Early changes in gene expression during direct somatic embryogenesis in alfalfa revealed by RAP-PCR

Abstract

A procedure for direct production of somatic embryos from alfalfa leaf explants has been developed. Wounding followed by 2,4-D treatment induces competent cells in the leaf to produce embryos. Changes in gene expression following induction of direct somatic embryogenesis were investigated by RNA arbitrarily primed PCR (RAP-PCR) with a view to identifying genes involved in controlling the onset of somatic embryo development. Amongst the induced sequences identified were a calnexin-like sequence (which was induced within 2 d), and a novel plant homologue of the yeast SNQ2 multi-drug resistance protein (which did not appear until 10 d after induction). RT-PCR amplification of these sequences confirmed the expression patterns revealed by RAP-PCR and was used to show that the calnexin-like gene is induced by 2,4-D, but that the level of expression is enhanced by wounding. The technique of RAP-PCR has the potential to isolate genes of interest by identifying specific expression patterns during complex developmental processes.