RESEARCH NOTE High-frequency in vitro propagation of the endangered species Tuberaria major Sandra Gonc ¸alves • Laura Fernandes • Anabela Romano Received: 23 June 2009 / Accepted: 15 January 2010 / Published online: 6 February 2010 Ó Springer Science+Business Media B.V. 2010 Abstract A novel protocol suitable for the micropropa- gation of the endangered species Tuberaria major using seedlings as explants is reported. Using this protocol, we studied the effects of explant type (apical shoots and nodal segments) and cytokinins [6-benzyladenine (BA), kinetin, and zeatin (ZEA)] on shoot proliferation. Explant type significantly influenced the proliferation frequency and mean number of shoots, with nodal segments showing a higher proliferation capacity. The mean number of shoots was significantly higher when the explants were cultured in half-strength (1/2) MS medium supplemented with 0.2 mg l -1 BA (6.83 ± 0.77 shoots) or ZEA (6.55 ± 0.71 shoots). The shoots showed a great rooting capacity that was significantly influenced by the concentration of MS macronutrients but not by the concentration of auxins. The highest rooting frequencies (97–100%) were obtained in 1/2 MS medium with or without plant growth regulators. The plants obtained were easily acclimatized to ex vitro conditions, with 97% surviving after 6 weeks. The micro- propagated plants were successfully reintroduced into their natural habitat and exhibited normal development. In conclusion, our culture protocol, with efficient seed ger- mination, subsequent multiplication of nodal explants using ZEA at 0.2 mg l -1 , and successful ex vitro estab- lishment of well-rooted plantlets on 1/2 MS medium, provides a simple and reliable methodology for the large- scale propagation of T. major, thereby contributing to germplasm preservation of this endangered species. Keywords Acclimatization Á Conservation Á Micropropagation Á Plantlet production Á Risk of extinction Abbreviations BA 6-Benzyladenine IBA Indole-3-butyric acid KIN Kinetin NAA a-Naphthaleneacetic acid PGR Plant growth regulators ZEA Zeatin Introduction Tuberaria major (Willk.) P. Silva & Rozeira (Cistaceae) is a perennial plant found on the coast of the Algarve region (Portugal), mainly in sandy places among xerophilous bushes. In the past, its distribution included a greater part of the coast of the Algarve; however, at present it is reduced to small nuclei located in the center of the region. This plant is geographically and ecologically rare because its distribution is restricted to a very small area worldwide and is consistent with a particular habitat at a regional comparison scale. T. major is considered a critically endangered and priority species by European legislation and is legally protected under the European Habitats Directive 92/43/CEE and by Portuguese law (reference 140/99 from April 24). As each conservation method has its own advantages and disadvantages, it is important to create strategies that integrate different conservation approaches, such as the combination of micropropagation and cryopreservation, with the aim of (1) increasing the number of individuals in the case of a declining, small population (Bowes 1999); (2) S. Gonc ¸alves Á L. Fernandes Á A. Romano (&) Faculty of Sciences and Technology, University of Algarve and Institute for Biotechnology and Bioengineering (IBB/CGB – UTAD), Campus de Gambelas, Ed. 8, 8005-139 Faro, Portugal e-mail: aromano@ualg.pt 123 Plant Cell Tiss Organ Cult (2010) 101:359–363 DOI 10.1007/s11240-010-9683-y