IDENTIFICATION OF MOLECULAR MARKERS OF EMBRYOGENESIS IN ARABIDOPSIS-THALIANA BY PROMOTER TRAPPING

The technique of promoter trapping has been exploited to identify mark ers of embryogenesis in Arabidopsis thaliana. A population of transgen ic A. thaliana was generated containing the promoter trap vector p Del ta gusBin19, following Agrobacterium tumefasciens-mediated transformat ion. This vector contains, at the T-DNA left border, a promoterless gu sA gene, which is activated following integration downstream of a nati ve gene promoter that directs transgene transcription. Screening of a population of 430 independent transgenic lines revealed that 74 lines (17.2%) exhibited GUS activity in siliques, as determined by fluorimet ric assay. Histochemical GUS analysis was used to identify lines that expressed GUS in embryos, and three lines that were demonstrated to co ntain single T-DNA inserts were analysed in detail. Each line showed a distinct pattern of GUS fusion activity. Fusion transcripts were iden tified, demonstrating that transcription was initiated in the genomic DNA flanking the T-DNA left border. Inverse PCR was used to clone the T-DNA flanking sequences, and for one line a corresponding cDNA was id entified, demonstrating that the tagged sequences are transcribed. The markers represent the earliest embryonic genes known to be expressed in plants.