Global gene expression analysis of in vitro root formation in Medicago truncatula

Medicago truncatula Gaertn. can generate roots in vitro through the formation of root stem cells from leaf explants cultured with auxin. To identify key genes involved in the early processes of root initiation, we compared gene expression in root-forming cultures (RFC) enriched for root stem cells with non-root-forming cultures (NRFC) and untreated leaves using the Affymetrix Medicago GeneChip. Comparing RFC (at 1 week, before root primordium formation) to normal leaf tissue, we identified 904 and 993 up- and downregulated probe sets. Comparing RFC and NRFC, we identified 92 and 182 up- and downregulated probe sets. By comparing all the samples we identified a set of 76 and 42 probe sets up- and downregulated that may be crucial to root stem cell formation and subsequent root initiation. Upregulated probe sets in RFC include Arabidopsis orthologs that are involved in root stem cell formation and root initiation. To validate the GeneChip results, quantitative real-time RTPCR analysis was used to examine the expression of specific up- and downregulated genes, all of which positively correlated with the microarray data. We used bioinformatic tools developed to functionally annotate the Medicago genome array. This showed significant changes in metabolism, signalling and the expression of transcription factors including some with described roles in root organogenesis and other genes not previously linked to this process. This data facilitates the mapping of regulatory and metabolic networks in M. truncatula and provides candidates for further functional analysis of root initiation in vitro and in planta.