The effect of various pretreatments, culture conditions, and storage time on in vitro germination of seeds, as well as the effect of explant origin and plant growth regulators on in vitro propagation of Teucrium capitatum L. (Teucrium polium sp. capitatum Arcang., Lamiaceae) were examined. Seeds, collected from native plants and stored at room temperature for 3, 7, and 12 months, were cultured for germination in vitro in petri dishes with solid half-strength (½) Murashige and Skoog (1962) growth medium (MS) at 5, 10, 15, 20, 25, and 30 °C, and 16 hours light or continuous darkness. Pretreatments, such as cold stratification, scarification with sandpaper, dipping in concentrated sulfuric acid (H2SO4 > 95%), or dipping in boiling water were tested. Seeds without any pretreatment germinated at lower than 10%. Dipping in concentrated H2SO4 for 15 or 20 minutes was the most effective pretreatment, but still seed germination achieved was low (36%). Seeds preserved their germination capacity for at least 1 year, and germinated satisfactorily at a wide temperature range, from 15 to 25 °C (optimum), while photoperiod did not affect seed germination. Explants excised from in vitro-grown seedlings were established in vitro on MS medium with 1.0 mg·L−1 6-benzyladenine (BA) at much higher rates (≈90%) compared with those collected from plants grown from cuttings in a greenhouse (25%), while explants collected from adult wild plants failed to do so. Explants from seedlings showed strong variability in their response; those excised from branched seedlings formed shoots at significantly higher percentage (90%) at the establishment stage (cultured on MS medium with 0.5–2.0 mg·L−1 BA) than explants excised from unbranched seedlings (36% to 43%), while during subcultures on MS medium with 1.0 mg·L−1 BA, explants from branched seedlings also showed higher multiplication rates than those from unbranched ones. BA at 0.5–2.0 mg·L−1 induced shoot multiplication during both establishment and multiplication stages (7–8 and 14–15 shoots per explant, respectively), while kinetin and 6-γ-γ-(dimethylallylamino)-purine (2iP) were less effective than BA, and zeatin the least appropriate. Microshoot rooting was enhanced by 1-week culture on (½) MS medium with 1.0–4.0 mg·L−1 indole-3-butyric acid (IBA), followed by transfer to auxin-free, (½) MS medium (93% to 98%, 7–8 roots per microshoot), compared with culture on the same medium continuously for 5 weeks (69% to 80%, 5–6 roots per microshoot) or at lower IBA concentrations. Plantlets were acclimatized to ex vitro conditions at 98% on a peat–perlite (1:1, v/v) mixture.
Influence of Culture Conditions on In Vitro Asymbiotic Germination of Anacamptis longicornu and Ophrys panormitana (Orchidaceae)
