The association of homeotic gene expression with stem cell formation and morphogenesis in cultured medicago truncatula

Research Doctorate - Doctor of Philosophy (PhD)Somatic embryogenesis (SE) can be induced in vitro in Medicago truncatula 2HA by auxin and cytokinin but not in wild type Jemalong. Wild-type Jemalong will only form callus in the presence of auxin and cytokinin and both Jemalong and 2HA will form roots in response to auxin alone. The F2 analysis of 2HA X Jemalong crosses suggest that a single gene may open the way to SE but additional genes are required to maximise the process. Auxin and cytokinin are required for 2-3 weeks for SE and for de novo root formation auxin is essential for about one week. Abscisic acid (ABA) and ethylene, both stress related hormones, enhance SE induction but inhibit callus and de novo root formation. The WUSCHEL (WUS) gene was investigated in M. truncatula (Mt) and identified by the similarity with Arabidopsis WUS in amino acid sequence, phylogeny, promoter element patterns, and expression patterns in planta. MtWUS is induced by cytokinin after 24-48 h in embryogenic cultures and maximum expression occurs after 1 week which coincides with totipotent stem cell induction. MtWUS expression, as illustrated by promoter-GUS studies, subsequently localises to the embryo and corresponds to the onset of MtCLV3 expression. RNAi studies show that MtWUS expression is essential for callus and somatic embryo production. There is evidence based on the presence of MtWUS promoter binding sites that MtWUS is required for the induction of MtSERF1 which appears to have a key role in the signalling required for SE induced in 2HA. MtWOX5, as for MtWUS, was identified by similarity to Arabidopsis WOX5 based on amino acid sequence, phylogeny, promoter element patterns, and expression patterns in planta. MtWOX5 expresses in the auxin induced root primordium and root meristem and appears to be involved in pluripotent stem cell induction. GA suppresses the MtWOX5 expression in the root apex and suppresses the root primordium induction, consistent with the importance of MtWOX5 in in vitro root formation. The evidence is discussed that the homeotic genes MtWUS and MtWOX5 are “hijacked for stem cell induction which is key to somatic embryo and de novo root induction. In relation to SE, a key role for WUS in the signalling involved in induction is discussed and a model developed