line #21-3: 1 bp insertion) by PPT leaf coating assay and PCR analysis. Analysis of soybean Gmipk1 gene-edited lines indicated a reduction in PA content in soybean T₂ seeds but did not show any defects in plant growth and seed development.     

Changes in Oil Content of Transgenic Soybeans Expressing the Yeast <Emphasis Type="Italic">SLC1</Emphasis> Gene

The wild type (Wt) and mutant form of yeast (sphingolipid compensation) genes, SLC1 and SLC1-1, have been shown to have lysophosphatidic acid acyltransferase (LPAT) activities (Nageic et al. in J Biol Chem 269:22156–22163, 1993). Expression of these LPAT genes was reported to increase oil content in transgenic Arabidopsis and Brassica napus. It is of interest to determine if the TAG content increase would also be seen in soybeans. Therefore, the wild type SLC1 was expressed in soybean somatic embryos under the control of seed specific phaseolin promoter. Some transgenic somatic embryos and in both T2 and T3 transgenic seeds showed higher oil contents. Compared to controls, the average increase in triglyceride values went up by 1.5% in transgenic somatic embryos. A maximum of 3.2% increase in seed oil content was observed in a T3 line. Expression of the yeast Wt LPAT gene did not alter the fatty acid composition of the seed oil.     

Low-palmitic,low-linolenic soybean development

Plant breeding research efforts are currently focused on developing breeding procedures to decrease the saturated FA palmitic acid (16∶0) and the PUFA linolenic acid (18∶3) in U.S. soybean cultivars. Soybean oil with lower 16∶0 may provide cardiovascular benefits to health-conscious consumers, and lower 18∶3 could contribute to better flavor and stability of the oil. The purpose of this study was to determine genetic parameters that indicate the potential for breeding success and to characterize the correlated effect of the incorporation of the modified oil traits on the agronomic and seed quality traits of a soybean breeding population formed from a cross between the soybean cultivar Anand (normal) and germplasm N97-3708-13 (low 16∶0, low 18∶3). Although lines with only one modified oil quality trait (low 16∶0 or 18∶3) are useful as parents, commercial utilization requires productive cultivars with the combination of both oil traits. This paper shows the ease with which they may be combined with seed yield and other traits. Measurements were obtained from 179 F₂ single plants grown in 1999 and 121 F_2∶4 lines grown in replicated plots in 2000. Modified FA lines were developed with ca. 4% 16∶0 and 18∶3, respectively. Very weak positive correlations were found between 16∶0 concentration and seed yield (r=0.12) and between 16∶0 and seed oil concentration (r=0.13). No correlation was found between 18∶3 levels and seed yield, or between 18∶3 levels and seed oil concentration. These results indicate that breeding for reduced 16∶0 and 18∶3 should not have a negative impact on seed yield or oil concentration. 16∶0 and 18∶3 had moderately high heritabilities of 0.65 and 0.73, respectively. This indicates that breeders using low 16∶0, low 18∶3 germplasm in crosses with normal, elite lines can expect to recover low 16∶0 and low 18∶3 in pure line progenies via selection and generation advancement of F₂ individuals that express low levels of these FA.     

Genomic Regions Governing the Biosynthesis of Unsaturated Fatty Acids in Recombinant Inbred Lines of Soybean Raised across Multiple Growing Years

The ratio of oleic acid to the combined value of linoleic and α-linolenic acids determines the oxidative stability, and the ratio of linoleic acid to α-linolenic acid is the key to the nutritional value of soybean oil. The present study was conducted to identify genomic regions associated with oleic, linoleic, and α-linolenic acids in recombinant inbred lines (RIL), developed from LSb1 × NRC7, across 5 cropping years. These RIL were genotyped using 105 polymorphic SSR markers across soybean genome and analyzed for fatty acid composition. SSR markers, namely, Satt245 (LGp M), Satt556 (LGpB2), Sat_042 (LGp C1), Staga002 (LGp D1b), Satt684 (LGp A1), and AI856415 (LGpD1b) showed significant (P < 0.05) association with oleic acid for all the 5 years, though this association was weak in the years when the growing temperature during active seed formation stage was high. Quantitative trait loci (QTL) linked to Satt684 (LGp A1), Satt556 (LGp B2), Sat_042 (LGp C1), and AI856415 (LGp D1b) showed pleiotropic influence on the levels of unsaturated fatty acids. Complementation of favorable QTL from LSb1 and NRC7 generated 60% oleic acid and less than 4% α-linolenic acid RIL, stable across 5 cropping years. New SSR markers, namely, Satt245, AI856415, and Staga002 identified to be associated with different unsaturated fatty acids may be useful in improving the efficiency of marker-assisted breeding for enhancing the monounsaturated to polyunsaturated fatty acids ratio of soybean oil.     

Soybean seed composition under high day and night growth temperatures

High diurnal temperatures often affect development of soybean [Glycine max (L.) Merr.], but little is known about the relative influence of high day and night temperatures on the chemical composition of the seed. This study was conducted to determine the effects of combinations of high day and night temperatures during flowering and pod set (R1–R5), seed fill and maturation (R5–R8), and continuously during the reproductive period (R1–R8) on soybean seed oil, protein, and fatty acid composition. Day/night temperatures of 30/20, 30/30, 35/20, and 35/30°C were imposed on the soybean cultivar Gnome 85 in growth chambers. The day/night temperature combinations during R1–R5 had little effect on the oil and protein concentration and the fatty acid composition of seed produced. As mean daily temperature increased from 25 (30/20) to 33 (35/30)°C during R5–R8 and 25 (30/20) to 33 (35/30)°C during R1–R8, and oil concentration decreased and protein concentration increased. Increased day temperature during R5–R8 and R1–R8, averaged across the two night temperatures, increased oleic acid and decreased linoleic and linolenic acids. When night temperature was increased at 30°C day temperature during R5–R8 and R1–R8, oleic acid decreased and linoleic acid increased. When night temperature was increased at 35°C day temperature during R1–R8, oleic acid increased, and linoleic and linolenic acids decreased. These results indicate the importance of high day and night temperatures during seed fill and maturation in the oil, protein, and fatty acid composition of soybean seed.     

Non-destructive Determination of High Oleic Acid Content in Single Soybean Seeds by Near Infrared Reflectance Spectroscopy

Soybean [Glycine max (L.) Merr] with increased oleic acid is desirable to improve oxidative stability and functionality of soybean seed oil. Recently, soybean genotypes with high oleic acid (≥70 %) were developed by breeding programs. Efficient and effective identification of high oleic acid soybean genotypes using non-destructive near infrared reflectance (NIR) on whole seeds would greatly enhance progress in breeding programs. The objective of this study was to develop a calibration equation for NIR determination of high oleic acid from single soybean seeds. A total of 600 intact, single F₂ seeds were scanned by NIR. Spectral data were collected between 400 and 2,500 nm at 2 nm intervals. The relationship between NIR spectral patterns of each soybean seed and its oleic acid content was examined. The best predicted equations for oleic acid were selected on the basis of minimizing the standard error of cross-validation and increasing the coefficient of determination. Validation demonstrated that the equations for determining total oleic acid and over 50 % oleic acid content had high predictive ability (r ² = 0.91 and r ² = 0.99, respectively). To validate the newly developed equation, F₂ seeds from a different genetic background were tested. Again, high oleic acid from single soybean seeds was accurately predicted from various genetic backgrounds. Therefore, applying the calibration equations to NIR will be useful to rapidly and efficiently select high oleic acid soybean genotypes in breeding programs.     

Improvement of Oil Quality in Soybean [<Emphasis Type="Italic">Glycine max </Emphasis>(L.) Merrill] by Mutation Breeding

Low oxidative stability, off-flavor and rancidity are the major drawbacks of soybean oil. Modification of the fatty acid composition of soybean [Glycine max (L.) Merrill] oil can improve its quality and value for processors and acceptability among consumers. Mutation breeding of soybean was therefore initiated with the objective of identifying stable soybean mutants with altered fatty acid composition for improved oxidative stability and nutritional quality. Seeds of soybean cultivar ‘MACS 450’ were treated with γ-radiation and/or ethyl methane sulfonate (EMS). The harvest of M₁ plants was evaluated for fatty acid composition by gas chromatography. Highly significant variation in all the fatty acids except palmitic acid was observed. Treatment of EMS in higher concentrations as well as combined treatment of both the mutagens, i.e., γ-radiation and EMS were effective in increasing the variability for the fatty acid content in soybean oil. The variability was skewed towards high levels of oleic (35–42%) and low levels of linolenic acid (3.77–5.00%). M₃ and M₄ generations of desirable variants were analyzed for the stability of the mutated trait_. Only high oleic variants were stable in M₃ and M₄ generations. Based on fatty acid values, oxidative stability index (OSI), nutritional quality index (NQI) and ratio of essential fatty acids (ω₆/ω₃) were calculated for the control and M₂, M₃ and M₄ generations. The ω₆/ω₃ ratio in all the high oleic mutants was within the World Health Organization (WHO) recommended value (5–10%). A significant positive correlation between OSI and oleic acid content (P < 0.001) indicated improved oxidative stability of the oil while retaining nutritional quality. These high oleic lines could be utilized further in breeding programs for improvement of soybean oil quality.