Abstract: Botryosphaeria dothidea is a major important pathogen infecting a wide range of woody plant species. Understanding infection and pathogenic processes of the pathogen could help reveal the interaction mechanism between the pathogen and host better. Pathogen with expressed gfp gene can be used as an effective approach to detect and analyze the infection process. B. dothidea is difficult to produce spores during naturally infecting process and in vitro culture process, therefore, preparation of high quality protoplasts is essential for gfp gene transformation and expression. In this study, six parameters influencing protoplast preparation were analyzed, including enzyme species, enzyme concentration, mycelial age, time and temperature of enzymolysis and osmotic stabilizer. The results showed that optimal condition for gaining maximum yields of viable protoplasts was of 42-hour-old mycelia age incubated in 0.7 mol·L^-1 NaCl solution with 1.5% driselase and 1.5% glucanase at 31℃ for 3.5 h. The prepared protoplasts showed a regeneration efficiency of 48.33% in yeast extract peptone sucrose (YPS) medium. A reporter gene gfp conferring green fluorescent protein was transformed successfully to B. dothidea mediated by PEG-CaCl₂. Polymerase chain reaction (PCR) analysis,fluorescent microscope observation and stability test of transformants indicated that the gfp gene was stable in heredity and effective expression. This protocol was the first report for protoplast preparation and gfp transformation of B. dothidea.

Key words: Botryosphaeria dothidea, protoplast preparation, regeneration, transformants, gfp