Condition optimization for protoplast preparation of Botrytis cinerea and the patho-genicity of regenerated strains
In order to obtain high-quality and sufficient protoplasts of Botrytis cinerea that can be used for ge-netic transformation,effects of multiple parameters including mycelial age,combinations of lytic enzymes,types of osmotic stabilizers,enzymatic hydrolysis temperature,and time of enzyme digestion on protoplasts prepara-tion were studied.The optimal lytic enzyme was determined to be the combination of driselase,snailase,and ly-sing enzyme at an active ingredient of 1% ,0.1% and 1% ,respectively,and the mycelial age,composition and concentration of osmotic stabilizer,enzyme digestion temperature,and enzyme digestion time were as follows:mycelia of JA-6 was cultivated on PDA at 25 ℃ for 36 h,osmotic stabilizer contained 0.6 mol·L-1 KCl and 50 mmol·L-1 CaCl2,and enzyme digestion time was 3 h at 120 r·min-1 at 28 ℃.Enzymatic hydrolysis of 5 g·mL-1 of B.cinerea mycelium can yield 1.06×107 protoplasts·mL-1 under above optimal protoplast preparation condi-tions.No significant differences of the colony morphology,growth rate,conidial production and pathogenicity were observed between the regenerated strain and the wild-type strain.The GFP(green fluorescent protein)gene was subsequently transformed into B.cinerea JA-6 by PEG mediated transformation.The fluorescence signal of the transformants can be stably inherited.The established protoplast preparation method in the present research would meet the requirements of genetic transformation of B.cinerea for further study.
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