Efficient insertion mutagenesis of Arabidopsis by tissue culture-induced activation of the tobacco retrotransposon Tto1.
The tobacco retrotransposon Tto1 is one of a few active retrotransposons in plants. Its transposition is activated by tissue culture and is primarily regulated at the transcriptional level. Here we demonstrate that Tto1 introduced in Arabidopsis is also activated by tissue culture. Transcription of Tto1 was induced by tissue culture and driven by its LTR promoter. Transposed copies of Tto1 were observed in almost all of the plants regenerated from the explants cultured for only 1 week. A total of 255 independent regenerated lines have been produced, and the average copy number of transposed Tto1 in these lines is estimated to be 3.2. Sequences flanking Tto1 were amplified by thermal asymmetric interlaced (TAIL)-PCR. Of 165 independent amplified products, 123 showed significant homology to known genes or hypothetical protein genes. The insertion sites of Tto1 are spread over all chromosomes and the target site sequence shows moderate consensus. Taken together, these results indicate that Tto1 can be used as a tool for efficient insertion mutagenesis of Arabidopsis which is especially suitable as a reverse genetics system.
Okamoto
H
H
Department of Molecular Genetics, National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan.
Hirochika
H
H
D017360:Arabidopsis / Q000235:genetics*; D046508:Culture Techniques; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D018745:Genome, Plant; D005966:Glucuronidase / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D018626:Retroelements; D014026:Nicotiana / Q000235:genetics*
D018626:Retroelements; D005966:Glucuronidase