Enhanced frequency of transposition of the maize transposable element Activator following excision from T-DNA in Petunia hybrida.

ABSTRACT

Many of the systems currently employed for heterologous transposon tagging in plants rely on an excision assay to monitor transposon activity. We have used the streptomycin phosphotransferase (SPT) reporter system to assay Ac activity in Petunia hybrida. In other species, such as tobacco or Arabidopsis, excision of Ac from the SPT gene in sporogenous tissue gives rise to streptomycin-resistant seedlings in the following generation. The frequency of fully streptomycin-resistant seedlings in petunia was low (0.4%) but molecular analysis of these indicated that the actual excision frequency may be as low as 0.05%. This indicates that the SPT assay is not a reliable selection criterion for germinal excision in petunia. Extensive molecular screening for reinsertion of Ac was consistent with a low primary transposition frequency (0%-0.6%). In contrast to these findings, the progeny of confirmed germinal transpositions for three independent transformants showed frequent transposition to new sites (9.5%-17.0%). This suggests a high frequency of secondary transposition compared with primary transposition from the T-DNA. Segregation analysis indicates that the high transposition activity is closely associated with transposed copies of Ac. No evidence was found for an altered methylation state for Ac following transposition. The implications of these results for heterologous transposon tagging in petunia are discussed in the context of the reliability of excision reporter systems in general.