Protoplasts were isolated from embryogenic cell suspensions of hybrid larch (Larix x eurolepis Henry) and subdivided into three populations by filtration through nylon filters with 20, 45 and 60 µm pore diameter. Flow cytometry analyses showed that cytoplasmic complexity increased with cell volume. The mixed protoplast population and the three purified populations were cultured for 25 days in MSG (Klimaszewska, K. 1989. Plant Cell Rep. 8: 440-444) medium, and the variation in their nuclear absolute DNA amount over time was studied with flow cytometry. Microcolonies were obtained in all populations, with frequencies from 14 to 21%. The highest frequency (11.6%) of somatic embryogenesis was obtained in the 45 µm population, while no somatic embryos were recovered in the 20 µm one. Regression analyses showed that DNA amount varied significantly over the whole culture period in the mixed and 45 µm populations and during the first week in the 60 µm population. However, the variation was not significant in the 20 µm population. Overall,the mixed and 20 µm populations were comparable with regards to their DNA amount, with values higher than those of the 45 and 60 µm populations. In addition, protoplast sub-populations differing in their DNA amount were detected sporadically in cultures. Quantitative variations in nuclear DNA in the course of protoplast culture might be related to morphogenetic processes and constitute a response of cells to the modification of in vitro environment over time.

Key words: Larix x eurolepis , flow cytometry, morphogenetic processes, nuclear absolute DNA amount, protoplast, somatic embryogenesis