Developing an effective Agrobacterium-mediated transformation system for Bryophyllum pinnatum

Bryophyllum pinnatum, which is well-known for its regenerative and pharmacological properties, is devoid of a well-established genetic transformation system that would enable the investigation of its genetic and metabolic pathways. This study aimed to develop a reliable Agrobacterium tumefaciens-mediated transformation protocol for Bryophyllum pinnatum. Leaf tissues demonstrated the maximum regeneration potential among the tested explants, which is consistent with the plant’s inherent ability to propagate vegetatively. Transformation efficiency was systematically optimized by adjusting key parameters, such as Agrobacterium density (OD₆₀₀ = 0.7), co-cultivation conditions (pH 5.6, three days in darkness), and regulated wounding and desiccation treatment. Hygromycin (5 mg/L) was efficacious for selection, reducing chimerism and ensuring regeneration while maintain explant viability. The protocol resulted in a high transient GUS expression frequency of approximately 88%, indicating efficient T-DNA delivery, while stable integration of the transgene was confirmed in about 7% of explants. The method demonstrated strong reproducibility, accurate selection of transformants, and reduced bacterial overgrowth. This optimized transformation system provides a reliable platform for functional genomics, metabolic engineering, and targeted gene editing in Bryophyllum pinnatum.