Cytometric and molecular techniques were used to verify genetic uniformity among somatic embryo-derived plantlets of Gentiana pannonica Scop. Cytometric analysis of regenerants revealed absence of chromosomal changes and alterations in ploidy. However, reverse phase high pressure liquid chromatography detected higher levels of methylation in regenerated plants than those of control plants. These changes were further investigated using a quantitative molecular marker-based approach. This revealed that numerous tissue culture-induced variations, ∼3% (epi)mutations, were observed, including sequence variation and changes in methylation patterns. Moreover, complex patterns of variation, including combinations of genetic and epigenetic changes, were relatively high (ca. 9%). Overall, tissue culture-induced variation reached 16%; while, demethylation was lower than de novo methylation in heterozygotic material and similar in all regenerated plantlets.
Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH 4 NO 3 was replaced with 3.0 g l -1 glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l -1 2,4-D + 1.0 mg l -1 kinetin or 2.0 mg l -1 BAP + 1.0 mg l -1 dicamba + 0.1 mg l -1 NAA + 80 mg l -1 adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l -1 2,4-D + 1.0 mg l -1 kinetin or 1.0 mg l -1 kinetin + 0.5 mg l -1 GA 3 + 80.0 mg l -1 adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to halfstrength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.
In this paper, we would like to show unexpected morphogenic potential of cell suspensions derived from seedling explants of Gentiana kurroo (Royle). Suspension cultures were established with the use of embryogenic callus derived from seedling explants (root, hypocotyl and cotyledons). Proembryogenic mass proliferated in liquid MS medium supplemented with 0.5 mg l -1 2,4-D and 1.0 mg l -1 Kin. The highest growth coefficient was achieved for root derived cell suspensions. The microscopic analysis showed differences in aggregate structure depending on their size. To assess the embryogenic capability of the particular culture, 100 mg of cell aggregates was implanted on MS agar medium supplemented with Kin (0.0-2.0 mg l -1 ), GA 3 (0.0-2.0 mg l -1 ) and AS (80.0 mg l -1 ). The highest number of somatic embryos was obtained for cotyledon-derived cell suspension on GA 3 -free medium, but the best morphological quality of embryos was observed in the presence of 0.5-1.0 mg l -1 Kin, 0.5 mg l -1 GA 3 and 80.0 mg l -1 AS. The morphogenic competence of cultures also depended on the size of the aggregate fraction and was lower when size of aggregates decreased. Flow cytometry analysis reveled luck of uniformity of regenerants derived from hypocotyl suspension and 100% of uniformity for cotyledon suspension.
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