| Summary: | Most cultivated orchids, contributing to a worldwide highly profitable industry, are originated from tropic regions. Conversely, a considerable number of spontaneous orchids, belonging to the terrestrial orchids and widely diffused throughout the European continent, are not considered for trading due to their less gorgeous appearance and for technical difficulties in seed propagation. However, a breeding programme was undertaken aimed at developing a new hybrid between <i>Himantoglossum adriaticum</i> H. Baumann and <i>H. robertianum</i> (Loisel.) P. Delforge [syn. <i>Barlia robertiana</i> (Loisel.) Greuter] by applying techniques of anther conservation, manual pollination and in vitro asymbiotic germination of the obtained seeds. The plantlets that originated from the protocorms after seed germination were successfully acclimatised after potting in a proper medium. The parentage of the progenies of the hybridisation experiment was assessed by sequencing the Internal Transcribed Spacer assembly (ITS) and plastid barcoding markers of the parental lines and of the hybrids. The method proved to be effective in revealing the origin of the hybrids and to validate the maternal inheritance of the plastid DNA.
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