Molecular characterization of several Brassica shoot apical meristem genes and the effect of their altered expression during in vitro morphogenesis
Elhiti, Mohamed Abdelsamad
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A common event during in vitro morphogenesis (either embryogenesis or shoot organogenesis) is the ability of somatic cells within the explants to de-differentiate and acquire “meristematic identity”. The developmental program of such meristematic cells can then be re-routed to form shoots or embryos depending on the imposed culture environment. The objective of this research is to investigate how the altered expression of Brassica genes regulating meristematic activity in vivo affects in vitro morphogenesis. It is predicted that ectopic expression of positive regulators of the shoot apical meristem, SHOOT MERISTEMLESS (STM) and ZWILLE (ZLL) which increase the pool of meristematic cells within the apical meristem, has a beneficial effect on somatic embryogenesis and shoot organogenesis. Conversely the over-expression of CLAVATA1 (CLV1), a negative regulator which depletes the pool of meristematic cells, should inhibit both processes. Over-expression of the Brassica STM in Arabidopsis enhanced the production of somatic embryos and shoots in vitro possibly by reducing the requirement of the tissue for exogenous auxin, which is the inductive signal for the production of embryogenic and organogenic cells. This was also accompanied by profound alterations in gene expression patterns affecting components of DNA methylation and glutathione metabolism, which are beneficial for embryo formation. The introduction of STM also enhanced Arabidopsis shoot organogenesis through profound transcriptional changes in cytokinin signalling. While the ectopic expression of the Brassica CLV1 inhibited both somatic embryogenesis and shoot organogenesis, the expression of ZLL had no effects on the production of somatic embryos but encouraged the formation of shoots. Taken together these results suggest the existence of similar genetic mechanisms regulating the formation of meristem cells in vivo and embryogenic/organogenic cells in vitro.