Transgenic Soybean Lines Research Articles

Utility of Arabidopsis KASII Promoter in Development of an Effective CRISPR/Cas9 System for Soybean Genome Editing and Its Application in Engineering of Soybean Seeds Producing Super-High Oleic and Low Saturated Oils.

This study reports the use of the Arabidopsis KASII promoter (AtKASII) to develop an efficient CRISPR/Cas9 system for soybean genome editing. When this promoter was paired with Arabidopsis U6 promoters to drive Cas9 and single guide RNA expression, respectively, simultaneous editing of the three fatty acid desaturase genes GmFAD2-1A, GmFAD2-1B, and GmFAD3A occurred in more than 60% of transgenic soybean lines at T2 generation, and all the triple mutants possessed desirable high-oleic traits. In sharp contrast, not a single line underwent simultaneous editing of the three target genes when AtKASII was replaced by the widely used AtEC1.2 promoter. Furthermore, our study showed that the stable and inheritable mutations in the high-oleic lines did not alter the overall contents of oil and protein or amino acid composition while increasing the oleic acid content up to 87.6% from approximately 23.8% for wild-type seeds, concomitant with 34.4- and 3.7-fold reductions in linoleic and linolenic acid, respectively. Collectively, this study demonstrates that the AtKASII promoter is highly promising for optimization of the CRISPR/Cas9 system for genome editing in soybean and possibly beyond.

Improvement of superior soybean variety, Biosoy-1 for aluminium tolerance character

Abstract Indonesia has lot of sub-optimal land, especially outside Java, but generally the land is acidic, contains lots of heavy metals and is poor in nutrients, making it less suitable for agriculture, including soybean cultivation. Efforts to improve land conditions through the application of liming are quite helpful, but are inefficient because they are easily washed out by rainwater and require expensive labor and costs for large areas. The use of tolerant and adaptive varieties for acidic land are effective solutions. Biosoy-1 and Biosoy-2 are new superior soybean varieties released by Ministry of Agriculture which have high yield potential (more than 3 tons/ha) and big grain, but are less adaptive to sub-optimal land so that the planting area is still limited. Efforts to improve the Biosoy variety for aluminum stress tolerant have been carried out through the introgression of the MaMt2 gene from transgenic soybean lines into the Biosoy variety. Thirty of F1 soybean seeds have been produced and 24 plants of them were positive for carrying the MaMt2 gene. Furthermore, the F1-MaMt2 plants were backcrossed to Biosoy several times in order to obtain a soybean line with high yield and tolerance to aluminum stress. Currently, a total of 16 number of BC3F1 seeds are being planting in confined screen house for molecular analysis and following by backcrossing to Biosoy-1 variety to produce BC4F1 seeds.

Overexpression of cry1c* Enhances Resistance against to Soybean Pod Borer (Leguminivora glycinivorella) in Soybean.

Soybean [Glycine max (L.) Merr.], an essential staple food and oil crop worldwide, boasts abundant vegetable proteins and fats beneficial for both human and animal consumption. However, the soybean pod borer (Leguminivora glycinivorella) (SPB) stands as the most destructive soybean insect pest in northeast China and other northeastern Asian regions, leading to significant annual losses in soybean yield and economic burden. Therefore, this study aims to investigate the introduction of a previously tested codon-optimized cry1c gene, cry1c*, into the soybean genome and assess its effect on the SPB infestation by generating and characterizing stable transgenic soybeans overexpressing cry1c*. The transgenic soybean lines that constitutively overexpressed cry1c* exhibited a significant reduction in the percentage of damaged seeds, reaching as low as 5% in plants under field conditions. Additionally, feeding transgenic leaves to the larvae of S. exigua, S. litura, and M. separta resulted in inhibited larval growth, decreased larval body weight, and lower survival rates compared to larvae fed on wild-type leaves. These findings showed that the transgenic lines maintained their resistance to SPB and other lepidopteran pests, especially the transgenic line KC1. Southern blotting and genome-wide resequencing analysis revealed that T-DNA integration occurred as a single copy between loci 50,868,122 and 50,868,123 of chromosome 10 in the transgenic line KC1. Therefore, the transgenic line KC1, overexpressing high levels of cry1c* in leaves and seeds, holds strong potential for commercial use in the integrated management of SPB and other lepidopteran pests.

The GmCYP2-GmHAL3 module regulates salt tolerance in soybean seedlings

Soybean (Glycine max L. Merr) production is severely affected by soil salinization as an important crop. The discovery of salt stress-responsive genes is essential for soybean breeding. To our knowledge, cyclophilins (CYPs) play important roles in salt stresses in crops such as rice and cotton, except in soybean. This study cloned GmCYP2 from soybean salt-tolerant cultivar Suxie No. 1 and obtained ten stable transgenic soybean lines overexpressing GmCYP2 by Agrobacterium rhizogenes-mediated transformation. Two of the higher-expressing transgenic lines (OE1 and OE2) were used to compare salt tolerance with the wild type (WT). Under the salt stress, the chlorophyll content, the maximum efficiency of PSII photochemistry (Fv/Fm) and actual photochemical quantum yield [Y(II)] of OE1 and OE2 lines were significantly higher than those of the WT. Meanwhile, the accumulation of malondialdehyde (MDA) in the OE1 and OE2 lines was significantly lower than that in the WT. In addition, the Na+/K+ ratio was also significantly lower than that of the WT in leaves and roots. The results also confirmed that the salt tolerance of GmCYP2-silencing soybean seedlings obtained by virus-induced gene silencing (VIGS) was reduced compared to the control. Subcellular localization showed that GmCYP2 was expressed in the plasma membrane and nucleus of Nicotiana benthamiana. RNA-seq results demonstrated that GmCYP2 is involved in key regulators of the ABA and Ca2+ signaling pathway. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and split-luciferase complementation assay demonstrated that GmCYP2 protein interacts with salt-associated halotolerance 3 (HAL3) protein. These results suggest that the GmCYP2-GmHAL3 module enhances salt tolerance of soybean seedlings by maintaining the Na+/K+ ratio and good photosynthetic state and it may be involved in the ABA and Ca2+ signaling pathway.

Evaluating salinity effects on transgenic soybean harboring a cytokinin dehydrogenase gene at early growth stage

Soil salinization, along with drought, is a natural threat that has a great impact on agricultural production in many locations throughout the world. The cytokinin oxidases/dehydrogenases (CKXs) play an essential role in determining plant CK levels and several of which have been modulated to improve abiotic stress tolerance. In this study, the aim was to investigate whether there were differential effects of salinity on wild-type (WT) soybean (Glycine max L.) plants and a transgenic soybean line harboring GmCKX13, which had previously been identified as a responsive gene to osmotic stress. The effects of salt stress were monitored by deploying different concentrations of NaCl. According to the obtained results, the transgenic plants could maintain a better germination rate and radicle growth than the WT plants after a 4-day treatment of 200 mM NaCl. In addition, the transgenic seedlings also displayed a more vigorous shoot and root growth with a larger biomass production under the adverse condition. The analytic data revealed that advantages acquired by the transformed plants at least came from the better capacity to reserve water in the tissue and lower stress damage, as indicated by a lower malondialdehyde content. Taking these findings together, it is suggested that modulating the expression of CKX13 could contribute to better salt tolerance in soybeans, providing the foundation for conducting more elaborate studies in the future and paving the pathway for the development of elite salinity-tolerant varieties.

Overexpression of the Peanut AhDGAT3 Gene Increases the Oil Content in Soybean

Soybean (Glycine max) is the main oilseed crop that provides vegetable oil for human nutrition. The main objective of its breeding research is to increase the total oil content. In the Kennedy pathway, Diacylglycerol acyltransferase (DGAT) is a rate-limiting enzyme that converts diacylglycerol (DAG) to triacylglycerol (TAG). Here, the AhDGAT3 gene was cloned from peanut and overexpressed in the wild-type (WT) Arabidopsis. The total fatty acid content in T3AhDGAT3 transgenic Arabidopsis seeds was 1.1 times higher on average than that of the WT. Therefore, AhDGAT3 was transferred into the WT (JACK), and four T3 transgenic soybean lines were obtained, which proved to be positive using molecular biological detection. Specific T-DNA insertion region location information was also obtained via genome re-sequencing. The results of high-performance gas chromatography showed that the contents of oleic acid (18:1) composition and total fatty acids in transgenic soybean plants were significantly higher than that of the WT. However, linoleic acid (18:2) was much lower compared to the WT. The agronomic trait survey showed that the quantitative and yield traits of AhDGAT3 transgenic soybean were better than those of the WT. These results suggest that fatty acids in transgenic soybeans, especially oleic acid and total fatty acid, are enhanced by the over-expression of AhDGAT3.

Walking the 'design-build-test-learn' cycle: flux analysis and genetic engineering reveal the pliability of plant central metabolism.

This article is a Commentary on Morley et al. (2023), 239: 1834–1851.

Arbuscular mycorrhizal fungus Rhizophagus irregularis alleviates drought stress in soybean with overexpressing the GmSPL9d gene by promoting photosynthetic apparatus and regulating the antioxidant system

Drought is the most destructive abiotic stress and negatively affects crop growth and productivity. Modern breeding efforts have produced numerous cultivars with distinct genetic traits that improve crop growth and drought stress tolerance. Arbuscular mycorrhizal fungi (AMF) can enhance drought tolerance in soybean plants by directly providing nutrients to plants, promoting photosynthesis, or influencing interspecific plant interactions in natural communities. However, the interactions between AMF and wild and transgenic soybean genotypes remain unclear. Therefore, in the present study, we evaluated the effect of arbuscular mycorrhizal fungi on the growth performance of drought-stressed transgenic soybean lines (ZXOE-11 and ZXOE-13) overexpressing GmSPL9d gene and their wild soybean Tianlong 1 (TL1) at the seedling stage (45 d after sowing). The results showed that colonization of wild and transgenic soybean with Rhizophagus irregularis significantly decreased the adverse effects of drought on plant growth. AMF inoculation significantly increased plant biomass, root activity, chlorophyll metabolism, photosynthesis, and chlorophyll fluorescence in wild-type and transgenic plants under both control and drought stress conditions. Drought causes the production of ROS, such as hydrogen peroxide, which enhances MDA, thereby decreasing the membrane stability index (MSI). However, AMF-inoculated plants exhibited decreased ROS accumulation and increased MSI. Moreover, AMF treatment significantly improved osmolyte, nitrogen, and nitrate reductase activity under control and drought conditions, which increased the relative water content. Furthermore, AMF treatment enhanced the antioxidant systems of drought-stressed plants by increasing the activities of peroxidase, superoxide dismutase, catalase, and ascorbate peroxidase. AMF improved the growth performance, photosynthesis, and antioxidant activity of transgenic plants under drought stress conditions. The present findings indicate that the AMF contribution to soybean seedling drought tolerance was more significant for the transgenic plants than for the wild plants under drought conditions. The current findings emphasize the possibility of growth and photosynthetic variation in the degree of AMF-associated drought resistance in soybean plants. Our findings suggest that future crop breeding challenges include developing cultivars for sustainable production and maximizing crop cultivar and fungal species (AMF) combinations in drought-stressed regions.

Overexpression of GmNF-YA14 produced multiple phenotypes in soybean

Soybean is an important oil crop, and more and more soybean gene functions have been reported; however, one soybean gene with multiple functions is rarely seen. The NF-YA subfamily plays a broad regulatory role in plant growth and development, and GmNF-YA14 (Glyma.14G010000) is one of them. We found that the expression level of GmNF-YA14 in the root of soybean seedlings was significantly increased after a one-hour treatment with 200 mM salt. The transgenic soybean was obtained by Agrobacterium-mediated method, and all experiments were carried out using transgenic soybean homozygous lines. Under a 200 mM salt treatment, the leaf chlorophyll content of overexpressed GmNF-YA14 transgenic soybean seedlings was significantly decreased compared to the control. Moreover, the first flowering time of overexpressed GmNF-YA14 transgenic soybean was shortened by more than six days compared to the control, and the growth period structure changed significantly. Overexpressed GmNF-YA14 transgenic soybean plants had two or three branches, whereas control soybean plants had eight or nine branches. Compared to the control, the number of main stem nodes and plant height of overexpressed GmNF-YA14 transgenic soybean plants were significantly reduced. The 100-seed weight of GmNF-YA14 transgenic soybean line OE-8 was significantly higher than that of the control. Transcriptome analysis indicated that the multiple phenotypes of the overexpressed GmNF-YA14 transgenic soybean may be related to the mitogen-activated protein kinase (MAPK) signaling pathway. These results indicated that compared to the control, overexpressed GmNF-YA14 transgenic soybeans had changes in salt stress tolerance, growth period, plant type, and 100-seed weight, and these changes may be related to the MAPK signaling pathway.

Overexpression of soybean trypsin inhibitor genes decreases defoliation by corn earworm (Helicoverpa zea) in soybean (Glycine max) and Arabidopsis thaliana.

Trypsin inhibitors (TIs) are widely distributed in plants and are known to play a protective role against herbivores. TIs reduce the biological activity of trypsin, an enzyme involved in the breakdown of many different proteins, by inhibiting the activation and catalytic reactions of proteins. Soybean (Glycine max) contains two major TI classes: Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Both genes encoding TI inactivate trypsin and chymotrypsin enzymes, which are the main digestive enzymes in the gut fluids of Lepidopteran larvae feeding on soybean. In this study, the possible role of soybean TIs in plant defense against insects and nematodes was investigated. A total of six TIs were tested, including three known soybean trypsin inhibitors (KTI1, KTI2 and KTI3) and three genes encoding novel inhibitors identified in soybean (KTI5, KTI7, and BBI5). Their functional role was further examined by overexpression of the individual TI genes in soybean and Arabidopsis. The endogenous expression patterns of these TI genes varied among soybean tissues, including leaf, stem, seed, and root. In vitro enzyme inhibitory assays showed significant increase in trypsin and chymotrypsin inhibitory activities in both transgenic soybean and Arabidopsis. Detached leaf-punch feeding bioassays detected significant reduction in corn earworm (Helicoverpa zea) larval weight when larvae fed on transgenic soybean and Arabidopsis lines, with the greatest reduction observed in KTI7 and BBI5 overexpressing lines. Whole soybean plant greenhouse feeding bioassays with H. zea on KTI7 and BBI5 overexpressing lines resulted in significantly reduced leaf defoliation compared to non-transgenic plants. However, bioassays of KTI7 and BBI5 overexpressing lines with soybean cyst nematode (SCN, Heterodera glycines) showed no differences in SCN female index between transgenic and non-transgenic control plants. There were no significant differences in growth and productivity between transgenic and non-transgenic plants grown in the absence of herbivores to full maturity under greenhouse conditions. The present study provides further insight into the potential applications of TI genes for insect resistance improvement in plants.

Overexpression of lncRNA77580 Regulates Drought and Salinity Stress Responses in Soybean

Emerging evidence indicates that long non-coding RNAs (lncRNAs) play important roles in diverse biological processes. However, the biological functions of most plant lncRNAs are still unknown. We previously discovered a soybean abiotic-stress-related lncRNA, lncRNA77580, and cloned the entire full-length sequence. Here, in order to fully identify the function of lncRNA77580 in soybean stress response, we created transgenic soybean lines overexpressing lncRNA77580. Compared with the wild type, overexpression of lncRNA77580 enhances the drought tolerance of soybean. However, the transgenic plants exhibit increased sensitivity to high salinity at the seedling stage. We found that lncRNA77580 modulates the transcription of different gene sets during salt and drought stress response. Under water deficit at the reproductive stage, lncRNA77580 overexpression increases the seed yield by increasing the seed number per plant. These results provide insight into the role of lncRNA77580 in soybean stress response.

The host niches of soybean rather than genetic modification or glyphosate application drive the assembly of root-associated microbial communities.

Plant roots significantly influence soil microbial diversity, and soil microorganisms play significant roles in both natural and agricultural ecosystems. Although the genetically modified (GM) crops with enhanced insect and herbicide resistance are thought to have unmatched yield and stress resistance advantages, thorough and in-depth case studies still need to be carried out in a real-world setting due to the potential effects of GM plants on soil microbial communities. In this study, three treatments were used: a recipient soybean variety Jack, a triple transgenic soybean line JD321, and the glyphosate-treated JD321 (JD321G). Three sampling stages (flowering, seed filling and maturing), as well as three host niches of soybean rhizosphere [intact roots (RT), rhizospheric soil (RS) and surrounding soil (SS)] were established. In comparison to Jack, the rhizospheric soil of JD321G had higher urease activity and lower nitrite reductase at the flowering stage. Different treatments and different sampling stages existed no significant effects on the compositions of microbial communities at different taxonomic levels. However, at the genus level, the relative abundance of three plant growth-promoting fungal genera (i.e. Mortierella, Chaetomium and Pseudombrophila) increased while endophytic bacteria Chryseobacterium and pathogenic bacteria Streptomyces decreased from the inside to the outside of the roots (i.e. RT → RS → SS). Moreover, two bacterial genera, Bradyrhizobium and Ensifer were more abundant in RT than in RS and SS, as well as three species, Agrobacterium radiobacter, Ensifer fredii and Ensifer meliloti, which are closely related to nitrogen-fixation. Furthermore, five clusters of orthologous groups (COGs) associated to nitrogen-fixation genes were higher in RT than in RS, whereas only one COG annotated as dinitrogenase iron-molybdenum cofactor biosynthesis protein was lower. Overall, the results imply that the rhizosphere host niches throughout the soil-plant continuum largely control the composition and function of the root-associated microbiome of triple transgenic soybean.

GmPAO-mediated polyamine catabolism enhances soybean Phytophthora resistance without growth penalty

Plant immunity is activated upon perception of pathogens and often affects growth when it is constitutively active. It is still a challenge to balance plant immunity and growth in disease resistance breeding. Here, we demonstrated that soybean (Glycine max) polyamine oxidase (GmPAO) confers resistance to multiple Phytophthora pathogens, but has no obvious adverse impact on agronomic traits. GmPAO produces H2O2 by oxidizing spermidine and spermine. Phytophthora sojae induces an increase in these two substrates, and thus promotes GmPAO-mediated polyamine catabolism specifically during infection. Interestingly, we found that the two substrates showed higher accumulation in transgenic soybean lines overexpressing GmPAO than in WT and CK after inoculation with P. sojae to ensure H2O2 production during infection, rather than directly inhibit P. sojae. In these transgenic soybean plants, the significantly enhanced resistance to different P. sojae isolates was achieved; PAMP-induced H2O2 accumulation was enhanced by GmPAO overexpression. Moreover, transient expression of GmPAO also significantly improved Nicotiana benthamiana resistance to Phytophthora capsici and Phytophthora parasitica in agroinfiltration assays. Our results provide a novel approach to allow rapid defense responses in plants upon pathogen infection while minimizing growth penalties under normal conditions, with a clear mechanism in which plant promotes H2O2 production via pathogen-activated substrates.

Functional characterization of the pUceS8.3 promoter and its potential use for ectopic gene overexpression.

The pUceS8.3 is a constitutive gene promoter with potential for ectopic and strong genes overexpression or active biomolecules in plant tissues attacked by pests, including nematode-induced giant cells or galls. Soybean (Glycine max) is one of the most important agricultural commodities worldwide and a major protein and oil source. Herein, we identified the soybean ubiquitin-conjugating (E2) enzyme gene (GmUBC4; Glyma.18G216000), which is significantly upregulated in response to Anticarsia gemmatalis attack and Meloidogyne incognita-induced galls during plant parasitism by plant nematode. The GmUBC4 promoter sequence and its different modules were functionally characterized in silico and in planta using transgenic Arabidopsis thaliana and G. max lines. Its full-length transcriptional regulatory region (promoter and 5´-UTR sequences, named pUceS8.3 promoter) was able to drive higher levels of uidA (β-glucuronidase) gene expression in different tissues of transgenic A. thaliana lines compared to its three shortened modules and the p35SdAMV promoter. Notably, higher β-glucuronidase (GUS) enzymatic activity was shown in M. incognita-induced giant cells when the full pUceS8.3 promoter drove the expression ofthis reporter gene. Furthermore, nematode-specific dsRNA molecules were successfully overexpressed under the control of the pUceS8.3 promoter in transgenic soybean lines. The RNAi gene construct used here was designed to post-transcriptionally downregulate the previously characterized pre-mRNA splicing factor genes from Heterodera glycines and M. incognita. A total of six transgenic soybean lines containing RNAi gene construct were selected for molecular characterization after infection with M. incognita pre-parasitic second-stage (ppJ2) nematodes. A strong reduction in the egg number produced by M. incognita after parasitism was observed in those transgenic soybean lines, ranging from 71 to 92% compared to wild-type control plants. The present data demonstrated that pUceS8.3 is a gene promoter capable of effectively driving dsRNA overexpression in nematode-induced giant cells of transgenic soybean lines and can be successfully applied as an important biotechnological asset to generate transgenic crops with improved resistance to root-knot nematodes as well as other pests.

Comparative analysis of nutritional composition of seeds of wild-type soybean and lines overexpressing GmWRI1a

Context With the widespread use of transgenic soybeans (Glycine max (L. ) Merr.), their nutritional assessment for human food and animal feed is an important aspect of safety evaluation. WRINKLED 1 (WRI1) is a transcriptional activator of genes involved in fatty acid synthesis. Aims We aimed to evaluate the effect of overexpression of GmWRI1a in soybean on the nutritional profile of soybean seeds. Methods Using molecular biology techniques, we identified three transgenic soybean lines stably overexpressing GmWRI1a (GmWRI1a-OE). We quantified the nutritional and anti-nutritional components in seeds of GmWRI1a-OE and wild-type (DN50) soybean, and compared them on the basis of the principle of substantial equivalence. Key results The GmWRI1a and Bar genes were stably inherited in the three GmWRI1a-OE lines, and GmWRI1a protein content was higher in transgenic soybean seeds than in wild-type seeds. Overexpression of GmWRI1a resulted in changes in fatty acid composition, a significant increase in oil content, and a significant decrease in stachyose content in seeds. Contents of other nutritional components (proximates, amino acids and isoflavones) and anti-nutritional factors (phytic acid, trypsin inhibitors and raffinose) in seeds were not significantly different between the wild-type and GmWRI1a-OE lines. Conclusions Contents of nutritional and anti-nutritional components of GmWRI1a-OE seeds were all within the reference ranges reported for commercially available soybeans and, therefore, are substantially equivalent to those of wild-type seeds. Implications GmWRI1a-OE soybean seeds are a high-quality product for health-conscious consumers.

The GmXTH1 gene improves drought stress resistance of soybean seedlings.

In order to study the role of GmXTH1 gene in alleviating drought stress, soybean seeds with GmXTH1 gene were transferred by T4 treated with PEG6000 concentration of 0%, 5%, 10%, and 15% respectively. The germination potential, germination rate, germination index, and other indicators were measured. The results showed that the germination potential, germination rate, and germination index of OEA1 and OEA2 strains overexpressed in T4 generation were significantly higher than those of the control material M18. After 0-day, 7-day, and 15-day drought stress, the analysis of seedling phenotypes and root-shoot of different T4 generation transgenic soybean lines showed that under stress conditions, the growth of GmXTH1 overexpression material was generally better than that of the control material M18. The growth of GmXTH1 interference expression material was generally worse than that of the control material M18, with significant differences in plant phenotypes. The root system of GmXTH1 overexpressed material was significantly developed compared with that of the control material M18. The analysis of physiological and biochemical indexes showed that the relative water content and the activity of antioxidant enzymes (superoxide dismutase and peroxidase) of GmXTH1 transgenic soybean material were significantly higher than those of the control material M18, and the accumulation of malondialdehyde was lower under the same stress conditions at seedling stage. Fluorescence quantitative PCR assay showed that the relative expression of GmXTH1 gene in transgenic soybean was significantly increased after drought stress. The results showed that the overexpression of GmXTH1 could increase the total root length, surface area, total projection area, root volume, average diameter, total cross number, and total root tip number, thereby increasing the water intake and reducing the transpiration of water content in leaves, thus reducing the accumulation of MDA and producing more protective enzymes in a more effective and prompt way, reducing cell membrane damage to improve drought resistance of soybean.

The overexpression of NCED results in waterlogging sensitivity in soybean

Soil waterlogging limits plant growth and has a major impact on agricultural productivity. Here, we tested the hypothesis that the overexpression of the NCED gene improves waterlogging and reoxygenation tolerance in soybean plants. This was tested using a transgenic soybean line overexpressing the NCED gene (2Ha11) and the conventional cultivar BRS184 (wild-type; WT), which is the genetic background of the 2Ha11 line. Plants at vegetative (V6) and reproductive (R2) stages were exposed to waterlogging for 60 h and posterior reoxygenation for an additional 60 h. Overall, the NCED overexpression increased the levels of lipid peroxidation in plants exposed to waterlogging and posterior reoxygenation while decreased the antioxidant activity and alcohol dehydrogenase activity. In addition, NCED overexpression decreased seed weight and grain yield in the 2Ha11 line exposed to waterlogging at the reproductive stage. Taken together, these results suggest that the overexpression of NCED triggers an increased waterlogging sensitivity in soybean plants especially when they are exposed to waterlogging at the reproductive stage.

A Fast, Visual, and Instrument-free Platform Involving Rapid DNA Extraction, Chemical Heating, and Recombinase Aided Amplification for On-Site Nucleic Acid Detection.

The nucleic acid-based technique has been widely utilized in many fields including for on-site detection. However, traditional molecular detection techniques encounter limitations like relying on instruments, time consuming or complex operation, and cannot meet the demands of on-site testing. In this study, a rapid DNA extraction method (RDEM), recombinase aided amplification (RAA), and chemical heating packet (CHP) are integrated and termed as RRC platform for on-site detection of nucleic acid. For demonstration purposes, SHZD32-1 (a new transgenic soybean line from China) was detected using the novel platform to demonstrate its feasibility and capability for on-site detection. Using the RDEM, high-quality DNA appropriate for molecular detection was quickly extracted in 3–5 min. The heat energy generated by CHP was met the temperature requirements of RAA. Using the RRC platform, the whole detection process can be accomplished within only 30 min, and the results can be visually detected with glasses under blue light. No special or expensive instrument was needed for the detection process. This study provides a novel approach for on-site detection of nucleic acids besides providing valuable insight on related future research.

Convergence of Bar and Cry1Ac Mutant Genes in Soybean Confers Synergistic Resistance to Herbicide and Lepidopteran Insects.

Soybean is a globally important crop species, which is subject to pressure by insects and weeds causing severe substantially reduce yield and quality. Despite the success of transgenic soybean in terms of Bacillus thuringiensis (Bt) and herbicide tolerance, unforeseen mitigated performances have still been inspected due to climate changes that favor the emergence of insect resistance. Therefore, there is a need to develop a biotech soybean with elaborated gene stacking to improve insect and herbicide tolerance in the field. In this study, new gene stacking soybean events, such as bialaphos resistance (bar) and pesticidal crystal protein (cry)1Ac mutant 2 (M#2), are being developed in Vietnamese soybean under field condition. Five transgenic plants were extensively studied in the herbicide effects, gene expression patterns, and insect mortality across generations. The increase in the expression of the bar gene by 100% in the leaves of putative transgenic plants was a determinant of herbicide tolerance. In an insect bioassay, the cry1Ac-M#2 protein tested yielded higher than expected larval mortality (86%), reflecting larval weight gain and weight of leaf consumed were less in the T1 generation. Similarly, in the field tests, the expression of cry1Ac-M#2 in the transgenic soybean lines was relatively stable from T0 to T3 generations that corresponded to a large reduction in the rate of leaves and pods damage caused by Lamprosema indicata and Helicoverpa armigera. The transgenic lines converged two genes, producing a soybean phenotype that was resistant to herbicide and lepidopteran insects. Furthermore, the expression of cry1Ac-M#2 was dominant in the T1 generation leading to the exhibit of better phenotypic traits. These results underscored the great potential of combining bar and cry1Ac mutation genes in transgenic soybean as pursuant of ensuring resistance to herbicide and lepidopteran insects.

Differential Assembly and Shifts of the Rhizosphere Bacterial Community by a Dual Transgenic Glyphosate-Tolerant Soybean Line with and without Glyphosate Application

Modern agriculture has gained significant economic benefits worldwide with the use of genetically modified (GM) technologies. While GM crops provide convenience to humans, their biosafety has attracted increasing concern. In this study, the Illumina MiSeq was used to perform a high-throughput sequencing of the V3-V4 hypervariable regions of 16S rRNA gene (16S rDNA) amplicons to compare the rhizosphere bacterial communities of the EPSPS/GAT dual transgenic glyphosate-tolerant soybean line Z106, its recipient variety ZH10, and Z106 with glyphosate application (Z106G) during flowering, seed filling, and maturing stages under field settings. At each of the three stages, the alpha and beta diversity of rhizosphere bacterial communities revealed no significant differences between ZH10, Z106, and Z106G. However, some bacterial taxa demonstrated a greater proportional contribution, particularly the nitrogen-fixing rhizobium Ensifer fredii, in the rhizospheric soil of Z106 at the seed filling and maturing stages, when compared to ZH10 and Z106G. The present study therefore suggests that the EPSPS/GAT dual transgenic line Z106 and exogenous glyphosate application have a minimal effect on the composition of the soybean rhizosphere bacterial community but have no impact on the structure of the rhizosphere microbial community during a single planting season.