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The genus  Stachys  is one of the largest in the Lamiaceae family. Representatives of the genus are among the most ancient medicinal plants used in the ethnomedicine. The Balkan endemic species  S. thracica ,  S. bulgarica  and  S. scardica  are included in The Red Data Book of Bulgaria and due to their endangered status are scarcely studied. The aim of the present work was to examine the genetic status of these three endemic  Stachys  species during the process of their  ex situ  conservation. To gain information about their taxonomic position in the genus  Stachys , we applied the DNA barcoding approach. Nuclear (ITS) and plastid ( rbcL ,  matK  and  trnH‑psbA ) DNA barcodes were generated and aligned with accessions available in the data base. In the constructed phylogenetic trees  S. thracica  was placed in a cluster together with  S. alpina ,  S. germanica  and  S. cretica , while  S. bulgarica  and  S. scardica  were clustered with  S. officinalis . The  ex situ  conservation was achieved by the initiation of  in vitro  shoot cultures and their subsequent adaptation in  ex vitro  conditions. To check the genomic stability of the plants during the acclimatisation from  in vitro  conditions to  ex vitro , analysis by sequence-related amplified polymorphism (SRAP) markers was performed. No difference was detected between the SRAP profiles of  in vitro  cultivated and  ex vitro  adapted  S. thracica  and  S. scardica  plants. In  S. bulgarica , only 0.4% fragment difference was detected. The obtained results indicated that the three  Stachys  species preserved their genetic stability during the process of  in vitro  multiplication, which is a prerequisite for conserved bioactive capacity.

Phylogenetic identification of Balkan endemic Stachys species and genomic stability during ex vitro conservation