imm partheno-sporophyte replicate 1
Partheno-sporophytes were cultivated in 90mm round petri dishes in natural sea water supplemented with 0.01% Provasoli enrichment, in standard light/temperature conditions, and harvested at maturity (3 to 4 weeks old). Before harvesting, fragments of 3 to 5 mature individuals from each Petri dish were transplanted into freshly prepared culture medium in a new Petri dish for maintenance and further harvesting. All petri dishes were rapidly checked under the binocular microscope for the presence of upright filaments, the presence of plurilocular sporangia and the absence of unilocular sporangia and diatoms. Some swimming zoids were observed. Younger individuals (without upright filaments and/or without sporangia) were also observed. Samples were asynchronous. Harvesting was carried out as follow: the medium was discarded from the Petri dish to eliminate a maximum of swimming zoids and to eliminate any unseen, floating gametophytes. Tissues were then harvested using a plastic squeegee and recovered in a 45µm sieve. The tissue from 5 individuals was bulked in the same sieve. The harvest was then rapidly dried on clean absorbent paper, put in a 1.5mL eppendorff tube, weighed and frozen in liquid nitrogen. Samples were kept at -80¡C until extraction. Total RNA was extracted from 0.05 to 0.1 g of bulked tissue.