Thymus sibthorpii Benth. (Lamiaceae), with accession number 01,1796-22, is a biotype of native Greek thyme with ascending stems and potential use as a new medicinal-aromatic crop and ornamental plant. An efficient and reliable protocol for in vitro clonal propagation of T. sibthorpii from nodes and meristem tip explants was developed. Shoot proliferation succeeded on a new basal medium (BB) without plant growth regulators, as prior experiments with 6-benzyladenine generated hyperhydricity. Eight different basal media were compared; on two formulations using the new BB 5.9 and 5.6 shoots per explant were produced. Regenerated single shoots were rooted in the BB medium, supplemented with 5 mu M of indole-3-butyric acid, and produced 3.1 roots along with 2.5 adventitious shoots. Three types of acclimatization were assessed: in vitro, using two different systems (no significant differences); ex vitro, using eight soil substrates under greenhouse and outdoor nursery conditions (in two of them, 100% of plantlets survived); and in field cultivations, established at eight geographically distant areas of Greece (100% survival rate at all locations). Molecular characterization of T. sibthorpii was evaluated with one nuclear ribosomal DNA and seven chloroplast DNA markers, followed by DNA sequence comparisons with a total of 30 different Thymus species, subspecies, and varieties. The trnH/psbA, trnL/trnF, and matK genes were the most efficient markers for molecular characterization of T. sibthorpii. The molecular markers rpoC1 and petB/petD did not match to any Thymus species and therefore, these DNA sequences provide new sequence information for entire Thymus taxa.
