Using in vitro culture for conservation of genetic resources: micropropagation of a monumental Prunus dulcis tree

The need to conserve biodiversity has been granted increasing political and social attention in the last years. Monumental or emblematic trees, both those >wild> trees living in forests and the century-old, >domesticated> agricultural trees, should be preserved in situ for its intrinsic value, its cultural legacy, the rich diversity of microhabitats they generate, and the quantity of organisms that depend on them to life. Also, they should be preserved ex situ for the study and the conservation of their genetic resources, for educational issues and for reintroducing plant material of high quality in their natural areas, most of them currently degraded or threatened. Within ex situ conservation methods, in vitro culture presents special advantages in the case of emblematic trees, such as the small quantity of plant material needed to begin the micropropagation procedure and the possibility of implementing long-term conservation techniques as cryopreservation. The aim of this study was to micropropagate mature material from an ancient almond tree, named ¿Gladiador¿, located in Membrilla (Central Spain), together with juvenile material proceeding from its seeds. This monumental tree, probably 300-years-old, dominates a landscape formed by hundreds of olive trees, and has a special emblematic meaning for the population of the area. For establishment of axillary shoot cultures, plant material was provided by the FIRE foundation. Three types of material were used: 1) shoots flushed at the tree at the beginning of spring, 2) shoots forced to flush in a phytotron from branch segments collected in late winter, and 3) seeds collected in autumn and stored at a cool place for six months before being germinated in vitro. Murashige and Skoog medium supplemented with 0.5 mg L-1 N6-benzyladenine and 0.5 mg L-1 indole-3-butyric acid was used for culture establishment and stabilization. Different combinations of plant growth regulators were evaluated for shoot proliferation, elongation and adventitious root formation. Rooted shoots from cultures obtained from the Gladiador mother tree and from lines originated from seeds were successfully acclimatized in the phytotron and the greenhouse. For mid-term conservation, shoots from mature and juvenile origins were submitted to cold storage at 4-6 ºC. So far, we have obtained 78 plantlets that are currently being acclimated, 70 corresponding to clonal material from the mother tree and 10 corresponding to clonal material from seeds