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Dedifferentiation-mediated hypomethylation leads to increased AC transposition in indica rice. Implications for saturation insertional mutagenesis

: Fiume, E.; Rodriguez, J.; Rafel, M.; Prynne, M.Q.; Gomonda, R.W.J.; Bassie, L.; Aguado, E.; Capell, T.; Kohli, A.; Pereira, A.; Christou, P.

Lupotto, E. ; Fondazione per le Biotecnologie:
ricEUconf. Dissemination conference of current European research on rice 2002. Book of abstracts : June 6-8, 2002
Torino, 2002
Dissemination Conference of Current European Research on Rice <2002, Torino>
Conference Paper
Fraunhofer IME ()
rice; functional genomic; tissue culture

Insertion mutagenesis is a powerful tool to identify and isolate genes. In rice, mobilization of endogenous retrotransposons, T-DNA tagging and heterologous transposon tagging have been used to this effect. The ultimate aim of these strategies is to achieve insertion of the tagging element in all genes, leading to saturation insertional mutagenesis. We had earlier reported the generation and molecular analysis of an A c tagged indica rice population (1). We now report identification of transgenic lines with high A c excision-reinsertion activity. We have used these lines to investigate their potential for implementing a strategy for saturation mutagenesis of the rice genome. Alteration in genomic DNA methylation status on A c excision-reinsertion frequency was recognized to be an important component of this approach. R1 seeds derived from lines with high Ac excision-reinsertion activity were directly germinated and grown to maturity. Simultaneously, R1 seeds from the same lines were induced to dedifferentiate in vitro, resulting in callus lines that were subsequently regenerated back to intact plants. Both populations were analyzed for the presence, active reinsertion and amplification of A c. We obtained multiple excision-reinsertion events of A c to linked and unlinked sites. These resulted in more than a nine-fold increase of unique A c inserts in plants recovered from cell lines compared to germinated seedlings. We demonstrated coincident changes in the level of cytosine methylation in the genome of callus-derived plantlets. Tagged Transcriptome Display (TTD) was used to identify A c amplification inserts in transcribed sequences (1). We recovered one variegated mutant phenotype that was heritable. Sequencing of the DNA flanking the insertion site in this plant identified transposon integration in the gene for 1-aminocyclopropane-1-carboxylic acid synthase (acs) an enzyme necessary for production of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Our findings indicate activation of A c in indica rice during dedifferentiation, demonstrating the potential for rapid genome saturation by insertion mutagenesis.