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.
Highlights
Gaeumannomyces tritici, one of the major soil-borne pathogens, survives saprophytically on crop debris
The results showed that a mixture of 0.25% snailase (S), 0.25% driselase (D), and 0.25% lysing enzyme (L) produced 2.68 × 107 protoplasts/mL, which was higher than the enzyme of 0.25% L
The enzyme of 0.25% L was significantly higher than the enzyme of 0.25% D (0.17 × 107 protoplasts/mL) and 0.25% S (0.03 × 107 protoplasts/mL)
Summary
Gaeumannomyces tritici, one of the major soil-borne pathogens, survives saprophytically on crop debris. G. tritici colonizes roots of susceptible cereals, especially wheat, and causes take-all disease in the affected areas [1]. G. tritici mycelium [2,3]. This devastating disease is widespread throughout wheat producing areas worldwide [4,5], resulting in yield losses of 40–60% [6,7,8]. Chemical fungicides are not always effective for soil-born fungi, and theses fungicides may have negative effects on the environment and human health [9,10]. The G. tritici genome sequence is available [1] and there is an urgent need for an efficient and rapid transformation system
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