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.

Soybean (Glycine max (L.) Merr.) is one of the most important crops with economic value as a source of protein and vegetable oil for human food and animal feed. In recent years, rapidly developed genome editing techniques have shown widespread application prospects for gene function studies and for improving important agronomic traits in many crops. Therefore, it is important to establish a highly efficient method for protoplast isolation and transient expression systems in soybeans. In this study, we established an efficient method for protoplast isolation and its application to transient gene expression in Korean soybean cultivars. The protoplasts were isolated from leaves, epicotyls, hypocotyls, cotyledons, and etiolated hypocotyls using various combinations of enzyme mixtures. We found that high-quality and large amounts of protoplasts were isolated from the etiolated hypocotyls when incubated for 8 h under conditions of 0.5% cellulase, 0.5% pectinase, and 1% viscozyme. In addition, we observed a high transfection efficiency of green fluorescent protein using etiolated hypocotyl protoplasts. Taken together, our protoplast isolation and transfection method is highly efficient and can be used for gene function and molecular analysis to better understand the biological and physiological processes in soybean.