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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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References

Deane, EE, Whipps, JM and Lynch, JM et al. . (1999) Transformation of Trichoderma reesei with a constitutively expressed heterologous fungal chitinase gene. Enzyme and Microbial Biotechnology 24: 419–424.CrossRefGoogle Scholar
Ding, WL, Cheng, HZ and Zhang, GZ (1997) Advantages of Trichoderma and its applying to control the medicinal plant disease. Chinese Traditional and Herbal Drugs 28: 505–507. (in Chinese with English abstract)Google Scholar
Elad, Y and Kirsher, B (1999) Role of Trichoderma harzianum protease in the biocontrol of Botrytis cinerea. European Journal of Plant Pathology 105: 77–189.CrossRefGoogle Scholar
Giczey, G, Kerenyi, Z and Dallmann, G et al. . (1998) Homologous transformation of Trichoderma hamatum with an endochitinase encoding gene, resulting in increased levels of chitinase activity. FEMS Microbiology Letters 165: 247–252.CrossRefGoogle ScholarPubMed
Groot, MJAD, Paul, B and Paul, HJJ (1998) Agrobacterium tumefaciens -mediated transformation of filamentous fungi. Nature Biotechnology 16: 839–842.CrossRefGoogle ScholarPubMed
Herrera, EA, Goldman, GH and Montagu, MV (1990) High-efficiency transformation system for the biocontrol agents, Trichoderma spp. Molecular Microbiology 4: 839–843.CrossRefGoogle Scholar
Krauss, U and Soberanis, W (2002) Effect of fertilization and biocontrol application frequency on cocoapod diseases. Biological Control 24: 82–89.CrossRefGoogle Scholar
Li, DB, Zhou, XP and Xu, JP et al. (1996) Gene Engineering Operation Technique. Shanghai: Technology Press (in Chinese).Google Scholar
Manczinger, L, Komonyi, OAntal, Z et al. . (1997) A method for high-frequency transformation of Trichoderma virides. Journal of Microbiological Methods 29: 207–210.CrossRefGoogle Scholar
Margolles, CE, Harman, GE and Penttila, M (1996) Enhanced expression of endochitinase in Trichoderma harzianum with the cbhI promoter of Trichoderma reesei. Applied and Enviromental Microbiology 62: 2152–2155.CrossRefGoogle Scholar
Moriguchi, M and Kotegawa, S (1985) Preparation and regeneration of protoplasts from mycelia of Morchella. Agricultural and Biological Chemistry 49: 2791–2793.Google Scholar
Oinonen, AM, Torkkeli, T and Polohejmo, M et al. . (1997) Overexpression of the Aspergillus niger pH?2.5 acid phosphatase gene in a heterologous host Trichoderma reesei. Journal of Biotechnology 58: 13–20.CrossRefGoogle Scholar
Sabatini, MA, Grazioso, P and Altomare, C et al. . (2002) Interactions between Onychiurus armatus and Trichoderma harzianum in take-all disease suppression in a simple experimental system. European Journal of Soil Biology 38: 71–74.CrossRefGoogle Scholar
Schiestl, RH and Gietz, RD (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Current Genetics 16: 339–346.CrossRefGoogle ScholarPubMed
Shi, Z, Christian, D and Leung, H (1995) Enhanced transformation in Magnaporthe grisea by restriction enzyme mediated integration of plasmid DNA. Phytopathology 85: 329–333.CrossRefGoogle Scholar
Wang, ZY and Li, DB (2001) Restriction-enzymed insertional mutagenesis and its application in filamentous fungi. Mycosystema 20: 142–147. (in Chinese).Google Scholar
Yang, Q and Zhao, XY (1998) Transformation of the Trichoderma harzianum with gene of the resistance to carbendazim. Chinese Science Bulletin 43: 2423–2426. (in Chinese).Google Scholar