Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis

A GFP excision assay was developed to monitor the excision of Ac introduced into rice by Agrobacterium-mediated transformation. The presence of a stro ng double enhancer element of the CaMV 35S promoter adjacent to the Ac prom oter induced very early excision, directly after transformation into the pl ant cell, exemplified by the absence of Ac in the T-DNA loci. Excision fing erprint analysis and characterization of transposition events from related regenerants revealed an inverse correlation between the number of excision events and transposed Ac copies, with single early excisions after transfor mation generating Ac amplification. New transpositions were generated at a frequency of 15-50% in different lines, yielding genotypes bearing multiple insertions, many of which were inherited in the progeny. The sequence of D NA flanking Ac in three representative lines provided a database of inserti on tagged sites suitable for the identification of mutants of sequenced gen es that can be examined for phenotypes in a reverse genetics strategy to el ucidate gene function. Remarkably, two-thirds of Ac tagged sites showing ho mology to sequences in public databases were in predicted genes. A clear pr eference of transposon insertions in genes that are either predicted by pro tein coding capacity or by similarity to ESTs suggests that the efficiency of recovering knockout mutants of genes could be about three times higher t han random. Linked Ac transposition, suitable for targeted tagging, was doc umented by segregation analysis of a crippled Ac element and by recovery of a set of six insertions in a contiguous sequence of 70 kb from chromosome 6 of rice.