The control of embryogenic competence in Alfalfa (Medicago falcata L.)
Somatic embryogenesis is a process by which somatic cells undergo a developmental sequence similar to that observed in zygotic embryos. This process is not only an important plant propagation technique, but it also provides an essential tool for basic research into plant embryo development and other aspects of plant physiology. One of the basic features of the initiation of somatic embryogenesis is the reactivation of the cell cycle and subsequent cell division in differentiated plant cells. The initiation of somatic embryogenesis effectively involves resetting an entire ontogenic program and presumably requires a substantial reprogramming of gene expression patterns. The reactivation of cell division may be an essential prerequisite for this genetic reprogramming. In this project, transgenic alfalfa plants carrying Arabidopsis cdc2a and, CycB1;1 promoter::gusA constructs, were used to investigate the activation of cell division during the induction of somatic embryogenesis. Promoter fragments of a sugar beet cdc2-like protein kinase gene (Bvcrk1-17. 26) fused to the gusA reporter gene were also introduced into alfalfa plants by Agrobaeterium-mediated transformation, to investigate their potential as molecular markers for re-entry into the cell division cycle. Histochemical assays of r,3 -glucuronidase (GUS) activity were carried out to investigate cell division activity patterns during direct somatic embryogenesis. The cell cycle inhibitors oryzalin and hydroxyurea were used to determine the relationship between the induction of cell division and somatic embryogenesis. The role of 2,4-D in the induction of cell division and somatic embryogenesis was also examined using these transgenic alfalfa lines. The expression patterns of the cell cycle promoter constructs in response to 2,4-D were compared with their behaviour in transgenic tobacco under the same conditions. Preliminary studies indicated that the response to 2,4-D induction treatment in activation of cell division was different in these two systems. The expression patterns of a group of clones isolated from induced alfalfa leaf explants by subtractive cloning were determined by dot blot array hybridisation. Some of these genes were found to be expressed during the very early stages of induction of somatic embryogenesis. Certain genes associated with signal transduction (protein phosphatase [PP2C]; lysophospholipase, [PLP]) and gene regulation (bZIP and HD-Zip class transcription factors) were selected for further characterisation. 5' RACE and 3' RACE products of the original clones were sub cloned in order to extend the DNA sequence information. These DNA sequence data were used to search the sequence databases by BLAST homology searching. These results indicated similarity to genes which function as regulatory elements during plant development. The HD-Zip transcription factor gene (Mfhb-l) was of particular interest because similar genes have been associated with somatic embryogenesis in other species. One feature of this gene was the presence of a highly conserved uORF sequence. In order to characterise the role of this gene in somatic embryogenesis, constructs carrying different fragments of the gene in sense and antisense orientations were established and introduced into alfalfa plants via Agrobacterium-mediated transformation and transformants with each of the constructs were successfully regenerated. The phenotypic effects of overexpression and antisense expression of the HD-Zip transcription factor gene during regeneration of the transformants and subsequent direct somatic embryogenesis in suspension culture, provided some insight into its biological function. It is proposed that the HD-Zip transcription factor gene Mfhb-l may be involved in the control of the auxin response (particularly auxin polar transport) during somatic embryogenesis in alfalfa. Expression of the HD-Zip gene enhances somatic embryogenesis, and the uORF may serve to enhance the expression or activity of the HD-Zip protein.
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