High rooting frequency and functional analysis of GUS and GFP expression in transgenic Medicago truncatula A17.

•  An effective transformation method is described for Medicago truncatula A17, verifying its suitability as a model legume for functional genomics. •  Media and culture methods are detailed that yielded an average frequency of 35% for recovery of transgenic shoots from cotyledonary node explants and 39% for root induction and regeneration of entire plants from 419 phosphinothricin-resistant shoots. •  Fertile plants transgenic for both 35S-GFP and phas-GUS were obtained in five of eight independent experiments. The presence and stable inheritance of transgenes was confirmed by GFP or GUS expression and by genomic DNA blots. GFP expression driven by the normally constitutive CaMV 35S promoter diminished as the leaves matured. Although GUS was very strongly and uniformly expressed in seed cotyledons of most lines, one line exhibited an aberrant, patchy pattern. Additionally, weak GUS expression was evident in leaf veins from the normally stringently spatially regulated phas promoter. •  Stably transformed, fertile, M. truncatula A17 plants were generated. The unconventional expression patterns for 35S-GFP and phas-GUS expression obtained in some transformants suggest the occurrence of novel epigenetic events.