Non-transgenic Soybean Seeds Research Articles

Quadruplex and q-PCR based diagnostic assay to delineate the major quarantine and other seed-borne fungal pathogens of soybean.

Soybean is one of the most important crops grown worldwide and accounting for significant global trade including transgenic soybean. The crop is attacked by several seed-borne fungal pathogens and some of them are of quarantine concern for India. Keeping in view of the risks associated with movement of soybean seeds, sensitive and reliable molecular diagnostics have been developed for precise and simultaneous detection of three pathogens of quarantine concern for India namely, Diaporthe phaseolorum (stem blight), D. longicolla (seed decay), Peronospora manshurica (downy mildew), along with Macrophomina phaseolina causing dry root rot. The targeted pathogens after isolation from imported transgenic and non-transgenic soybean seeds were identified. Quadruplex and qPCR assays were developed targeting the sequences of different genes such as Histone-3 for detection of D. longicolla and M. phaseolina. The markers DlHisF2&R2 and MpHisF1&R1 produced 265 and 309bp amplicons for D. longicolla and M. phaseolina, respectively. Actin gene based marker DpActF1&R2 was developed for D. phaseolorum which provided 113bp amplicon whereas, COX2 based marker PmCoxF2&R2 was developed for P. manshurica with amplified product of 152bp. During qPCR analysis, these markers proved highly specific and sensitive for detection of these pathogens up to 0.1 pg of template DNA. Quadruplex PCR protocol was also developed by combining these specific markers which could distinguish all the targeted pathogens simultaneously in a single reaction. The developed diagnostic protocols are extremely valuable for quarantine clearance and to ensure the safe transboundary exchange and healthy conservation of germplasm in the National Genebank.

On-line coupling of physiologically relevant bioaccessibility testing to inductively coupled plasma spectrometry: Proof of concept for fast assessment of gastrointestinal bioaccessibility of micronutrients from soybeans

In-vitro physiologically relevant gastrointestinal extraction based on the validated Unified BARGE Method (UBM) is in this work hyphenated to inductively coupled plasma optical emission spectrometry in a batch-flow configuration for real-time monitoring of oral bioaccessibility assays with high temporal resolution. A fully automated flow analyzer is designed to foster in-line filtration of gastrointestinal extracts at predefined times (≤15 min) followed by on-line multi-elemental analysis of bioaccessible micro-nutrients, viz., Cu, Fe and Mn, in well-defined volumes of extracts (300 μL) of transgenic and non-transgenic soybean seeds taken as model samples.The hyphenated flow setup allows for recording of temporal extraction profiles to gain full knowledge of the kinetics of the gastrointestinal digestion processes, including element leaching and concomitant precipitation and complexation reactions hindering bioavailability. Simplification of the overall standard procedure is also feasible by identification of steady-state extraction conditions. Our findings indicate that reliable measurement of oral bioaccessible pools of Cu, Fe and Mn in soybean might be obtained in less than 180 min rather than 240 min as endorsed by UBM. Using a matrix-matched external calibration, limits of detection according to the 3s criteria were 0.5 μg/g for Mn, 0.6 μg/g for Cu and 2.3 μg/g for Fe. Trueness of the automatic bioaccessibility method was confirmed by mass balance validation with recoveries ranging from 87 to 116% regardless of the target element and sample. Cu was the micronutrient with the highest oral bioaccessibility ranging from 73% to 83% (7.5–7.9 μg/g) for non-transgenic and transgenic soybeans, respectively, followed by Mn and Fe within the ranges of 29–31% (10.8–11.4 μg/g) and 11–15% (8–14 μg/g), respectively, regardless of transgenesis. The proposed kinetic method is proven suitable for fast and expedient estimation of the nutritional value of soybeans and elucidation of the potential effect of transgenesis onto bioaccessible fractions of elements.

Comparative analysis of chemical compositions between non-transgenic soybean seeds and those from plants over-expressing AtJMT, the gene for jasmonic acid carboxyl methyltransferase

Transgenic overexpression of the Arabidopsis gene for jasmonic acid carboxyl methyltransferase (AtJMT) is involved in regulating jasmonate-related plant responses. To examine its role in the compositional profile of soybean (Glycine max), we compared the seeds from field-grown plants that over-express AtJMT with those of the non-transgenic, wild-type (WT) counterpart. Our analysis of chemical compositions included proximates, amino acids, fatty acids, isoflavones, and antinutrients. Overexpression of AtJMT in the seeds resulted in decreased amounts of tryptophan, palmitic acid, linolenic acid, and stachyose, but increased levels of gadoleic acid and genistein. In particular, seeds from the transgenic soybeans contained 120.0–130.5% more genistein and 60.5–82.1% less stachyose than the WT. A separate evaluation of ingredient values showed that all were within the reference ranges reported for commercially available soybeans, thereby demonstrating the substantial equivalence of these transgenic and non-transgenic seeds.

Enhanced levels of nicotianamine promote iron accumulation and tolerance to calcareous soil in soybean.

Iron (Fe) is an essential nutrient in both plants and humans. Fe deficiency on calcareous soil with low Fe availability is a major agricultural problem. Nicotianamine (NA) is one of the Fe chelator in plants, which is involved in metal translocation into seeds, and serves as an antihypertensive substance in humans. In this study, soybean plants overexpressing the barley NA synthase 1 (HvNAS1) gene driven by the constitutive CaMV 35S promoter were produced using Agrobacterium-mediated transformation. The transgenic soybean showed no growth defect and grew normally. The NA content of transgenic soybean seeds was up to four-fold greater than that of non-transgenic (NT) soybean seeds. The level of HvNAS1 expression was positively correlated with the amount of NA, and a high concentration of NA was maintained in the seeds in succeeding generations. The Fe concentration was approximately two-fold greater in transgenic soybean seeds than in NT soybean seeds. Furthermore, the transgenic soybeans showed tolerance to low Fe availability in calcareous soil. Our results suggested that increasing the NA content in soybean seeds by the overexpression of HvNAS1 offers potential benefits for both human health and agricultural productivity.

Improving metallomics information related to transgenic and non-transgenic soybean seeds using 2D-HPLC-ICP-MS and ESI-MS/MS

This work reports the use of 2D-HPLC-ICP-MS to enlarge metallomics information when considering soybean seeds. Separations using size exclusion chromatography (SEC) allowed the identification of three metal fractions: the first corresponding to molecular weights from 38.1 to 181.1 kDa, the second from 8.2 to 17.2 kDa and the third from 0.4 to 3.8 kDa. In a second dimension, using anion exchange chromatography (AEX), three sub-fractions containing Fe, Mg and Mn, one containing Cu, and three containing Co, Cu, Mg, Mn and Zn were obtained. After these separations, 33 proteins were identified using the ESI-MS/MS technique, and divided into four functional categories: plant growth/cell division, protein destination and storage, metabolism and unclassified proteins. Among the identified proteins, proteins previously related to metals were found.

New insights on proteomics of transgenic soybean seeds: evaluation of differential expressions of enzymes and proteins

This work reports the evaluation of differentially expressed enzymes and proteins from transgenic and nontransgenic soybean seeds. Analysis of malondialdehyde, ascorbate peroxidase (EC 1.11.1.11), glutathione reductase (EC 1.6.4.2), and catalase (EC 1.11.1.6) revealed higher levels (29.8, 30.6, 71.4, and 35.3%, respectively) in transgenic seeds than in nontransgenic seeds. Separation of soybean seed proteins was done by two-dimensional polyacrylamide gel electrophoresis, and 192 proteins were identified by matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) and electrospray ionization (ESI) QTOF MS. Additionally, the enzyme CP4 EPSPS, involved in the genetic modification, was identified by enzymatic digestions using either trypsin or chymotrypsin and ESI-QTOF MS/MS for identification. From the proteins identified, actin fragment, cytosolic glutamine synthetase, glycinin subunit G1, and glycine-rich RNA-binding protein were shown to be differentially expressed after analysis using the two-dimensional difference gel electrophoresis technique, and applying a regulator factor of 1.5 or greater.

Image analysis of two-dimensional gel electrophoresis for comparative proteomics of transgenic and non-transgenic soybean seeds

Considering the importance of bidimensional electrophoresis and image analysis in comparative proteomics, the parameters that influence the analysis of protein expression of transgenic and non-transgenic soybean seeds were evaluated. The loaded mass of the proteins (150-500 microg), the pH separation range (3-10 or 4-7), and manual/automatic image editing were evaluated. Additionally, after optimizing the conditions, histograms and matchings were obtained in order to accurately analyze the variations (90%) in protein expression. From this, 10 proteins displayed significant differences in expression, and eight of them were characterized and identified by mass spectrometry.

A comparative study of element concentrations and binding in transgenic and non-transgenic soybean seeds

Transgenic and non-transgenic soybean seeds were compared in terms of total element concentrations, behavior of elements during sequential extraction fractionation and element bioaccessibility using an in vitro simulated gastrointestinal digestion. The analysis were carried out by ICP-sector field-MS or size-exclusion ICP-MS (25 elements in concentrations varying from ng g⁻¹ to the % level). It seems that transgenic and non-transgenic soybean seeds exhibit statistically significant differences in concentrations of Cu, Fe and Sr, which are also reflected by element contents in water extracts and residues. Additionally, contributions of bioaccessible fractions of Cu, Fe and other elements (Mn, S, Zn) for transgenic soybean seeds appear to be larger than those found in non-transgenic soybean seeds.

Analysis of the composition and concentration of fatty acids in transgenic soybean (cp4-epsps1) oil

The transgenic and non-transgenic soybean seeds oils were extracted and the composition and concentration of the fatty acids were analyzed by gas chromatography. Sixteen different fatty acids were detected in both transgenic and non-transgenic soybean oil: C14:0, C16:0, C18:1, C18:2, C18:3, C20:0, C20:1, C20:2, C22:0, C20:5, C22:1, C24:0 and C22:6. The concentration of 12 of 16 fatty acids (C14:0, C16:0, C18:1, C18:2, C20:0, C20:1, C20:2, C22:0, C20:5, C22:1, C24:0 and C22:6) in transgenic soybean oil were higher than those in non-transgenic one (P < 0.05). The concentration of C17:0 and C18:3 were lower than those in non-transgenic one (P < 0.05). The total fatty acids concentration increases by 12.6 % (P < 0.01). The degree of saturation of transgenic soybean oil increased from 58.7 to 69.8 % (P < 0.01). All the data above show that the insertion of foreign genes can change the concentration of fatty acids in plant oil. The mechanism for these changes should be studied.