GENE IDENTIFICATION WITH SEQUENCED T-DNA TAGS GENERATED BY TRANSFORMATION OF ARABIDOPSIS CELL-SUSPENSION

A protocol for establishment and high-frequency Agrobacterium mediated transformation of morphogenic Arabidopsis cell suspensions was develo ped to facilitate saturation mutagenesis and identification of plant g enes by sequenced T-DNA tags. Thirty-two self-circularized T-DNA tagge d chromosomal loci were isolated from 21 transgenic plants by plasmid rescue and long-range inverse polymerase chain reaction (LR-iPCR). By bidirectional sequencing of the ends of T-DNA-linked plant DNA segment s, nine T-DNA inserts were thus localized in genes coding for the Arab idopsis ASK1 kinase, cyclin 3b, J-domain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxi dase, a peripheral Golgi protein and a maize pollen-specific transcrip t. In addition, 16 genes were identified in the vicinity of sequenced T-DNA tags illustrating the efficiency of genome analysis by insertion al mutagenesis.