Development of auxotrophic agrobacterium tumefaciens for gene transfer in plant tissue culture.
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.
Collens
Jason I
JI
Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Lee
Dae R
DR
Seeman
Amy M
AM
Curtis
Wayne R
WR
D016960:Agrobacterium tumefaciens / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D005240:Feasibility Studies; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013045:Species Specificity; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000382:microbiology*
D011994:Recombinant Proteins; D005966:Glucuronidase