Development of a stable genetic transformation system in Echinochloa phyllopogon

Abstract Echinochloa phyllopogon is a major C4 grass weed that causes serious yield losses in paddy fields and has evolved resistance to multiple herbicides. Understanding the molecular mechanisms underlying this resistance requires functional analysis of candidate genes involved in herbicide metabolic detoxification. However, the lack of a stable transformation system has limited such approaches. In this study, we established a particle bombardment-mediated transformation system for E. phyllopogon using embryogenic calli and plasmid pAHC25 carrying the GUS reporter gene and the bialaphos resistance gene ( bar ) selectable marker. Three morphologically distinct callus types were identified, among which Type 1 (compact) and Type 2 (friable) embryogenic calli with exhibited significantly higher regeneration ability and transient GUS expression than Type 3 calli (wet and viscous). This finding indicates that callus quality is a critical determinant of transformation efficiency. In addition, callus culture duration significantly affected transformation outcomes. Calli cultured for 90 days showed the highest transformation efficiency (5.0%) and complete regeneration capacity, whereas prolonged culture (300 and 630 days) resulted in reduced transformation efficiency and regeneration. Stable transgene integration and expression were confirmed by Southern blot analysis and histochemical GUS staining. Furthermore, transgenic plants exhibited stable inheritance of the introduced gene and strong resistance to Basta in the T 1 generation. These results demonstrate that both callus type and culture duration are key factors for efficient transformation. The transformation system established in this study provides a valuable platform for functional analysis of genes involved in herbicide metabolic detoxification in E. phyllopogon .