Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method.
The efficiency of soybean [Glycine max (L.) Merrill] transformation was significantly increased from an average of 0.7% to 16.4% by combining strategies to enhance Agrobacterium tumefaciens-mediated T-DNA delivery into cotyledonary-node cells with the development of a rapid, efficient selection protocol based on hygromycin B. Wounded cotyledonary-node explants were inoculated with A. tumefaciens carrying either a standard-binary or super-binary plasmid and co-cultivated in the presence of mixtures of the thiol compounds, L-cysteine, dithiothreitol, and sodium thiosulfate. Transformed shoots began elongating only 8 weeks after co-cultivation. Southern analysis confirmed integration of the T-DNA into genomic DNA and revealed no correlation between the complexity of the integration pattern and thiol treatment applied at co-cultivation. All T(0) plants were fertile and the majority of the lines transmitted the beta-glucuronidase (GUS) phenotype in 3:1 or 15:1 ratios to their progenies.
Olhoft
Paula M
PM
Department of Agronomy and Plant Genetics, University of Minnesota, 411 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108, USA.
Flagel
Lex E
LE
Donovan
Christopher M
CM
Somers
David A
DA
D016960:Agrobacterium tumefaciens / Q000235:genetics; D018548:Cotyledon / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D046508:Culture Techniques; D003545:Cysteine / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics; D006921:Hygromycin B / Q000494:pharmacology*; D010641:Phenotype; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D013025:Glycine max / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D013885:Thiosulfates / Q000494:pharmacology; D014162:Transfection / Q000379:methods
D004269:DNA, Bacterial; C036483:T-DNA; D013885:Thiosulfates; D006921:Hygromycin B; C017717:sodium thiosulfate; D003545:Cysteine; D004229:Dithiothreitol