Kim Kihwan, Shin Junseop, Lee Jeong-Dong, Kim Won-Chan
Department of Applied Biosciences, Kyungpook National University, Daegu, Republic of Korea.
Upland Field Machinery Research Center, Kyungpook National University, Daegu, Republic of Korea.
Front Plant Sci. 2025 May 20;16:1587927. doi: 10.3389/fpls.2025.1587927. eCollection 2025.
Soybean is important crop species in agriculture, food science, and biotechnology due to their valuable components. The exploration of soybean genetic traits is being highlighted for the advancement of research in various aspects. The utilization of plant biotechnology, plant protoplast techniques, for the study of genetic characteristics is being extended to various agricultural crop species. The quintessential goal of genetic characterization utilizing plant protoplasts encompasses the provision of stable plant protoplasts alongside the establishment of transfection condition. Despite the numerous studies on protoplast isolation, standardized and reliable soybean protoplasts protocols for comprehensive investigations into the intricate regulatory mechanisms governing immune responses, cellular processes, and developmental pathways remain insufficiently established. In this study, we propose an efficient methodology for the protoplast isolation and the PEG-Ca mediated transfection of soybean [ (L.) Merr.] cultivar (Williams 82). The protoplast isolation entailed the evaluation of variables including mannitol concentration, enzyme mixture composition, and enzymatic digestion duration. The optimal conditions for hypocotyl-derived protoplast isolation were identified as 0.4 M mannitol, an enzyme mixture containing 1.5% (w/v) cellulase and 0.4% (w/v) macerozyme, and an 8-hour enzymatic digestion period, resulting in high viability and protoplast yield (>3.0 × 10/g FW). For the PEG-Ca mediated transfection process, the parameters assessed including PEG concentration, plasmid quantify or purified recombinant proteins, and PEG-Ca incubation duration. The validation of the reliability of hypocotyl-derived protoplast system through transient gene expression demonstrates its utility as a robust platform for analysis of genetic traits in soybean. This could extend the scope of application to understanding the cell-to-cell interactions for physiological responses in soybean.
大豆因其具有重要成分，在农业、食品科学和生物技术领域是重要的作物品种。大豆遗传特性的探索在各个方面的研究进展中受到重视。利用植物生物技术——植物原生质体技术来研究遗传特性，正被推广到各种农作物品种。利用植物原生质体进行遗传特性鉴定的核心目标包括提供稳定的植物原生质体以及建立转染条件。尽管对原生质体分离进行了大量研究，但用于全面研究免疫反应、细胞过程和发育途径等复杂调控机制的标准化且可靠的大豆原生质体方案仍未充分建立。在本研究中，我们提出了一种高效的方法，用于大豆[（L.）Merr.]品种（Williams 82）的原生质体分离和聚乙二醇 - 钙（PEG - Ca）介导的转染。原生质体分离涉及对甘露醇浓度、酶混合物组成和酶解持续时间等变量的评估。确定了从下胚轴分离原生质体的最佳条件为0.4 M甘露醇、含有1.5%（w/v）纤维素酶和0.4%（w/v）离析酶的酶混合物以及8小时的酶解时间，从而获得了高活力和原生质体产量（>3.0×10/g鲜重）。对于PEG - Ca介导的转染过程，评估的参数包括PEG浓度、质粒定量或纯化的重组蛋白以及PEG - Ca孵育持续时间。通过瞬时基因表达对下胚轴来源的原生质体系统可靠性的验证表明，它可作为分析大豆遗传特性的强大平台。这可能会扩大应用范围，以了解大豆生理反应中的细胞间相互作用。
