DEFINITION AND CHARACTERIZATION OF AN ARTIFICIAL EN-BASED SPM-BASED TRANSPOSON TAGGING SYSTEM IN TRANSGENIC TOBACCO

A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for exc ision are expressed by fusing their cDNAs to the CaMV 35S promoter. Th e dSpm receptor component is inserted in the 5'-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed g ermination on L-PPT-containing medium or by spraying seedlings with th e herbicide Basta. Using this bar-based excision reporter construct, a n average frequency of germinal excision of 10.1% was estimated for dS pm-S, an En/Spm native internal deletion derivative. Insertion of En-f oreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSp m-DHFR has a lower frequency of somatic and germinal excision than dSp m-S. Revertants carrying a transposed dSpm-DHFR element can be selecte d with methotrexate. Germinal excision is frequently associated with r einsertion but, as in maize, dSpm has a tendency to integrate at chrom osomal locations linked to the donor site. Concerning the timing of ex cision, independent germinal transpositions are often found within a s ingle seed capsule. All activity parameters analysed suggest that tran sposon tagging with this system in heterologous hosts should be feasib le.