Isolation and Characterization of Proximal Promoters Regulating Ethylene-Induced Ripening in Banana

Fruit ripening is a key developmental phase with biochemical and physiological changes. In climacteric fruits (ethylene responsive), such as bananas, ethylene predominantly regulates these changes. This study presents a protocol to isolate and functionally analyse proximal promoters of banana ripening genes. Using genome walking, we identify promoter fragments of key ripening genes. We then characterize their ethylene responsiveness. Promoter-GUS fusion constructs are prepared for functional testing. We deliver these constructs transiently into banana fruit tissues. Promoter activity is quantified by histochemical (color-based) and fluorometric (fluorescence-based) β-glucuronidase (GUS) assays. Findings clarify the regulation of ripening-associated pathways. Therefore, they suggest genetic engineering options to extend shelf life and improve banana nutritional quality. This protocol provides a practical guide for researchers to study and manipulate the molecular mechanisms underlying fruit ripening.