Efficient Micropropagation Using Different Types of Explant and Addressing the Hyperhydricity of Ballota acetabulosa, a Mediterranean Plant with High Xeriscaping Potential

Ballota acetabulosa (L.) Benth. (syn. Pseudodictamnus acetabulosus (L.) Salmaki and Siadati), f. Lamiaceae, the Greek horehound, is a compact evergreen small shrub native to Greece, with hairy grey-green leaves, that bears small pink-purple flowers with green conical calyxes along its erect stems in late spring. The species stands out for its high resistance in xerothermic conditions and therefore it is advisable to promote its use in xeriscaping. The aim of this study was to develop an efficient protocol for in vitro propagation of B. acetabulosa for introduction into the horticultural and pharmaceutical industries. Shoot tip and single node explants derived from in vitro seedlings were cultured on MS medium with various cytokinin types and concentrations. Explants responded at almost 100% to produce high number of shoots on a medium with 1.0 mg L−1 zeatin or 6-benzyladenine. However, there was intense hyperhydricity in the cultures, which was addressed in further experiments by increasing agar concentration from 8 to 12 g L−1, preserving high multiplication indices (92% response, 10.2 shoots per explant). Microcuttings with 2–3 visible nodes, either from the apical part, including the apical meristem, or from the basal part of microshoots, as well as microshoot clusters, rooted 100% on full- or half-strength MS medium, respectively, regardless of the addition of indole-3-butyric acid (ΙΒA, 0.5–4.0 mg L−1) in the rooting medium. However, middle level concentrations of IBA increased the number and length of roots produced, while the higher its concentration, the more and longer axillary shoots developed in the microcuttings during the rooting period. The acclimatization of all plantlets was completely successful (100%) in ex vitro conditions on peat/perlite substrate (1:1, v/v). Thus, efficient methods of producing propagation material to promote Ballota acetabulosa as a horticultural and medicinal plant were developed. In particular, rooting of microshoot clusters or microcuttings without the shoot tip, in the presence of 1.0 mg L−1 IBA, leads to a plant of suitable shape for the floricultural market, without the need for further manipulation (pruning) in the nursery.