Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici

Abstract

Take-all, caused by Gaeumannomyces tritici, is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors-temperature, enzyme lysis time, and concentration of the lysing enzyme-were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 10⁷ protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46⁻54 transformants/μg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici. An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.

Keywords: Gaeumannomyces tritici; PEG-mediated; protoplast; response surface methodology (RSM); transformation.

Figure 1

(a) Effects of media on protoplast yield; (b) effects of digesting temperature on protoplast yield; and (c) effects of enzyme lysis time on protoplast yield. Data represent the mean ± standard errors (SE) of three independent experiments. Different lower-case letters indicate a significant difference (p < 0.05).

Figure 2

(a) Effects of mycelia weight on protoplast yield; (b) effects of rotational speed on protoplast isolation; and (c) effects of concentration of lysing enzyme on protoplast yield. Data represent the mean ± standard errors (SE) of three independent experiments. Different lower-case letters indicate significant difference (p < 0.05).

Figure 3

Response surfaces and contour plots showing: (a) the combined effect of temperature and enzyme lysis time on the protoplast yield; (b) the combined effect of temperature and lysing enzyme concentration on the protoplast yield; and (c) the combined effect of enzyme lysis time and lysing enzyme concentration on the protoplast yield.

Figure 4

Southern blot hybridization analysis of strains using the region of hygromycin phosphotransferase gene as a probe. (a) Strains were identified by polymerase chain reaction (PCR) with primers of Yzb-F/R using genomic DNA as the template; (b) Southern blot hybridization analysis of strains using the region of hygromycin phosphotransferase gene as a probe, which was amplified with primers of Yzb-F/R. Lane M: DNA Marker 2000; Lane 1: G. tritici; Lanes 2–5: hph transformants.