Two-dimensional gel electrophoretic analysis and histological studies were performed on somatic embryos in cypress. Embryogenic cultures were obtained from in vitro culture of immature seeds. On a modified Murashige and Skoog (MS) medium they showed an intense and repetitive cleavage polyembryogenesis phenomenon which maintained them in a continuous proliferating status instead of undergoing a complete embryogenic development. Only the addition of bovine serum albumin to the culture allowed somatic embryo development and maturation. Major histological differences were noticed between developing and nondeveloping embryogenic cultures. Attempts to find proteins that could be associated with developmental stages of somatic embryos have been achieved. Proteins were extracted and analyzed by two-dimensional electrophoresis from nondeveloping embryogenic cultures (S0) and from embryogenic cultures at three different stages of somatic embryo development: small size and rounded shape embryos (S1), increased size embryos with a well-developed suspensor (S2) and embryos with two well-separated cotyledons (S3). The results revealed some qualitative and quantitative protein variations between the two cultures. Some could be connected with the induction of pro-embryo differentiation whereas others should be more related to the mechanisms involved in somatic embryo development and maturation. Specific polypeptides associated with the presence of bovine serum albumin (BSA) in the medium have been detected.
Biochemical changes that characterize megagametophyte and zygotic embryo development in the conifer Cupressus sempervirens L. (Cupressaceae) were studied by complementary methods of cytochemistry and two-dimensional electrophoresis (2-DE). These analyses revealed that early in their development megagametophytes and embryos were characterized by the predominant elaboration of starch in association with a low protein content. As their development proceeded, starch content gradually decreased while protein body synthesis progressively intensified, both in the megagametophyte and the embryo. In parallel, an increase in protein level as well as an accumulation of specific polypeptides could be observed in the two tissues. During maturation, protein bodies accumulated to high levels both in megagametophyte and embryo cells, whereas starch could no longer be detected. Protein levels were high in mature seeds and reached 12% and 8% of the megagametophyte and embryo DW, respectively. Some sets of polypeptides accumulated more specifically at this time in both megagametophyte and embryo. Some of these began to first accumulate in the megagametophyte during embryo development before their concentration rose in the embryo at cotyledonary stage. Others displayed a more specific-embryo accumulation pattern.
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