Preparation and Regeneration of Protoplast from Verticillium dahliae

doi:10.13560/j.cnki.biotech.bull.1985.2018-0160

Abstract

Abstract: In order to establish optimal conditions on preparation and regeneration of protoplast from Verticillium dahlia,the single-factor experiment was used to analyze the effects of culture time,concentration of lyase,enzymatic digesting time and temperature,type and concentration of osmotic stabilizer,and pH on the release of protoplasts. The conditions of regeneration medium,agar concentration,and enzymatic lysing time on protoplast regeneration were optimized. In order to observe protoplast visually,the recombinant plasmid containing GFP was transformed by PEG-CaCl₂-mediated method. The results showed that the yield of protoplast was the highest with 3.3×10⁷cells/mL under the optimal conditions：collecting the mycelia when conidia cultured for 18 h,using 1.2 mol/L KCl as osmotic stabilizer,then adding 10 mg/mL lyase at pH 6.0,and lysing for 4 h at 30℃. Protoplast regeneration was achieved in TB3 medium with 0.5% agar,and the regeneration rate was up to 22.45%. The result from GFP transient-expression indicated that the protoplasts under optimal conditions may be used for genetic transformation.

Key words: Verticillium dahliae, protoplast preparation, protoplast regeneration, transformation

ZHAO Xiao-qiang, CHEN Zhi-rong, HE Fang, SHEN Nan, GAO Feng, HUANG Jia-feng. Preparation and Regeneration of Protoplast from Verticillium dahliae[J]. Biotechnology Bulletin, 2018, 34(7): 166-173.

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