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Full Length Research Paper
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Protoplast formation,
regeneration and transformation from the taxol-producing
fungus Ozonium sp.
Xuanwei Zhou1*,
Yamin Wei2, Huifang Zhu1, Zinan Wang2,
Juan Lin2, Lu Liu2 and Kexuan Tang1,2*
1Plant
Biotechnology Research Center, Shanghai Key Laboratory of
Agrobiotechnology, School of Agriculture and Biology,
Fudan-SJTU-Nottingham Plant Biotechnology R and D Center,
Shanghai Jiao Tong University, Shanghai 200240, People’s
Republic of China.
2State
Key Laboratory of Genetic Engineering, School of Life
Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R and D
Center, Morgan-Tan International Center for Life Sciences,
Fudan University, Shanghai 200433, People’s Republic of
China.
*Corresponding authors. E-mail:
xuanweizhou@sjtu.edu.cn,
kxtang@sjtu.edu.cn.
Tel: +86-21-34205778. Fax: +86-21-34206916.
Accepted
12 May, 2008 |
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Abstract |
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The effects of some factors on protoplast isolation and
regeneration from taxol-producing fungus Ozonium sp.
BT2 were investigated, including the enzymolysis time
and temperature, the osmotic pressure stabilizer, mycelial
incubation time, the culture medium, the culture methods and
preprocessing. The mycelia were digested by enzyme
combination of 1.5% lywallzyme, 0.5% snailase, 1.5%
cellulase and 1.0% lysozyme for 3 h at 30°C, and a high
yield of protoplasts (1.31 × 108/g moist mycelia)
were obtained. The protoplasts were purified, and then
regenerated by different culture methods. The results showed
that regeneration frequency was not different. The
protoplasts could regenerate on both PDA and Czapek medium
with 0.6 M sucrose, 0.6 M sodium chloride and 0.6 M mannitol,
respectively. The regeneration rate was about 2.56% under
25°C on Czapek medium using 0.6 M sodium chloride as osmotic
pressure stabilizer. Furthermore, transformants were
obtained by transforming the protoplasts with plasmid pAN7-1
carrying hygromycin B phosphotransferase gene (hph) conferring hygromycin resistance. This study
provides the foundation to develop an engineered strain of
taxol-producing fungus by protoplast mutagenesis, fusion and
genetic transformation.
Key
words:
Endophytic fungus, enzyme combination, regeneration,
protoplast, transformation, Ozonium sp. |
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