Analysis of protoplasts and somatic embryogenesis in Medicago truncatula
Abstract
This thesis examined protoplast proliferation and somatic embryogenesis, by
comparing a highly with a poorly embryogenic Medicago truncatula line through microscopic, proteomic and in situ hybridization analysis. Proteome analysis of M. truncatula was used to identify proteins involved in protoplast proliferation and the
initiation of somatic embryogenesis. Furthermore, an in situ hybridization study was done to compare the expression of genes known to be involved in zygotic embryogenesis with the expression during somatic embryogenesis. A large number
of proteins were up-, and down-regulated during the first 5 days of protoplast culture
indicating that cellular reorganization took place. An up-regUlation of PR 1 O-like
proteins and flavonoid synthesis proteins and a down-regulation of energy
metabolism proteins were observed, indicating an initiation of a stress response. The observed stress response in protoplasts was down-regulated before the first cell divisions at 5-7 d. A stress-inducing bioassay on protoplasts showed that the ability of protoplasts to overcome stress and to proliferate under stress conditions depended on the level of stress and density of the protoplast culture, whereby more stress or a lower culture density resulted in higher levels of cell death. Proteomic analysis of the initiation of somatic embryogenesis showed that similar metabolic pathways were involved in the initiation of somatic embryogenesis and protoplast proliferation. By using a highly embryogenic (2HA) line, and a poorly embryogenic (A 17) line of M. truncatu!a, it was shown that particular proteins were specifically accumulated during the initiation of somatic embryogenesis. A high accumulation of a peroxidase was observed only in At7 tissue at the time of initiation of somatic embryogenesis and might be the reason why the initiation of somatic embryogenesis is inhibited in A 17 tissue. The specific accumulation of flavonoid synthesis proteins might also indicate that flavonoids are involved during the initiation of somatic embryogenesis.
In situ hybridization with probes to genes known to be involved in zygotic embryogenesis, showed that M. truncatula somatic and Arabidopsis thaliana zygotic
embryogenesis both followed similar developmental pathways. However, a few
genes showed distinct patterns of gene expression in M. truncatula somatic embryos.