Seed ecophysiology and micropropagation of Clinopodium nepeta, an aromatic Mediterranean plant with pharmaceutical and horticultural uses was investigated. The optimum germination temperature of seeds stored at room temperature for 0, 6, or 12 months was 15 to 20 °C (100% germination completed in 10 to14 days) and cardinal temperatures were defined at 10 and 30 °C (80% to 82% and 62% to 76% germination, respectively). Six or 12 months of storage did not seem to affect germination, although 12-month-old seeds germinated at higher percentage and completed germination earlier at 15 °C than at 20 °C. Concerning micropropagation, shoot multiplication at subcultures of both adult plant- and seedling-origin nodal explants was tested on Murashige and Skoog (MS) medium supplemented with various cytokinin types, i.e., zeatin (ZEA), 6-benzyladenine (BA), kinetin (KIN), and 6-γ-γ-(dimethylallylamino)-purine (2IP), at various concentrations from 0.0 to 8.0 mg·L−1. Both explant types presented a rather similar response during in vitro culture. Increasing concentration of all cytokinin types resulted in an increase in shoot number per responding explant (1.1–5.3) and in most cases a decrease in shoot length (0.6–3.4 cm). Increasing cytokinin concentration induced hyperhydricity to a number of shoots (0.1–6.5) per explant, mostly when ZEA and BA were used. Supplementing the MS medium with 8.0 mg·L−1 BA combined with 0.1 mg·L−1 1-naphthaleneacetic acid (NAA) led to almost elimination of hyperhydricity and very satisfactory shoot production (80%/88% explant response and 6.5/7.5 shoot number per responding explant for seedling- / adult-origin explants, respectively). Alternatively, increasing the agar concentration to 12.0 g·L−1 and supplementing the medium with 8.0 mg·L−1 BA only, resulted in the same effect on eliminating hyperhydricity, such as the addition of NAA, and in the best shoot multiplication response achieved in this study (100% explant response, 9.4/9.9 shoots per explant for seedling-/adult-origin explants, respectively). Microshoots rooted abundantly (92% to 100%) on half-strength MS medium, either Hf or supplemented with 0.5 mg·L−1 to 4.0 mg·L−1 indole-3-butyric acid (IBA). The addition of IBA to the rooting medium, regardless of its concentration, affected only the root length by increasing it 2- to 3-fold. Microshoot clusters produced on multiplication media rooted at 96% when cultured on Hf half-strength MS medium. Rooted microshoots and shoot clusters survived at 80% to 100%, respectively, after ex vitro acclimatization in peat:perlite 1:1 (v/v).
Micropropagation of Dianthus deltoides L. through shoot tip and nodal cuttings culture
