Methods for Cercospora coffeicola protoplast isolation and genetic transformation with the green fluorescent protein

Abstract

Cercospora coffeicola is the causal agent of brown eye spot on coffee leaves. Although the disease has significant importance, few molecular studies have been done with C. coffeicola. Here we report a protocol for isolating protoplasts as well as development of a genetic transformation system using Green Fluorescent Protein. High yields of protoplasts (≈108/ml) were obtained from mycelial cultures from five isolates of C. coffeicola. One isolate was transformed with a vector encoding hygromycin resistance and Green Fluorescent Protein. Out of 43 hygromycin-resistant transformants obtained, Green Fluorescent Protein was highly expressed in one (2.3 %).