Hormone-optimized hypocotyl-epicotyl-cotyledonary tri-complex explant system enables efficient soybean transformation...

A composite hypocotyl-epicotyl-cotyledonary tri-complex (HECC) explant significantly improves soybean regeneration and Agrobacterium-mediated transformation efficiency (40.3%) while reducing genotype dependence, providing a rapid and robust platform for functional genomics. Soybean genetic transformation is hindered by low regeneration efficiency and considerable genotype dependency. Here, we present a hormone-optimized Agrobacterium-mediated transformation system employing a composite hypocotyl-epicotyl-cotyledonary tri-complex (HECC) explant that integrates multiple meristematic regions within a single explant and significantly improves regeneration and transformation efficiency. Compared to conventional single-meristem explant systems such as cotyledonary node and half-seed explants, HECC explants exhibited substantially higher regeneration frequencies and achieved an average transformation efficiency of 40.3%, calculated as the number of PCR-positive shoots obtained per infected explant in the JS-335 cultivar. Optimization of hormone combinations, particularly trans-zeatin riboside in shoot induction and elongation and indole-3-butyric acid (IBA) during rooting, promoted robust organogenic responses and reduced tissue damage. The system supported efficient transformation (26-41%) across multiple soybean cultivars, including JS-335, JS-2034, JS-2069 and PUSA-9712, indicating reduced genotype dependence compared with conventional explant systems. To demonstrate the applicability of this system for genome engineering, a CRISPR/Cas9 construct targeting GmSPL9c (Glycine max SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9c), a transcription factor involved in regulating plant developmental transitions and shoot architecture, was introduced. Molecular analyses by PCR and qRT-PCR confirmed the integration and expression of cas9 and bar genes, while segregation analysis in T₁ progeny demonstrated heritable transmission consistent with a 3:1 Mendelian segregation ratio. Functional selection by BASTA^® spraying in the T₁ generation further validated glufosinate tolerance and inheritance of the bar transgene. Collectively, this HECC-based system provides a reliable and reproducible platform for efficient soybean transformation and delivery of genome-editing constructs for functional genomics applications.