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Development of Auxotrophic Agrobacterium tumefaciens for Gene Transfer in Plant Tissue Culture
Author(s) -
Collens Jason I.,
Lee Dae R.,
Seeman Amy M.,
Curtis Wayne R.
Publication year - 2004
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp034306w
Subject(s) - auxotrophy , biology , agrobacterium tumefaciens , transformation (genetics) , agrobacterium , insertional mutagenesis , transposable element , mutant , microbiology and biotechnology , expression vector , dna , gene , genetics , recombinant dna
Auxotrophic strains of Agrobacterium tumefaciens were generated for use in liquid co‐culture with plant tissue for transient gene expression. Twenty‐one auxotrophs were recovered from 1,900 tetracycline‐resistant insertional mutants generated with a suicide vector transposon mutagenesis system. Twelve of these auxotrophs were characterized on a nutrient matrix. Isolates were screened for growth in plant cell and root culture, and three auxotrophs were identified that had limited growth: adenine (ade‐24), leucine (leu‐27), and cysteine (cys‐32). Ade‐24 displayed poor T‐DNA delivery in a transient expression test delivering GUS from a binary vector, while cys‐32 displayed the best ability to deliver DNA of these three auxotrophs. The growth yield of cys‐32 on cysteine was assessed to provide a quantitative basis for co‐culture nutrient supplementation. The utility of cys‐32 for delivering T‐DNA to plant tissues is demonstrated, where an 85‐fold enhancement in GUS expression over wild‐type A. tumefaciens was achieved.