In Vitro Seed and Clonal Propagation of the Mediterranean Aromatic and Medicinal Plant Teucrium capitatum
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 (1/2) Murashige and Skoog (1962) growth medium (MS) at 5, 10, 15, 20, 25, and 30 8C, 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 8C (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 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 BA) than explants excised from unbranched seedlings (36% to 43% while during subcultures on MS medium with 1.0 mg*L BA, explants from branched seedlings also showed higher multiplication rates than those from unbranched ones. BA at 0.5 2.0 mg*L induced shoot multiplication during both establishment and multiplication stages (7 8 and 14 15 shoots per explant, respectively) while kinetin and 6 g g (dimethylallylamino) purine (2iP) were less effective than BA, and zeatin the least appropriate. Microshoot rooting was enhanced by 1 week culture on (1/2) MS medium with 1.0 4.0 mg*L indole 3 butyric acid (IBA) followed by transfer to auxin free, (1/2) MSmedium (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. Teucrium capitatum (T. polium sp. capitatum Arcang. is a strongly aromatic evergreen dwarf shrub, grown in dry areas of the Mediterranean region from the sea level up to 2000 m altitude. This xerophytic herb has characteristic gray green hairy stems and bears white flowers in compound heads from April until June (Blamey and Grey Wilson, 1993) being a pollen source for bees and other insects. Chemical composition of the essential oils of T. capitatum has been investigated (Antunes et al. 2004; Cozzani et al. 2005; Djabou et al. 2012; Menichini et al. 2009; Miti c et al. 2012) showing antiproliferative activity during in vitro cytotoxic assay on human cancer cell lines (Menichini et al. 2009) and antioxidant and anti inflammatory activities in vitro (Kerbouche et al. 2015) Extracts from aerial parts of this plant were also found to contain flavonoids with insulinotropic and antihyperglycemic effects on the lipid and carbohydrate metabolism in rats (Stefkov et al. 2011) Teucrium polium has also been traditionally used for different pathological conditions such as gastrointestinal disorders, inflammations, diabetes, and rheumatism, while various compounds have been isolated from various parts of it, mainly terpenoids and flavonoids, which possess a broad spectrum of pharmacological properties including antioxidant, anticancer, anti inflammatory, hypoglycemic, hepatoprotective, hypolipidemic, antibacterial, and antifungal (Bahramikia and Yazdanparast, 2012) Apart from medicinal uses, T. capitatum could be introduced as an ornamental landscape plant, suitable for xeriscaping, particularly for dry, rocky places, in calcareous and gypsum soils (Rom~ao and Escudero, 2005) or for urban green roofs in arid and semiarid areas. It could also be used in landscape restoration of East Mediterranean archaeological sites, due to its continuous use since ancient times in the area, as a medicinal and insect repelling plant (Theophrastus,Historia Plantarum, vol. 9, c. 350 BC c. 287 BC) Wild populations of T. capitatum are subjected to pressure because of selective collection for use in traditional medicine and overgrazing, especially from domestic goats, which very frequently select it in their diet (Barroso et al. 1995) as well as of deforestation and habitat encroachment by urban and agricultural development. Thus, propagation protocols should be developed before expanding the species use. Seeds of T. capitatum have low (less than 40% germination capacity (Luna andMoreno, 2009; Luna et al. 2007; Moreira et al. 2010; Rom~ao and Escudero, 2005) and appears to be nondependent on factors that may change with fire, such as heat and smoke (Luna et al. 2007; Moreira et al. 2010) increased light and soil nitrate (Luna and Moreno, 2009) for germination, while there is no report found in the literature about the effect of temperature, photoperiod, or cold stratification and mechanical scarification pretreatments on its germination. Similarly, T. polium has low seed germination (Nadjafi et al. 2006) The latter, being an endangered species in many countries of the Middle East, where it is widely used in traditional medicine, has been studied as for its possibility for in vitro propagation from ex vitro axillary buds (Al Qudah et al. 2011) and cryopreservation (Rabba'a et al. 2012) It has also been reported that micropropagation of the ornamental medicinal Teucrium fruticans from shoot tip explants (Frabetti et al. 2009) and of the medicinal Teucrium stocksianum from hypocotyl explants excised from seedlings germinated in vitro (Bouhouche and Ksiksi, 2007) To facilitate the use of T. capitatum as a medicinal and ornamental plant, an efficient propagation protocol should be developed. Propagation by cuttings from native plants would apply extra pressure to wild populations and is limited to certain periods of the year when this species roots satisfactorily (Papafotiou et al. 2013) Neither propagation by seed would be satisfactory, because of the low germination ability. Micropropagation is appropriate for species that cannot be efficiently propagated by conventional horticultural techniques and an important tool to select, multiply, and conserve the critical Received for publication 23 Oct. 2015. Accepted for publication 28 Feb. 2016. This research was funded by NSRF 2007 2013, Operational Program 'Education and Lifelong Learning' funded research project: Thales 'Integratedmanagement of vegetation at archaeological sites to protect monuments and enhance the historical landscape' 'ARCHAEOSCAPE, MIS code 380 237. Corresponding author. E mail: mpapaf@aua.gr. HORTSCIENCE VOL. 51(4) APRIL 2016 403 genotypes of medicinal plants in particular (Debnath et al. 2006; Tripathi and Tripathi, 2003) Various medicinal plants and plants of Lamiaceae family in particular have been cloned through micropropagation protocols (Aicha et al. 2013; Chaturvedi et al. 2007; Debnath et al. 2006; Gonc xalves and Romano, 2013; Santoro et al. 2013; Shtereva et al. 2015; Zuzarte et al. 2010) Moreover, germination of seeds in some species, which show low or no germination using conventional techniques, due to dormancy or specific germination requirements, could be greatly increased using in vitro methods (Fay, 1994) In the present study, seed germination and clonal propagation of the species were investigated in vitro. Seed pretreatments, such as cold stratification, mechanical and chemical scarification, and incubation conditions, such as temperature and light, as well as storage period, were investigated as for their effect on germination. Moreover, micropropagation of the species was studied using explants of different origin (seedlings grown in vitro, mature, native, and greenhouse plants) for culture establishment, testing various cytokinins for shoot growth and proliferation, as well as examining various IBA concentrations and culture times for root induction, to develop an efficient micropropagation protocol for commercial use. Materials and Methods In vitro seed germination. Seeds were collected in Aug. 2012 from selected T. capitatum wild plants in the region of 'Diomedes Botanic Garden' (38 00#39.24##B, 23 38#11.32##E, altitude 157 m) in Haidari, a western suburb of Athens, Greece, and were stored at room temperature. Two months after harvesting, they were surface sterilized by 15% v/v commercial bleach (4.5% w/v sodium hypochloride) water solution with one to two drops of Tween 20 (polyxyethylenesorbitan monolaurate, Merck KGaA, Darmstadt, Germany) for 15 min followed by three 3 min rinses with sterile distilled water and were cultured for germination in vitro, in petri dishes, with 1/2 MS medium (Murashige and Skoog, 1962) with 20 g*L sucrose, at 5, 10, 15, 20, and 25 C and 16 h cool white fluorescent light (37.5 mmol*m*s)/8 h dark photoperiod. Stratification for 2 or 3 months at 5 C, before culture at the above temperatures (excluding 5 C) was also tested. Seeds were also collected in Aug. 2013 from the same wild plants and stored at room temperature. Three months after harvesting, seeds were scarified either using sandpaper (suitable for metal surfaces) for 1 min or by dipping in concentrated H2SO4 >95% Fisher Scientific, Loughborough, UK) for 15 min, or they did not receive any treatment (control) before surface sterilization