At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for complex genome editing, especially the deletion of large DNA fragments in soybean, has not been systematically explored. In this study, we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2a. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2a and 15.8% for target fragments ranging from 1069 to 1161 bp in GmFT5a. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 ‘transgene-free’ homozygous ft2a mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean.

As a genetically modified crop, transgenic soybean occupies the largest global scale with its food, nutritional, industrial, and pharmaceutical uses.Efficient transformation is a key factor for the improvement of genetically modified soybean. At present, the Agrobacterium-mediated method is primarily used for soybean transformation, but the efficiency of this method is still relatively low (below 5%) compared with rice (above 90%). In this study, we examined the influence of l-glutamine and/or l-asparagine on Agrobacterium-mediated transformation in soybean and explored the probable role in the process of Agrobacterium-mediated transformation. The results showed that when the amino acids l-glutamine and l-asparagine were added separately or together to the culture medium, the shoot induction frequency, elongation rate, and transformation frequency were improved. The combined effects of l-glutamine and l-asparagine were better than those of l-glutamine and l-asparagine alone. The 50 mg/L l-glutamine and 50 mg/L l-asparagine together can enhance the transformation frequency of soybean by attenuating the expression level of GmPRs (GmPR1, GmPR4, GmPR5, and GmPR10) and suppression of the plant defense response. The transgene was successfully transmitted to the T1 generation. This study will be useful in genetic engineering of soybean.

Symbiotic nitrogen fixation boosts legume growth and production in nitrogen-poor soils. It has long been assumed that fixed nitrogen increases reproductive success, but until now, the regulatory mechanism was unknown. Here, we report a symbiotic flowering pathway that couples symbiotic and nutrient signals to the flowering induction pathway in legumes. We show that the symbiotic microRNA–microRNA172c (miR172c) and fixed nitrogen systemically and synergistically convey symbiotic and nutritional cues from roots to leaves to promote soybean ( Glycine max ) flowering. The combinations of symbiotic miR172c and local miR172c elicited by fixed nitrogen and development in leaves activate florigen-encoding FLOWERING LOCUS T ( FT ) homologs ( GmFT2a/5a ) by repressing TARGET OF EAT1-like 4a ( GmTOE4a ). Thus, FTs trigger reproductive development, which allows legumes to survive and reproduce under low-nitrogen conditions.

Soybean (Glycine max (L.) Merr.) oil is an important source of vegetable oil for supporting the human diet. However, the high level of polyunsaturated fatty acids in natural soybean oil renders the oil unstable and thus susceptible to the development of unpalatable flavors and trans fatty acids. Therefore, reducing the content of polyunsaturated fatty acids and increasing the content of monounsaturated fatty acids is a longstanding and important target for soybean breeding. However, soybean varieties with a high oleic acid content are rare in soybean germplasm resources, which introduces substantial difficulties in the cultivation of high-oleic-acid soybeans. In this study, CRISPR/Cas9-mediated gene-editing technology was used to create targeted knockout of the soybean fatty acid desaturase encoding genes GmFAD2-1A and GmFAD2-1B that contribute to the formation of polyunsaturated fatty acids. We obtained fad2-1a, fad2-1b, and fad2-1a/fad2-1b homozygous mutants using two sgRNAs. We found that the oleic acid content increased from 11% to 40-50% in the fad2-1a and fad2-1b mutants and to 85% in the fad2-1a/fad2-1b mutants. We also generated transgene-free double mutants that conferred higher oleic acid, and the fad2-1a/fad2-1b mutant had no adverse phenotyping compared with the wild type. Our study provided new materials for the selection and breeding of high-oleic-acid soybean varieties.

Current systems to screen for transgenic soybeans (Glycine max) involve laborious molecular assays or the expression of fluorescent markers that are difficult to see in soybean plants. Therefore, a visual system for early screening of transgenic plants would increase the efficiency of crop improvement by genome editing. The RUBY reporter system, which consists of three genes encoding betalain biosynthetic enzymes, leading to the accumulation of purple pigment in transgenic tissue, has been employed in some plants and dikaryon fungi. Here, we assessed the RUBY reporter for visual verification during soybean transformation. We show that RUBY can be expressed in soybean, allowing for visual confirmation of transgenic events without the need for specialized equipment. Plants with visible accumulation of purple pigment in any tissue were successfully transformed, confirming the accuracy of the RUBY system as a visual indicator. We also assessed the genetic stability of the transgene across generations, which can be performed very early, using the cotyledons of the progeny. Transgene-free seedlings have a distinct green color, facilitating the selection of genome-edited but transgene-free soybean seedlings for harvest. Using the RUBY system, we quickly identified a transgene-free Gmwaxy mutant in the T1 generation. This system thus provides an efficient and convenient tool for soybean genome editing.

Flowering time and growth period are key agronomic traits which directly affect soybean (Glycine max (L.) Merr.) adaptation to diverse latitudes and farming systems. The FLOWERING LOCUS T (FT) homologs GmFT2a and GmFT5a integrate multiple flowering regulation pathways and significantly advance flowering and maturity in soybean. Pinpointing the genes responsible for regulating GmFT2a and GmFT5a will improve our understanding of the molecular mechanisms governing growth period in soybean. In this study, we identified the Nuclear Factor Y‐C (NFY‐C) protein GmNF‐YC4 as a novel flowering suppressor in soybean under long‐day (LD) conditions. GmNF‐YC4 delays flowering and maturation by directly repressing the expression of GmFT2a and GmFT5a. In addition, we found that a strong selective sweep event occurred in the chromosomal region harboring the GmNF‐YC4 gene during soybean domestication. The GmNF‐YC4^Hap3 allele was mainly found in wild soybean (Glycine soja Siebold & Zucc.) and has been eliminated from G. max landraces and improved cultivars, which predominantly contain the GmNF‐YC4^Hap1 allele. Furthermore, the Gmnf‐yc4 mutants displayed notably accelerated flowering and maturation under LD conditions. These alleles may prove to be valuable genetic resources for enhancing soybean adaptability to higher latitudes.