• Journal of Marine Bioscience and Biotechnology
• /
• v.12 no.1
• /
• pp.50-58
• /
• 2020

Sphacelaria is a filamentous brown algal genus that can be epibiotic on macroalgae, marine plants, and sea turtles. Its important role in benthic ecosystems, exposure to different stressors (e.g., grazing), and use as a model organism make Sphacelaria ideal for assessing physiological responses of organisms to environmental inputs. Single-cell RNA sequencing is a powerful new probe for understanding environmental responses of organisms at the molecular (transcriptome) level, capable of delineating gene regulation in different cell types. In the case of plants, this technique requires protoplasts ("naked" plant cells). The existing protoplast isolation protocols for Sphacelaria use non-commercial enzymes and are low-yielding. This study is the first to report the production of protoplasts from Sphacelaria fusca (Hudson) S.F. Gray, using a combination of commercial enzymes, chelation, and osmolarity treatment. A simple combination of commercial enzymes (cellulase Onozuka RS, alginate lyase, and driselase) with chelation pretreatment and an increased osmolarity (2512 mOsm/L H₂O) gave a protoplast yield of 15.08 ± 5.31 × 10⁴ protoplasts/g fresh weight, with all the Sphacelaria cell types represented. Driselase had no crucial effect on the protoplast isolation. However, the increased osmolarity had a highly significant and positive effect on the protoplast isolation, and chelation pretreatment was essential for optimal protoplast yield. The protocol represents a significant step forward for studies on Sphacelaria by efficiently generating protoplasts suitable for cellular studies, including single-cell RNA sequencing and expression profiling.

• ALGAE
• /
• v.20 no.1
• /
• pp.1-13
• /
• 2005

The taxonomic status of three closely related species of the genus Sphacelaria, S. rigidula Kützing, S. fusca (Hudson) C.F Gray and S. didichotoma Saunders was reassessed on the basis of morphological observations from herbarium specimens and phylogenetic analysis of RUBISCO spacer sequences. Sphacelaria rigidula was characterized by having only 2-armed propagules and somewhat slender filaments. Culture experiments revealed that its propagules commonly formed secondary arms, even though they were rarely produced in the field. It is concluded that S. divaricata Montagne characterized by dichotomously branched arms in propagules shoud be synonymized with S. rigidula, based on propagule morphology and measurements. Sphacelaria fusca clearly be separated from S. rigidula by having 2-4 armed propagules. These morphological differences were highly supported by the differences in the RUBISCO spacer sequences. S. didichotoma resembles S. rigidula in having propagules with two dichotomously branched arms. However, it commonly produces the secondary arms in the field. Additionally, this species has shorter primary arms than S. rigidula. Phylogenetic analyses supported the distinction of S. didichotoma from S. rigidula. The northwestern Pacific plants, previously recorded as S. divaricata, were recognized as S. didichotoma.