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United States Department of Agriculture

Agricultural Research Service

Title: Conditions for Efficient Agrobacterium Tumefaciens-Mediated Transformation of Ascochyta Rabiei.

Authors
item White, David
item Chen, Yung Chun
item Chen, Weidong

Submitted to: North American Pulse Improvement Association
Publication Type: Abstract Only
Publication Acceptance Date: March 31, 2005
Publication Date: N/A

Technical Abstract: In order to develop an insertional mutagenesis transformation system to study pathogenicity factors of Ascochyta rabie, the causal agent of Ascochyta blight of chickpea, the conditions for efficient transformation of A. rabiei using Agrobacterium tumefaciens-Mediated Transformation have been determined. Two selection markers and two expression promoters were compared. Hygromycin B resistance was found to be superior to geneticin resistance in transforming A. rabiei, and the Aspergillus nidulans trpC promoter was more efficient than the Cauliflower mosaic virus 35S promoter CaMV35S. Extended co-cultivation times were optimal and acetocsyringone concentrations >100 µM were required to generate transformants, whereas increasing the ratio of bacterial cells to conidia did not affect transformation efficiency. Hygromycin-resistant transformants carried the T-DNA as determined by PCR and the majority of insertions appeared to be random and in single copy per genome as detected by Southern hybridization. All transformants tested remained mitotically stable, maintaining their resistance to hygromycin even without continuous selection. During screening transformants for pathogenicity, two transformants were unable to infect chickpea. This technique will provide a useful tool for studying pathogenicity factors of A. rabiei.

Last Modified: 11/23/2014
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