Gene expression during adventitious root formation in apple.

Abstract

A model system for adventitious root formation in woody plants was used to identify transcripts that are up-regulated during this process. 1 mm stem-disks from micropropagated shoots of the apple cultivar Jork 9 can be induced to form roots by treatment with the auxin indole butyric acid (IBA). Stem discs are placed on medium containing IBA for 24 hours and then transferred to IBA-free medium. Root initials become visible after 5-6 days and root elongation occurs within 7-9 days. The first visible cell divisions, which will give rise to the adventitious roots, are detectable 48 hours following IBA treatment. We have used this system to identify transcripts that are induced during adventitious root formation. Two techniques were employed in this analysis: differential messenger RNA display (DDRT) and mRNA representational difference analysis (RDA), a technique that couples PCR and subtractive hybridisation. Using both of these techniques a number of clones have been isolated that exhibit differential expression during auxin induced root formation. Both up-regulated and down-regulated transcripts have been identified. Expression of these genes has initially been verified by reverse northern blot analysis. Northern blot analysis with individual clones has confirmed the expression pattern observed in the reverse northern analysis. Tentative identities of some of the clones have been established by sequencing the partial cDNAs. Among the up-regulated transcripts are clones that share sequence homology with polygalacturonase and MAP kinases. A full-length cDNA for the most abundant up-regulated mRNA, a 2-oxoacid dependent dioxygenase, was characterised. This mRNA is expressed between 24 and 72 hours following IBA treatment of apple stem disks.