During the establishment of an embryogenic cell line from a carrot hypocotyl explant, processes closely resembling meiotic divisions are seen. A microdensitometric analysis revealed that the amount of cellular DNA diminished in the majority of cells to the haploid level. However, the diploid level was re-established in a matter of a few days. The genetic consequences of this segregation were studied by analyzing restriction fragment length polymorphisms (RFLP) and randomly amplified polymorphic DNAs (RAPD). The results showed that the great majority of embryos regenerated from segregants and that different segregants had different genetic constitutions.
Carrot cell lines Wl and W2 express permanently in culture a meiotic-like phenotype, with apparent pairing and chiasmata formation comparable to meiosis during carrot microsporogenesis. The variant lines also show several variants of division in relation to the presence or absence of cytokinesis, nuclear fusion or spindle disturbance.The meiotic-like divisions can also be found in the abnormal structures, which are regenerated from these spontaneous variant lines. A possible role of the chromosome reducing mechanisms on carrot embryogenesis capacity and somaclonal variability is postulated.
Serine and proline, when added in a wide range of concentrations to Daucus carota cultures during pre-growth in the presence of 2,4-D(2,4-dichlorophenoxyacetic acid), extended in time and quantity the mitotic divisions and stimulated significantly the number of embryos regenerated when the hormone subsequently was omitted from the medium. A specific effect of these amino acids on regeneration is suggested, since proline (as hydroxyproline) and serine are the two major constituents of the cell wall glycoprotein, extensin, which thus may have a morphoregulatory function. The significant role of the conditions during pregrowth of the cultures in the presence of hormone is underlined by the effect of hydroxyproline and D-proline which also stimulate embryogenesis, but alter markedly the development of the embryos.
Cytological analysis of different carrot cell lines in culture has shown various cytogenetic anomalies generating new levels of ploidy and novel chromosome numbers. Polyploidy may be considered a reservoir of variability that can be released in the form of distinct new segregants of different ploidy. Mechanisms alternative to mitosis (reductional grouping, prophase chromosome reduction ) operate from a polyploid state (possibly reached by means of endopolyploidy, endomitosis, nuclear fusion, or restitution nuclei) to generate new levels of ploidy and novel chromosome numbers necessary for selection to operate in vitro. The segregational phenomena require chromosome recognition in haploid set complements and abnormal behaviour of mitoses; the resulting chromosome variability suggests that chromosomes are arranged, in the resting nuclei, in an orderly and predictable manner.The knowledge of the molecular events governing these mechanisms, and how to control them, would be of great help for future applications of plant cell culture.
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