EARLY CHANGES IN GENE-EXPRESSION DURING DIRECT SOMATIC EMBRYOGENESIS IN ALFALFA REVEALED BY RAP-PCR

A procedure for direct production of somatic embryos from alfalfa leaf explants has been developed. Wounding followed by 2,4-D treatment ind uces competent cells in the leaf to produce embryos. Changes in gene e xpression following induction of direct somatic embryogenesis were inv estigated by RNA arbitrarily primed PCR (RAP-PCR) with a view to ident ifying genes involved in controlling the onset of somatic embryo devel opment. Amongst the induced sequences identified were a calnexin-like sequence (which was induced within 2 d), and a novel plant homologue o f the yeast SNQ2 multi-drug resistance protein (which did not appear u ntil 10 d after induction). RT-PCR amplification of these sequences co nfirmed the expression patterns revealed by RAP-PCR and was used to sh ow 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 t he potential to isolate genes of interest by identifying specific expr ession patterns during complex developmental processes.