Micropropagation and reintroduction of Nepeta rtanjensis, an endemic and critically endangered perennial of Serbia

Abstract

A micropropagation protocol was developed for the conservation of critically endangered Serbian perennial Nepeta rtanjensis (Lamiaceae). Rooted shoots were obtained from one-node stem segments and shoot tips on a half-strength Murashige and Skoog (MS) medium without growth regulators. The best pH of the medium for axillary buds induction and for rooting of shoots was found to be at 7 and/or 7.2 respectively. The addition of cytokinins to the culture medium did not significantly stimulated auxillary bud production as compared to the control. On the contrary, on media supplemented with high cytokinin concentrations, only dwarf shoots with rudimentary roots were obtained. All tested concentrations of 6-benzylaminopyrine (BAP) and kinetine (Kn) in combination with 0.1 mg 1(-1) indole-3-acetic acid (IAA) negatively affected the elongation and rooting of shoots. Plants micropropagated on hormone free medium and rooted in vitro were successfully acclimatized in greenhouse and in open field conditions. The result of successful acclimatization was the production of more than 7000 plantlets with normal sexually reproduction. They flowered, fruited and produced seeds which exhibited 47% germination. The survival rate of plants that were transferred to the open field for the acclimatization and exposed to the winter chill was 99%. The reintroduction of N. rtanjensis occurred in May 2004. One thousand plantlets were planted within the historic range of this plant species. The survival rate was also 99%.