Use of 4-methylindole or 7-methyl-DL-tryptophan in a transformant selection system based on the feedback-insensitive anthranilate synthase alpha-subunit of tobacco (ASA2).
Effective selectable markers are needed for basic research and commercial applications that do not involve antibiotic or herbicide resistance. A novel selection system based on a feedback-insensitive anthranilate synthase alpha-subunit of tobacco (ASA2) as selectable marker using either 4-methylindole (4MI) or 7-methyl-DL-tryptophan (7MT) as the selection agent was developed. We found that these two components were able to discriminate better between ASA2 expressing and untransformed lines than the most commonly used analog 5-methyltryptopan (5MT) in the seedling growth inhibition test. We successfully integrated an expression cassette containing an ASA2 cDNA driven by a cauliflower mosaic virus 35S promoter into tobacco leaf discs by A. tumefaciens and selected transgenic plants on medium supplemented with 300 microM of 7MT or 4MI. Due to the expression of the feedback-insensitive ASA2, the transgenic lines produced showed higher free tryptophan (Trp) concentrations than the untransformed WT control. These results demonstrate the feasibility of the selection system with the ASA2 gene in combination with the use of Trp or indole analogs as selective agent.
Department of Crop Sciences, Edward R Madigan Laboratory, University of Illinois, Urbana, IL 61801, USA.
P
P
Barone
Jack M
JM
Widholm
D016960:Agrobacterium tumefaciens / Q000235:genetics; D000878:Anthranilate Synthase / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D018515:Plant Leaves / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D018520:Plant Shoots / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction; D036226:Seedlings / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D014026:Nicotiana / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic / Q000187:drug effects; D014364:Tryptophan / Q000737:chemistry / Q000494:pharmacology*
D007211:Indoles; D014364:Tryptophan; D000878:Anthranilate Synthase