We have developed an effective protocol for in vitro micropropagation in order to obtain large numbers of identical plants and another protocol for in vitro polyploidization of Ajuga reptans, based on the use of oryzalin. Two donor plants of A. reptans (AR 4, AR 7) were treated with 0, 1, 5, 10 μM oryzalin for 2 weeks. The analysis of the ploidy level of these plants was verified by flow cytometric analysis using the internal standardization method. The effects of polyploidization on growth as well as morphological and stomatal size were also measured. After in vitro polyploidization, some plants became tetraploids or octoploids. The most efficient conditions for inducing tetraploidy were the treatments with 10 μM oryzalin.
This paper is the first published report describing micropropagation of Carlina onopordifolia, using shoot tip and hypocotyl explants. The explants were excised from 10-day-old seedlings and transferred to proliferation medium supplemented with 6-benzylaminopurine (BA; 1.0 or 3.0 mg l-1), kinetin (Kn; 1.0 or 3.0 mg l-1) or zeatin (ZEA; 1.0 or 3.0 mg l-1) in combination with naphthaleneacetic acid (NAA; 0.1 mg l-1). The shoot tips were significantly better than hypocotyls as initial material for shoot regeneration. For shoot multiplication, MS medium supplemented with BA proved superior to the other cytokinins tested. Medium supplemented with 1.0 mg l-1 BA gave the highest shoot propagation frequency (66.9%) and number of shoots per explant (2.5). Single shoots were separated from each other and rooted on MS supplemented with IBA for the whole period of culture, with longor short-pulse IBA application. The highest rooting frequency (84.8%) and root number (18.8) were for shortpulse (1 min) 1000 mg l-1 IBA solution. The higher IBA concentration stimulated callus formation and the development of short roots. The shoots were transferred to MS medium without growth regulators. Survival was highest (54.4%) for the plants from the short-pulse 100 mg l-1 IBA treatment. After 8 weeks of acclimatization the plantlets were removed to field conditions and grew normally.
Micropropagation of Plantago media L. and the presence of phenolic compounds in organs of multiplied plants were investigated for the first time. Multiplication of plant material was achieved in shoot-tip cultures and via direct organogenesis on Murashige and Skoog (MS) medium with four variants of plant growth regulators (M1–M4). The best multiplication coefficient – 9.2 was obtained in seedling shoot-tip cultures on MS medium M3 with BA 0.2 mg/L and IAA 1.0 mg/L. Methanol extracts prepared separately from shoots and roots of in vitroderived plantlets were found to contain typical of the genus Plantago L. phenylethanoid glycosides as the only phenolics. Acteoside and plantamajoside were the major compounds – both known to possess a wide range of promising biological activities applicable for medicinal (therapeutic) and cosmetic uses. Martynoside, as a trace constituent, was also found for the first time in the studied species. The quantitative screening of the extracts by TLC video densitometric method showed a higher content of acteoside in shoots (range 62.43–93.03 mg/g, dry weight) and plantamajoside in roots (range 22.45–44.08 mg/g); the highest recorded values – 93.03 mg/g and 44.08 mg/g, respectively, were found in the organs obtained on MS medium M4 with BA 2.0 mg/L.