Calafate berry (Berberis microphylla G. Forst) is an evergreen Patagonian wild berry with high polyphenol content and antioxidant capacity, distributed in Chile from Curicò (34°59'0"Lat S) to Tierra del Fuego (53°28'33"Lat S), also in Argentina. Furthermore, traditional breeding programs in wild species have had limited success due to low seed germination rate and in vitro growth problem. In order to speed up the breeding program in Magellan's Calafate berry, we studied the germination capacity and in vitro establishment of 10 segregant lines of Berberis microphylla accessions from different areas of Magellan's region; CALF = elch-4, CALF = IRiesco-5, CALF = Kamp-21, CALF = elch-1, CALF = elch-2, CALF = elch-3, CALF = Magall-6, CALF = Magall-7, CALF = Magall-8, CALF = Magall-9. A total of five pre-germination treatments (Control, 1 week low temperature, 10 min sulfuric acid, 20 min sulfuric acid and 60 min sulfuric acid) were evaluated under light (16/8 h day/night) and dark conditions, after that the seedless were transferred to in vitro medium for further evaluations. The experimental design was a BCA, with 4 replications in time; Also, in vitro vegetative propagation was evaluated in these segregant lines. All evaluated parameters were analyzed by descriptive statistical analysis and significant differences among treatments and lines were analyzed by Duncan's test analysis; significant differences (<0.05) were observed for the most evaluated parameter, some seedling such as show better germination capacity and in vitro growth.