Recent innovation in the field of genome engineering encompasses numerous levels of plant genome engineering which attract the substantial excitement of plant biologist worldwide. RNA-guided CRISPR Cas9 system has appeared a promising tool in site-directed mutagenesis due to its innovative utilization in different branches of biology. CRISPR-Cas9 nuclease system have supersedes all previously existed strategies and their associated pitfalls encountered with site-specific mutagenesis. Here we demonstrated an efficient sequence specific integration/mutation of FAD2–2 gene in soybean using CRISPR-Cas9 nuclease system. A single guided RNA sequence was designed with the help of a number of bioinformatics tools aimed to target distinct sites of FAD2–2 loci in soybean. The binary vector (pCas9-AtU6-sgRNA) has been successfully transformed into soybean cotyledon using Agrobacterium tumafacien. Taken together our findings complies soybean transgenic mutants subjected to targeted mutation were surprisingly detected in our target gene. Furthermore, the detection of Cas9 gene, BAR gene, and NOS terminator were carried out respectively. Southern blot analysis confirmed the stable transformation of Cas9 gene into soybean. Real time expression with qRT-PCR and Sanger sequencing analysis confirmed the efficient CRISPR-Cas9/sgRNA induced mutation within the target sequence of FAD2–2 loci. The integration of FAD2–2 target region in the form of substitution, deletions and insertions were achieved with notably high frequency and rare off-target mutagenesis. High frequent mutation efficiency was recorded as 21% out of all transgenic soybean plants subjected to targeted mutagenesis. Furthermore, Near-infrared spectroscopy (NIR) indicates the entire fatty acid profiling obtained from the mutants seeds of soybean. A considerable modulation in oleic acid content up to (65.58%) whereas the least level of linoleic acid is (16.08%) were recorded. Based on these finding CRISPR-Cas9 system can possibly sum up recent development and future challenges in producing agronomically important crops.

The RWP-RK is a small family of plant-specific transcription factors that are mainly involved in nitrate starvation responses, gametogenesis, and root nodulation. To date, the molecular mechanisms underpinning nitrate-regulated gene expression in many plant species have been extensively studied. However, the regulation of nodulation-specific NIN proteins during nodulation and rhizobial infection under nitrogen starvation in soybean still remain unclear. Here, we investigated the genome-wide identification of RWP-RK transcription factors and their essential role in nitrate-inducible and stress-responsive gene expression in soybean. In total, 28 RWP-RK genes were identified from the soybean genome, which were unevenly distributed on 20 chromosomes from 5 distinct groups during phylogeny classification. The conserved topology of RWP-RK protein motifs, cis-acting elements, and functional annotation has led to their potential as key regulators during plant growth, development, and diverse stress responses. The RNA-seq data revealed that the up-regulation of GmRWP-RK genes in the nodules indicated that these genes might play crucial roles during root nodulation in soybean. Furthermore, qRT-PCR analysis revealed that most GmRWP-RK genes under Phytophthora sojae infection and diverse environmental conditions (such as heat, nitrogen, and salt) were significantly induced, thus opening a new window of possibilities into their regulatory roles in adaptation mechanisms that allow soybean to tolerate biotic and abiotic stress. In addition, the dual luciferase assay indicated that GmRWP-RK1 and GmRWP-RK2 efficiently bind to the promoters of GmYUC2, GmSPL9, and GmNIN, highlighting their possible involvement in nodule formation. Together, our findings provide novel insights into the functional role of the RWP-RK family during defense responses and root nodulation in soybean.

Pathogenesis-related proteins, often used as molecular markers of disease resistance in plants, can enable plants to obtain systemic resistance. In this study, a gene encoding a pathogenesis-related protein was identified via RNA-seq sequencing analysis performed at different stages of soybean seedling development. Because the gene sequence showed the highest similarity with PR1L sequence in soybean, the gene was named GmPR1-9-like (GmPR1L). GmPR1L was either overexpressed or silenced in soybean seedlings through Agrobacterium-mediated transformation to examine the resistance of soybean to infection caused by Cercospora sojina Hara. The results revealed that GmPR1L-overexpressing soybean plants had a smaller lesion area and improved resistance to C. sojina infection, whereas GmPR1L-silenced plants had low resistance to C. sojina infection. Fluorescent real-time PCR indicated that overexpression of GmPR1L induced the expression of genes such as WRKY, PR9, and PR14, which are more likely to be co-expressed during C. sojina infection. Furthermore, the activities of SOD, POD, CAT, and PAL were significantly increased in GmPR1L-overexpressing soybean plants after seven days of infection. The resistance of the GmPR1L-overexpressing lines OEA1 and OEA2 to C. sojina infection was significantly increased from a neutral level in wild-type plants to a moderate level. These findings predominantly reveal the positive role of GmPR1L in inducing resistance to C. sojina infection in soybean, which may facilitate the production of improved disease-resistant soybean cultivars in the future.

Drought stress is a major limiting factor that adversely affects both the yield and quality of soybean crops. Transcription factors (TFs) play a pivotal role in regulating gene expression, facilitating plant adaptation and response to various abiotic stresses. Among the 179 NAC TFs encoded in the soybean genome, several are differentially expressed under stress conditions; however, the functional role of GmNAC3 in drought tolerance remains largely unknown. In this study, we cloned the 840 bp coding sequence of GmNAC3 and developed transgenic soybean hairy roots via Agrobacterium-mediated transformation to explore its role in drought response. The physiological and molecular responses of GmNAC3 overexpression (OE) chimeric soybean plants were assessed under polyethylene glycol (PEG)-simulated drought stress using Hoagland nutrient solution. Compared to empty vector (EV) controls, OE plants exhibited enhanced drought tolerance, including improved phenotypic traits, better root development, and stress resilience. Notably, OE plants showed a 23.9% reduction in hydrogen peroxide accumulation and a 31.25% decrease in superoxide anion levels. Biomass analysis on MS medium revealed significantly higher fresh and dry weights of OE hairy roots across different mannitol concentrations compared to EV roots. Furthermore, GmNAC3 overexpression led to the upregulation of key downstream genes involved in stress response, particularly GmLAC5 and GmLAC7. These findings suggest that GmNAC3 enhances drought tolerance in soybean by regulating both physiological and molecular pathways. Overall, GmNAC3 represents a promising target for genetic engineering aimed at improving drought resistance in soybean and potentially other crops.