In vitro regeneration of <Emphasis Type="Italic">Carlina acaulis</Emphasis> subsp. <Emphasis Type="Italic">simplex</Emphasis> from seedling explants

The aim of the study was to obtain an efficient system for Carlina acaulis subsp. simplex propagation. The experimental materials were shoot tips, fragments of hipocotyls, cotyledons and roots isolated from 10-day-old seedlings. The explants were transferred to the proliferation medium supplemented with different types of cytokinin: BA (13.3 μM), kinetin (13.9 μM) and zeatin (13.7 μM) in combination with NAA (0.54 μM). The best morphogenetic response was observed when explants were cultured on the BA supplemented medium. The maximum shoot organogenesis frequency was observed for shoot tip (nearly 94%). On average 8.6 axillary shoots were induced per explant. Multiplication rate increased during the first three subcultures. The shoots revealed a wide range of morphogenetic responses. Differences were observed in the presence or absence of hair on the surface of lamina. These changes had epigenetic character and were the effect of changes in DNA methylation, which is shown by differences in methylation pattern between 18S rRNA and 25S rRNA genes in the analyzed regenerated plants. Nearly 94% of plantlets were rooted on auxin lacking medium. Addition of auxin (NAA or IAA) increased both the rooting percentage (100%) and the number of roots per shoot, but their growth was inhibited. Shortening of the auxin exposition time reduced the number of roots. Moreover, high efficiency (90%) was observed for ex vitro rooting. Plantlets with a large number of roots survived better than the ones with only a few roots. Plants were able to flower and gave viable seeds.