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Isolation and transformation of Trichoderma viride protoplasts

Published online by Cambridge University Press:  12 February 2007

Liu Shi-Wang ,
Wang Zheng-Yi  and
Guo Ze-Jian
Liu Shi-Wang
Affiliation:
Biotechnology Institute, Zhejiang University, Hangzhou 310029, China College of Life Science, Xuzhou Normal University, Xuzhou 221116, China
Wang Zheng-Yi
Affiliation:
Biotechnology Institute, Zhejiang University, Hangzhou 310029, China
Guo Ze-Jian*
Affiliation:
College of Agronomy and Biotechnology, China Agricultural University, Beijing 100094, China
*
*Corresponding author: Email: [email protected]
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Abstract

The conditions for protoplast isolation and regeneration from Trichoderma viride were studied. Protoplasts were optimally isolated when mycelia of T. viride that had been cultured for 24 h were digested with 4 mg/ml Glucanex in phosphate buffer (pH 6.98) for 4 h at 30°C, resulting in a protoplast yield of 4.7×107 cfu/mg. The maximum regeneration ratio (14.5%) was obtained in mycelia culture medium containing 0.3 mol/l KCl and 0.3 mol/l inositol. In addition, a plasmid pCSSNCC1 carrying a hygromycin resistance gene and an elicitor-producing gene was transformed into T. viride protoplasts, with an efficiency of 1–2 transformants/μg DNA. The hygromycin-resistant transformants were determined by PCR and the elicitor protein was detected by ELISA. The results indicate that the elicitor protein was expressed stably in T. viride.

Keywords

Trichoderma virideprotoplastrestriction enzyme-mediated integration
Type
Research Article
Information
Chinese Journal of Agricultural Biotechnology , Volume 1 , Issue 2 , August 2004 , pp. 67 - 72
Copyright
Copyright © China Agricultural University and Cambridge University Press 2004

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