title: Expanding the Symbiodinium (Dinophyceae, Suessiales) Toolkit Through Protoplast Technology.
abstract: Dinoflagellates within the genus Symbiodinium are photosymbionts of many tropical reef invertebrates, including corals, making them central to the health of coral reefs. Symbiodinium have therefore gained significant research attention, though studies have been constrained by technical limitations. In particular, the generation of viable cells with their cell walls removed (termed protoplasts) has enabled a wide range of experimental techniques for bacteria, fungi, plants, and algae such as ultrastructure studies, virus infection studies, patch clamping, genetic transformation, and protoplast fusion. However, previous studies have struggled to remove the cell walls from armored dinoflagellates, potentially due to the internal placement of their cell walls. Here, we produce the first Symbiodinium protoplasts from three genetically and physiologically distinct strains via incubation with cellulase and osmotic agents. Digestion of the cell walls was verified by a lack of Calcofluor White fluorescence signal and by cell swelling in hypotonic culture medium. Fused protoplasts were also observed, motivating future investigation into intra- and inter-specific somatic hybridization of Symbiodinium. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Generation of Symbiodinium protoplasts opens exciting, new avenues for researching these crucial symbiotic dinoflagellates, including genetic modification.
authors.affiliation: Centre for Marine Bio-Innovation, The University of New South Wales, Sydney, NSW, 2052, Australia.|School of Biological, Earth and Environmental Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia.|Climate Change Cluster, University of Technology Sydney, Sydney, NSW, 2007, Australia.
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Levin
authors.affiliation: Climate Change Cluster, University of Technology Sydney, Sydney, NSW, 2007, Australia.
authors.forename: David J
authors.initials: DJ
authors.lastname: Suggett
authors.affiliation: Climate Change Cluster, University of Technology Sydney, Sydney, NSW, 2007, Australia.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Nitschke
authors.affiliation: Australian Institute of Marine Science, Townsville MC, Qld, 4810, Australia.|School of BioSciences, The University of Melbourne, Parkville, Vic., 3010, Australia.
authors.forename: Madeleine J H
authors.initials: MJH
authors.lastname: van Oppen
authors.affiliation: Centre for Marine Bio-Innovation, The University of New South Wales, Sydney, NSW, 2052, Australia.|School of Biological, Earth and Environmental Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia.|Sydney Institute of Marine Science, Chowder Bay Road, Mosman, NSW, 2088, Australia.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Steinberg
mesh_terms: D002473:Cell Wall / Q000378:metabolism; D002480:Cellulase / Q000378:metabolism*; D057894:Coral Reefs; D004141:Dinoflagellida / Q000648:ultrastructure*; D011523:Protoplasts / Q000378:metabolism / Q000648:ultrastructure*; D013559:Symbiosis
chemical_list: D002480:Cellulase
keywords: Cell wall; cellulase; cellulose; dinoflagellate; genetic modification; protoplast fusion; protoplast generation; somatic hybridization; zooxanthellae