A protocol for establishment and high‐frequency Agrobacterium‐mediated transformation of morphogenic Arabidopsis cell suspensions was developed to facilitate saturation mutagenesis and identification of plant genes by sequenced T‐DNA tags. Thirty‐two self‐circularized T‐DNA tagged 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 segments, nine T‐DNA inserts were thus localized in genes coding for the Arabidopsis ASK1 kinase, cyclin 3b, J‐domain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxidase, a peripheral Golgi protein and a maize pollen‐specific transcript. In addition, 16 genes were identified in the vicinity of sequenced T‐DNA tags illustrating the efficiency of genome analysis by insertional mutagenesis.
The Plant Journal – Wiley
Published: Mar 1, 1998
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