<FONT face="Verdana">Establishment of Agrobacterium tumefaciensMediated Transformation System for <EM>Rhizoctonia solani</EM> AG-1 ⅠA, the Causal Agent of Rice Sheath Blight</FONT>

YANG  Ying-qing#,YANG  Mei#,LI  Ming-hai,LI  Yong,HE  Xiao-xia,ZHOU  Er-xun*

doi:10.3969/j.issn.1001-7216.2010.06.009

2010 , Vol. 24 >Issue 6: 617 - 622

DOI: https://doi.org/10.3969/j.issn.1001-7216.2010.06.009

研究报告

Establishment of Agrobacterium tumefaciensMediated Transformation System for Rhizoctonia solani AG-1 ⅠA, the Causal Agent of Rice Sheath Blight

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Department of Plant Pathology, South China Agricultural University, Guangzhou 510642, China; ^#These authors contributed equally to this paper; *Corresponding author, E-mail: exzhou@scau.edu.cn

Received date: 1900-01-01

Revised date: 1900-01-01

Online published: 2010-11-10

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Abstract

In order to construct the TDNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG1 ⅠA, the virulent isolate GD118 of this pathogen was selected as the initial isolate for transformation. The conditions for the transformation of GD118 were optimized in 5 aspects, i.e. preinduction time, coculture time, acetosyringone concentration at coculture stage, coculture temperature and pH values of solid induction medium (SIM) at coculture phase. Finally, an Agrobacterium tumefaciensmediated transformation (ATMT) system for R. solani AG1 ⅠA was established successfully. The optimal conditions for this ATMT system are as follows: the concentration of hygromycin B at 30 μg/mL for transformant screening, 8 h of preinduction time, 20 h of coculture time, 200 μmol/L of acetosyringone in SIM at coculture stage, coculture temperature at 25℃ and pH 5.6 to 5.8 of SIM at coculture phase. The transformants still showed high resistance to hygromycin B after 5 generations’ subcultures. Ten transformants were randomly picked out for PCR verification using the specific primers designed from the hph gene, and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants. Meantime, PCR amplification for these 10 transformants was carried out using specific primers designed from the Vir gene of A. tumefaciens, with 4 strains of A. tumefaciens as positive controls for eliminating the falsepositive caused by A. tumefaciens′s contamination, and the results showed that an expected band of 730 bp could be amplified from the 4 strains of A. tumefaciens, whereas no corresponding DNA band could be amplified from the 10 transformants. The results of above two PCR amplifications clearly showed that TDNA was indeed inserted into the targeted isolate GD118.

Key words： rice sheath blight; Rhizoctonia solani; Agrobacterium tumefaciensmediated transformation; TDNA insertional mutagenesis; methodology

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YANG Ying-qing#,YANG Mei#,LI Ming-hai,LI Yong,HE Xiao-xia,ZHOU Er-xun* . Establishment of Agrobacterium tumefaciensMediated Transformation System for Rhizoctonia solani AG-1 ⅠA, the Causal Agent of Rice Sheath Blight[J]. Chinese Journal OF Rice Science, 2010 , 24(6) : 617 -622 . DOI: 10.3969/j.issn.1001-7216.2010.06.009

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