Protoplast from β-carotene-producing fungus Blakeslea trispora: Preparation, regeneration and validation

Abstract

The effects of key parameters on the preparation and regeneration of protoplast from the β-carotene-producing fungus Blakeslea trispora were discussed in this paper, including the combination of various enzymes, mycelial age, digesting time and temperature, pH value, osmotic stabilizers, pretreatment, culture medium and culture method. Under the condition of mixed enzymes in osmotic stabilizer (0.6 M NaCl) combined with 2% lysozyme, 3% cellulase and 3% snailase, the highest protoplast yield, as high as 7.48×10 6 protoplasts/mL, was obtained when mycelial age was 60 h at pH 5.0-6.0 with digesting for 14-16 h at 28 o C. After purification of the obtained protoplasts, they were regenerated in PDA regenerative medium using bilayer plate culture method. To validate the usability of the protoplasts, a novel plasmid with green fluorescent protein (GFP) was used in transformation for easy visual observation. The results showed that the protoplasts prepared by the optimized method were active and applicable in further gene manipulation experiments.

Preparation and Regeneration of Protoplasts from the Ethyl Vincamine Producing Fungus CH1 (Geomyces sp.)

“…The effect, in decreasing order, was: cellulase, driselase, and glusulase. The results indicate that the combination of the three enzymes obtained higher protoplast yields than that of any of them used alone; these results agree with those of other authors [6,10]. This may be due to the fungus having a complicated structure and the three enzymes have different functions, and the combination of them may have a synergistic effect.…”

“…However, the protoplast yield decreased in the mixture of pretreatment solution and enzyme solution. A similar phenomenon was also found by Xu et al [13] and Li et al [6]. The reason may be that enzymolysis of the cell wall and the cell membrane after pretreatment of the mycelium caused the yield of protoplast to be lower than that of direct enzymolysis.…”

Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae

“…The efficiency of protoplast isolation, in addition to the digestion enzyme and other factors, also depends on the age of the mycelia. Young and exponentially growing mycelia are the best choices for protoplast isolation [ 36 ], but the optimal age of the mycelia varies for different species, e.g., 60 h for Blakeslea trispora and 2 days for Pleurotus pulmonarius and Pleurotus florida [ 40 , 41 ]. For V. dahliae , previous studies have used 24-h-old mycelia for protoplast isolation [ 18 , 19 ], but here we obtained more protoplasts from the 20-h culture than from the 24-h culture.…”

“…The probable reason for this can be the sensitivity of the younger mycelia to the digesting enzymes, with the increase in growth the cell wall becomes thicker and the mycelia would be digested more difficultly leading to decreased protoplast yield [ 36 , 38 , 39 ]. In addition to the age of mycelia, the protoplast yield also depends on the duration of the enzyme digestion, which has ranged from 3 h to 16 h for maximum protoplast release in other fungal species [ 37 , 40 , 42 ]. The optimum time of enzymolysis in our study was 2.5 h. Less time is presumably not enough for all the mycelia to be digested by the enzyme, while prolonged enzymolyis can result in the breaking of protoplasts [ 37 , 40 – 42 ].…”

“…In addition to the age of mycelia, the protoplast yield also depends on the duration of the enzyme digestion, which has ranged from 3 h to 16 h for maximum protoplast release in other fungal species [ 37 , 40 , 42 ]. The optimum time of enzymolysis in our study was 2.5 h. Less time is presumably not enough for all the mycelia to be digested by the enzyme, while prolonged enzymolyis can result in the breaking of protoplasts [ 37 , 40 – 42 ].…”