The efficient genetic transformation of Cordyceps militaris by using mononuclear protoplasts

Abstract As a highly valued edible mushroom, Cordyceps militaris has attracted considerable attention because it contains a variety of bioactive ingredients. However, the unknown number of cell nuclei and lack of optimized protoplast preparation conditions have hindered the studies of protoplast fusion breeding and genetic engineering to increase the content of bioactive constituents. To further promote protoplast fusion and understand the function of genes involved in the synthesis of active ingredients, this study aimed to investigate the number of nuclei in C. militaris and increase the yield of protoplasts derived from mononuclear mycelia as well as the regeneration rate. The results observed with a fluorescence microscope showed that the conidia, blastospores, and mycelia of C. militaris were all mononuclear. The maximum protoplast yield of 2.25 × 10 7 protoplasts/g fresh weight (FW) was achieved when 4-day-old mononuclear C. militaris mycelia were incubated in an enzymolysis solution (pH 6.5) composed of 1.00% lysing enzyme, 1.00% lywallzyme, and 0.8 M KCl at 32 °C for 3.0 h. The maximum regeneration rate (36.5%) was obtained when protoplasts prepared under the above optimal conditions were spread onto regeneration media (RM) containing 1.0 M sorbitol. Linear DNA fragments containing the bar gene were efficiently transformed into protoplasts by polyethylene glycol (PEG)-mediated transformation, and 87 PCR-positive mutants containing the bar gene were obtained. These results provide effective methods for the preparation and regeneration of protoplasts derived from mononuclear mycelia, which lays an important foundation for efficient genetic transformation in C. militaris .