In-vitro germination of two endemic species from Berlengas Archipelago, Pulicaria microcephala and Armeria berlengensis

• ¹ MARE – Marine and Environmental Sciences Centre, ESTM, Instituto Politécnico de Leiria, Portugal

The Berlengas archipelago is located in the Atlantic Ocean, about 7 miles from Peniche. It is a protected area since 1981, included in the Portuguese protected areas network and has been classified as a Biosphere Reserve by UNESCO in 2011, aiming to ensure the conservation of the terrestrial and marine ecosystems representative of the island. Due to the characteristics of this area, three species of endemic plants are identified, which only appear in this territory. The species Pulicaria microcephala is present in this island, and although not classified at the moment as being in danger of extinction, constitutes one of the endemisms of the island. Human presence, pressure due to invasive species and herbivory may, if conservation measures are not taken, endanger this species in the future, similar to that presently found in Armeria berlengensis, another endemic species which is currently classified as critically endangered (Caldas, FB, 2011). Therefore, the main goal of this work was to establish seed germination protocols for P. microcephala and A. berlengensis, and to determine the viability and natural regeneration capacity of these species through seed propagation. In this sense, during the summer season of 2015, the infructescences of the two species under study were collected from natural wild population, from different individuals, at stages of similar maturation, no more than 5 infructescences removed from each plant, thus ensuring the viability of the natural propagation process of these species in the wild. In P. microcephala, the flowers were extracted from the infructescences and the achenes were collected. In Armeria berlengensis, due to their larger size it was possible to open the flowers and extract the achenes and proceed to the collection of the seeds. The values of: (a) fertility rate, (b) mean number of seeds, (c) mean number of empty calyxes, (d) average number of calyxes with unfertilized ovary, and (e) unfertilized necrotic ovary were registered. The collected seeds were sterilized at varying concentrations and times of exposure to ethanol and / or sodium hypochlorite. Additional steps of immersion for 24 hours in distilled water or fungicidal solution were also tested. The seeds were placed in Murashige and Skoog (MS, 1962) culture medium supplemented with B5 vitamins (Duchefa Biochemie BV, Netherlands), 0.3% (w/v) sucrose and 1% (w/v) Phyto-agar (Duchefa Biochemie BV, Netherlands). The seeds were maintained in a 16h photoperiod growing chamber under white fluorescent lamps (60 μmol.m-2.s-1) at a temperature of 21°C. The best results in P. microcephala, with success rates of 73%, were obtained by immersing the seeds one minute in 96% ethanol solution, followed by 20 minutes in 20% sodium hypochlorite solution, and one minute in solution of the fungicide Ridomil Gold (2.5g / L). As to A. berlengensis, the two sterilization processes that presented the best results consisted in: (a) immersing the seeds one minute in absolute ethanol, followed by 20 minutes in 10% sodium hypochlorite; (b) immersion of the seeds in distilled water for 2 hours followed by immersion in 70% ethanol for one minute and in 10% sodium hypochlorite solution for five minutes. The results of the two methods were identical, with a success rate of 20%. Regarding seed production, A. berlengensis exhibited a remarkably low fertility rate: having opened 4721 flowers, we obtained only 164 seeds (mean values of 1,673 seeds per inflorescence, each inflorescence having 48,173 flowers). The results indicate that it is possible to germinate P. microcephala in vitro with high success rates (up to 70%) whereas for Armeria berlengensis more studies need to be carried out to circumvent the low germination rates observed (up to 20%), and to try to determine the causes of the reduced fertility rate of this species. These findings are probably correlated with the critically endangered status this species currently holds. Overall, this very slow growing shrub is being unable to cope with anthropogenic disturbance.