Soybean is one of the most important crops because of its high protein and oil content. Previous studies have refined protoplast isolation methods for soybeans to enhance transfection efficiency. However, these methods have limitations due to the inconsistent number of viable protoplasts for various applications. In this study, we propose an optimized protoplast isolation method to overcome this challenge. Hypocotyls grown in the dark were selected to ensure rapid growth and a steady supply of plant materials. The hypocotyls were cut to 1–2 cm in length and halved longitudinally to achieve consistent protoplast yields. Our new hypocotyl cutting method demonstrated 1.5 times improved protoplast yield and improved protoplast viability compared to that of previous methods. The isolated protoplasts were purified using the sucrose density gradient purification method to remove residues while stacking viable protoplasts. Fluorescein diacetate (FDA) staining was performed to determine the proportion of healthy protoplasts throughout the process. Consequently, we propose a new protoplast isolation protocol that ensures a higher yield, better viability, and healthier conditions. This enhancement is expected to improve the efficiency of soybean transfection.

Phytophthora root rot attributable to infection by the soil-borne oomycete Phytophthora sojae causes serious damage to susceptible soybeans grown in poorly drained soil. Management of this disease depends primarily on Rps (resistance to P. sojae) resistance genes. The
objective
of this study was to map resistance to two P. sojae isolates (40412 and 2457) in a Daepung×Socheong2 recombinant inbred line population. Of these two varieties, Socheong2 is resistant to the two isolates, whereas Daepung is susceptible. Single-marker analysis of variance and linkage analyses using a high-density genetic map identified different resistance loci for each isolate. A genomic region of 36.2~37.4 Mbp on chromosome 3 was identified as being associated with resistance to isolate 40412, explaining 18% of the phenotypic variance (PV), whereas, a 2.1~2.6-Mbp region on chromosome 18 was significantly associated with resistance to isolate 2457, accounting for approximately 26% of the PV. An additional region of 53.1~53.3 Mbp on chromosome 18 was also significantly associated with resistance to isolate 2457. All three loci coincide with genomic regions where an Rps gene or partial resistance have been mapped in previous studies. The respective locus showed significance for only one or the other of the isolates, indicating an isolate-specific interaction. From this finding, it can be inferred that isolates 40412 and 2457 are characterized by different avirulence genes, and that Socheong2 has at least two Rps genes that interact with each isolate. The finding of multiple Rps loci specific to an isolate within a single soybean genotype is a unique discovery. Socheong2 will accordingly be a useful genetic source for breeding resistance to multiple P. sojae isolates.