https://doi.org/10.15433/ksmb.2020.12.1.050 ISSN 2383-5400 (Online)

Protoplast Production from Sphacelaria fusca (, Phaeophyceae) Using Commercial Enzymes

Jose Avila-Peltroche 1, Boo Yeon Won 2*

1Graduate student, Department of Life Science, Chosun University, Gwangju 61452, Republic of Korea 2Researcher, Department of Life Science, Chosun University, Gwangju 61452, Republic of Korea

(Received 14 May 2020, Revised 3 June 2020, Accepted 3 June 2020)

Abstract 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 H 2O) gave a protoplast yield of 15.08 ± 5.31 × 10 4 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.

Keywords : Phaeophyceae , Commercial enzymes, Sphacelaria fusca , Protoplast isolation

as the isopod Dynamene magnitorata [7], spider crab Leucippa pentagona [2], and the “key herbivore” The filamentous brown alga genus, Sphacelaria, is Diadema antillarum [8]. It is known that grazers can a common epiphyte on macroalgae and marine plants control the proliferation of epiphytic algae and thus they [1,2], as well as epizoic on sea turtles [3]. It provides help to avoid a decrease in the performance of the host food and habitat for animals, playing an important role [9]. Sphacelaria can be also be infected in the wild by in coastal benthic communities [4-6]. Sphacelaria repre- fungal parasites such as the chytrid Chytridium poly- sents an important component of the diet of grazers such siphonae [10]. Abiotic factors (e.g. intertidal elevation)

* Corresponding author This is an open-access journal distributed under the terms of the Creative Phone: +82-62-230-7161 Fax: +82-62-230-7467 Commons Attribution Non-Commercial License E-mail: [email protected] (http://creativecommons.org/licenses/by-nc/4.0/ )

- 50 - can affect the distribution of Sphacelaria in its host, as it has been reported for other epiphytic filamentous Isolation and culture of the strain [11]. Besides Sphacelaria has been used Sphacelaria fusca (MBRB0073TC18209C1) was as a model organism for plant morphogenesis due to collected by hand from Sargassum muticum in Jindo its apical growth and ease of cultivation [12-14]. Thus, Island, Jeollanam-do, Korea, on July 27, 2017. Apical Sphacelaria is a suitable organism for exploring differ- filaments of S. fusca were cultured in 12 well plates ent stressors and environmental inputs, both in con- containing PES medium [29] under 14:10-h light/dark trolled and non-controlled conditions. photoperiod at 20°C with light intensity 40 μ mol Transcriptome analysis can help to understand the mo- photons/m 2/s of blue LED (DyneBioCo.Korea). The lecular basis of physiological responses to environmental medium was renewed weekly. After 3 months , plants stressors [15]. Recently, single-cell RNA sequencing has were transferred to 100×40 mm Petri dishes and were emerged as a novel approach to measure transcriptome cultured under the same culture conditions. Two months with high resolution using different cell types of animal later, plants accumulated much biomass and were cells [16]. However, the presence of cell walls has hin- transferred into 1-L flat-bottomed round flasks filled with dered the application of this approach to plant cells [17]. 1-L PES medium under aeration. Light intensity was One way to overcome this problem is using protoplasts, 40-60 μ mol photons/m 2/s of white fluorescent light. The which are plant cells whose cell wall has been removed air was sterilized using 0.22-μ m surfactant-free cellulose by enzymatic methods [18]. The high amounts of proto- acetate (SFCA) syringe filters (Corning,Germany). The plasts are more important than their regeneration ability medium was renewed every 2 weeks. in single-cell RNA sequencing [17,19]. Moreover, they represent an important biotechnological tool for ge- Identification of the culture strain nome-editing and gene-silencing technologies [20], as Taxonomic identification was performed using well as, for the production of useful secondary metabo- morphological characters [30] from cultures maintained lites, such as dictyopterenes from the seaweed in 60×15 mm Petri dishes without agitation. Dictyopteris prolifera [21]. Photomicrographs were taken using a Leica inverted In brown algae, protoplast isolation has been reported microscope (DMi8; Leica, Germany) equipped with a in 29 species [18,22-25]. To date, protoplasts from Leica DFC450C camera. Genomic DNA extraction, Sphacelaria have only been isolated in low amounts us- PCR amplification, DNA purification, and sequencing ing commercial and non-commercial enzymes for mi- were performed as previously described [31,32] using crotubule analysis during regeneration [26,27]. The pro- cultured samples. The plastid-encoded RuBisCo spacer toplast isolation becomes more expensive and time-con- region was amplified using the primer combinations G1 suming because these non-commercial enzymes need to and G2 [33]. The amplified region sequences were be extracted from marine herbivores or microorganisms compared to the GenBank nucleotide database using the [28]. Thus, protoplast isolation protocols using commer- BLAST program [34]. cial enzymes are important for the application of sin- gle-cell RNA sequencing in Sphacelaria . Protoplast isolation In this study, we report for the first time the protoplast The commercially available cell wall lytic enzymes isolation from Sphacelaria fusca using a simple mix of used for this study included cellulase Onozuka RS commercial enzymes. Also, we tested the effect of os- (Yakult Co. Ltd., Japan), alginate lyase, and driselase molarity and driselase inclusion on protoplast pro- from Basidiomycetes sp. (Sigma-Aldrich, USA). duction to determine the best conditions for this process. Different enzyme combinations and conditions are

- 51 - shown in Table 1. measurements for each repetition. In protoplast preparations, cell types were identified by protoplast Table 1. Combinations and concentrations of enzyme mixtures for protoplast isolation from Sphacelaria fusca. size and the presence or absence of physodes [26].

Composition of enzyme Commercial mixtures Viability and cell wall removal enzymes A B C D The viability of protoplasts and cell wall removal was assessed by the red chlorophyll autofluorescence and Cellulase Onozuka RS 1 1 1 1 (%) staining with calcofluor white M2R (Sigma-Aldrich, Alginate lyase (U/mL) 4 4 4 4 Driselase (%) 1 - 1 - USA), respectively, as previously described [25]. Osmolarity 1X 1X 1.6X 1.6X

1X : 1570 mOsm/L H 2O; 1.6X: 2512 mOsm/LH 2O Portoplast regeneration Protoplasts were washed three times with enzymatic Protoplast isolation was carried out as previously solution by centrifugation for 10 min at 100× g. Cells described [35,36] with some modifications [25]. were dispensed into 2 mL of regeneration media (PES

Briefly, approximately 100– 300 mg plants from 1-L with 285 mM NaCl and 5 mM CaCl 2) and cultured round flasks (3-day-old cultures) were incubated in a at 20°C in the dark and at initial protoplast density of 0.22-μ m filter-sterilized enzymatic solution (400 mM 9 x 10 3 protoplasts/mL plus antibiotic mix (50 mg/L

NaCl, 130 mM MgCl 2·6H 2O, 22 mM MgSO 4, 160 mM penicillin G, 25 mg/L streptomycin and 5 mg/L KCl, 2 mM CaCl 2, and 10 mM MES; pH6; 1570 chloramphenicol). After 1 day in the dark, osmotic mOsm/L H 2O) containing cellulase Onozuka RS and pressure was reduced slowly by adding 400 µL of PES. alginate lyase, either with or without driselase, at 20°C Cultures were exposed to 1-2 μ mol photons m 2/s with shaking at 70 rpm for 6h in the dark. The 14:10-h light/dark photoperiod. Osmotic pressure was osmolarity of the enzymatic solution was tested in two further reduced during the next 2 days by adding 800 levels: normal osmolarity (1X=1570 mOsm/L H 2O) and µL of PES each day. Cultures were finally exposed to 2 increased osmolarity (1.6X=2512 mOsm/L H 2O). 10 μ mol photons m /s under the same photoperiod. The Osmolarity was increased by increasing the component medium was renewed every week thereafter. concentrations in the enzymatic solution keeping their same proportions. Chelation pre-treatment was Statistical analysis conducted with a calcium-chelating solution [665 mM To evaluate the effect of driselase addition and NaCl, 30 mM MgCl 2·6H 2O, 30 mM MgSO 4, 20 mM osmolarity on protoplast yield a generalized linear KCl, and 20 mM ethyleneglycol– bis(β– amino – model with a binomial negative error distribution ethylether)–N,N,N ′,N ′–tetraacetic acid tetrasodium salt (GLM.nb) was used as a traditional method for (EGTA Na– 4) as the calcium chelator; pH5.5] for 20 handling overdispersed data. The analyses were min before enzymatic digestion. performed using “MASS” package in R [38]. Protoplasts were filtered by using a 25-μ m nylon The proportion of cell types on protoplast mesh to remove undigested filaments and concentrated preparations were compared to controls (undigested S. by centrifugation at 100× g for 10 min. Protoplast yields fusca filaments) using beta regressions since beta were estimated by using a hemocytometer (Marienfeld, distribution provides a flexible model for continuous Germany) with an Olympus microscope (BX51TRF; variables restricted to the interval (0, 1) [39]. The Olympus, Japan) and expressed as protoplasts/g fresh analyses were performed using “betareg” package in weight (FW). Protoplast size was calculated by using R [40]. The p-value was corrected by the Bonferroni ImageJ 1.46r software [37] based on 100 cell method to compensate for the effect of multiple

- 52 - hypotheses testing. tion) did not present reproductive structures during the The significance threshold was set at p=0.01 to study. They showed three-armed propagules, typical reduce the true Type I error rate (at least 7% but morphological feature of this species (Figure 1B) [31]. typically close to 15%) [41]. Protoplast isolation was Our morphological identification of S. fusca was con- repeated four times in each treatment. firmed by molecular analysis. A 585-bp portion was sequenced for the strain (CUK18209 (= MBRB0073TC18209C1) in Chosun University Herbarium in Korea; MT009225 in GenBank) of S. Identification fusca . The RuBisCo spacer region of our strain was The vegetative characteristics of Sphacelaria fusca 100% identical to S. fusca clone A2 (FJ710148) [42]. are shown in Fig. 1A, C. Cultures (with or without aera-

Figure 1. Protoplast production from Sphacelaria fusca . (A) Piece of Sphacelaria fusca from unialgal cultures without aeration. (B) Three-armed propagule. (C) A 1-month old culture in 1-L flat-bottomed round flask with aeration. (D) Control (non-digested apex) showing apical (Ap) and subapical (Sap) cells. (E) Protoplast (p) release from an apical portion. Notice that the apical cell wall is intact (Ap). (F) Nodal-cell protoplast (p) being released from the middle part of a filament. Notice the “cell-wall ghost” (CWG). (G) Apical-cell protoplast. (H) Subapical-cell protoplast. (I) Nodal (N) and internodal-cell (IN) protoplasts. (J) True and viable protoplast showing red chlorophyll autofluorescence.

- 53 - Protoplast isolation using enzymes shape and very heterogeneous in size and pigmentation, Protoplast yields ranged from 0-15.08 x 10 4 corresponding to the various cell types in Sphacelaria . protoplasts/g FW. Mixture C (cellulase RS, alginate Protoplasts from apical cells were 30-50 µm in diameter lyase and driselase with 1.6X osmolarity) with chelation and contained numerous physodes (Figure 1G). pre-treatment produced the highest number of Protoplasts from subapical cells were similar in size but protoplasts (15.08±5.31 × 10 4 protoplasts/g FW), did not contain large numbers of physodes (Figure 1H). followed by mixture D (cellulase RS and alginate Smaller protoplasts (10-30 µm) were from nodal and lyasewith 1.6X osmolarity) with chelation pre-treatment internodal cells (Figure 1I). Compared with the (7.55 ± 4.16 x 10 4 protoplasts/g FW). The effect of proportions of the different cell types in the osmolarity and driselase inclusion is shown in Figure non-digested filaments (Figure 1D), protoplast 2. Osmolarity increase had a highly significant and preparations were enriched in subapical, nodal, and positive effect on protoplast production (p<0.001). intermodal-cell protoplasts. These values are shown in Under normal osmolarity (1X), protoplast yields were Table 2. True protoplast percentages were 98– 100% low (<100 protoplasts/g FW) and inconsistent. By with calcofluor white staining, while the viability of elevating the osmolarity, protoplast could be isolated freshly isolated protoplasts was approximately 98% in larger and consistent amounts. The inclusion of with red chlorophyll autofluorescence (Figure 1J). driselase to the enzymatic mix did not affect protoplast Protoplasts were able to regenerate whole new plants yield (p=0.030) however, it slightly increased the after 2-3 weeks in culture at 20°C. protoplast number. There was no interaction between both factors (p=0.454). As an effort to simplify our Table 2. Relative proportions (%) of the various cell protoplast isolation protocol, chelation pre-treatement types in the controls (undigested Sphacelaria fusca was skipped. However, this resulted in more than 10 filaments) and protoplast preparations. Superscript letters indicate highly significant differences among treatments times reduction of protoplast yield (p<0.001). Values are presented as mean ± SD (n=4). (<10 4protoplasts/gFW). Apical Subapical Nodal Internodal

20.38± 1.62± 0.44± 77.56± Controls a a a a 17.55 1.46 0.43 18.25

27.16± 19.62± 36.08± 17.14± Protoplasts 2.74 a 2.79 b 1.69 b 5.33 b

Figure 2. Effect of osmolarity and driselase inclusion on pro- The highest protoplast amount obtained in this study toplast yield from Sphacelaria fusca. Independent data points was 15.08 x 10 4 protoplasts/g FW, which represents and averages (horizontal lines) are shown (n=4). Error bars more than 30 times the highest value reported pre- represent 95% confidence intervals. IY: inconsistent yield; ns: no significant difference (p>0.01). viously for Sphacelaria by Ducreux and Kloareg [26]. These authors used two commercial enzymes and one Cell wall digestion was not complete and "cell-wall non-commercial enzyme for the isolation process ghosts" were still visible at the end of the enzymatic (Table 3). Similarly, Coelho et al. [36] used a combina- treatment (Figure 1F). However, protoplasts were tion of commercial and non-commercial enzymes for released through holes in the cell walls due to filament protoplast isolation from Ectocarpus . Our protocol used fragmentation (Figure 1E, F). Protoplasts were spherical commercial enzymes: cellulase Onozuka RS, alginate

- 54 - lyase, and driselase from Sigma. Using only commer- establishing protoplast systems [43]. Therefore our re- cial enzymes is desirable when developing protocols for sults will helpful for future studies.

Table 3. Comparison of protoplast isolation between the previous study and this study. Ducreux and Kloareg (1988) This study Species Sphacelaria sp. Sphacelaria fusca Chelation pre-treatment No Yes Commercial enzymes 2% Cellulysin, 0.5% pectolyase Y23 1% cellulose Onozuka RS, 1% driselase, 4 U/mL alginate lyase Non-commercial enzymes 2% alginate lyase from Haliotis Not used tuberculata , Patella vulgata or Aplysia punctata Incubation time 12 h 6 h

Osmolarity (mOsm/LH 2O) 1800 2512 pH 5.8 6 Protoplast yield 0.46 15.8 (x 10 4 protoplasts/g fresh weight) Cell types* in protoplast All, but apical-cell protoplast enriched All, but subapical and nodal-cell preparation protoplast enriched *Apical, subapical, nodal and internodal cells

Cell walls in brown algae are composed of alginate, Unlike other filamentous brown algae, plants of fucoidans, fuco-glucorono-xylans, and a small amount Sphacelaria present four different cell types: apical, of cellulose (1-8%) [44,45]. Recently, the presence of subapical, nodal and internodal. Differences among mixed-linked glucan (MLG) in brown algal cell walls, types are not only reflected by their morphology but including the related species Stypocaulon scoparium , also their morphogenetic competences. Sphacelaria has been demonstrated [46]. Although MLG can be de- growth is mainly directed by the large apical cell, which graded by driselase [47], its inclusion on the enzymatic has the ability to regenerate a whole plant [26]. mixture was not crucial for improving protoplast yields. Protoplast preparations obtained in this study showed The combination of cellulase and alginate lyase was all the cell types reported for Sphacelaria , with higher sufficient for releasing true protoplasts from all cell proportions of subapical and nodal-cell protoplasts. types. Chelation pre-treatment was necessary for im- Interestingly, our enzymatic combination could not de- proving protoplast yield. The use of cation chelators graded apical cell walls, which have been reported to has shown positive effect in protoplast isolation from be more digestible by other authors [24,49]. Protoplasts [25,36] and Laminariales [48,49]. from different cell types could allow to explore the tran- Our results also showed that high osmolarity (2512 scriptome profile in different cell populations [17]. mOsm/L H 2O) favored the isolation process, probably Protoplast isolation process inevitably results in mul- due to the stimulation of alginate lyase activity by in- tiple type of stress for vegetative cells due to cell wall creasing salt concentrations [50]; promotion of proto- removal, considerable cell death, and loss of cell to cell plast release through the holes in the cell wall; or pro- communication. In protoplasts from the red seaweed tection of the protoplast membrane [51]. Even though Chondrus crispus and the brown seaweed Laminaria protoplast isolation protocols commonly use lower os- digitata , expression of stress genes, such as heat shock molarities [26,35,36], the increased osmolarity did not proteins and enzymes involved with detoxification, damage the protoplasts. These changes give better re- were enhanced [53,54]. Although we did not assess sults in protoplast isolation. gene expression levels in the protoplast of S. fusca , we

- 55 - expect a similar trend. These changes in gene ex- and Veléz-Rubio, G. M. 2016. Epibiota of juvenile pression do not limit the use protoplasts in single-cell Hawksbill Sea Turtles Eretmochelys imbricata stranded in the coast of Rocha Department, Uruguay. Rev. Biol. RNA sequencing as protoplast-inducible genes are fil- Mar. Oceanogr. 51 , 449-453. tered out during the analysis [17]. 4. Pavia, H., Carr, H. and Åberg, P. 1999. Habitat and feeding preferences of crustacean mesoherbivores inhabiting the brown seaweed Ascophyllum nodosum (L.) Le Jol. and its epiphytic macroalgae. J. Exp. Mar. Biol. This is the first report of protoplast isolation from Ecol. 236 , 15-32. 5. Karez, R., Engelbert, S. and Sommer, U. 2000. ‘Co‐ S. fusca . Our result showed that a simple mix of com- consumption’ and ‘protective coating’: two new mercial enzymes (cellulase Onozuka RS, alginate lyase, proposed effects of epiphytes on their macroalgal hosts and driselase) with increased osmolarity (2512 mOsm/L in mesograzer ‐epiphyte ‐host interactions. Mar. Ecol. Prog. Ser. 205 , 85-93. H O) and chelation pre-treatment was capable of pro- 2 6. Viejo, R. and Åberg, P. 2003. Temporal and spatial ducing high amounts of true and viable protoplasts from variation in the density of mobile epifauna and grazing all cell types from S. fusca . These features make the damage on the seaweed Ascophyllum nodosum . Mar. present protocol useful for single-cell RNA sequencing, Biol. 142 , 1229-1241. 7. Arrontes, J. 1990. Diet, food preference and digestive transcriptomics, gene editing, gene silencing technol- efficiency in intertidal isopods inhabiting macroalgae. J. ogy, microtubule analysis and natural production from Exp. Mar. Biol. Ecol. 139 , 231-249. algae. 8. Hernández, J. C., Gil-Rodríguez, M. A., Herrera-López, G. and Brito, A. 2007. Diet of the “key herbivore” Diadema antillarum in two contrasting habitats in the Canary Islands (Eastern-Atlantic). Vieraea 35 , 109-120. 9. Whalen, M. A., Emmett, J. D. and Grace, J. B. 2013. Temporal shifts in top-down vs. bottom-up control of This research was supported by the Basic Science epiphytic algae in a seagrass ecosystem. Ecology 94 , Research Program through the National Research 510-520. 10. Raghukumar, C. 1987. Fungal parasites of marine algae Foundation of Korea (NRF) funded by the Ministry of from Mandapam (South India). Dis. Aquat. Org. 3, Education (2015R1D1A1A01058359); the National 137-145. Institute of Biological Resources (NIBR) funded by the 11. Longtin, C. M., Scrosati, R. A., Whalen, G. B. and Ministry of Environment (MOE) of Korea (NIBR No. Garbary, D. J. 2009. Distribution of algal epiphytes across environmental gradients at different scales: intertidal 2015-01-204); and by Marine Biotechnology Program elevation, host canopies, and host fronds. J. Phycol. 45 , grants (20170431 and PJT200669) funded by the 820-827. Ministry of Oceans and Fisheries of Korea to Tae Oh 12. Dworetzky, B., Klein, R. M. and Cook, P. W. 1980. Effect of growth substances on “apical dominance” in Cho. Sphacelaria furcigera (Phaeophyta). J. Phycol. 16 , 239-242. 13. Charrier, B., Bail, A. L. and de Reviers, B. 2012. Plant Proteus: brown algal morphological plasticity and underlying developmental mechanisms. Trends Plant Sci. 1. Piazzi, L., Balata, D. and Ceccherelli G. 2015. Epiphyte 17 , 468-477. assemblages of the Mediterranean seagrass Posidonia 14. Bogaert, K. A., Arun, A., Coelho, S. M. and De Clerck, oceanica : an overview. Mar. Ecol. 37 , 3-41. O. 2013. Brown Algae as a Model for Plant 2. Varisco, M., Martín, L., Zaixso, H., Velasquez, C. and Organogenesis. In: De Smet, I. (ed), Plant Vinuesa, J. 2015. Food and habitat choice in the spider Organogenesis: Methods and Protocols, Methods in crab Leucippa pentagona (Majoidea: Epialtidae) in Bahía Molecular Biology. Springer Science + Business Media, Bustamante, Patagonia, Argentina. Scientia Marina 79 , New York, vol. 959, pp. 97-125. 107-116. 15. Imadi, S. R., Kazi, A. G., Ahanger, M. A., Gucel, S. 3. Velasco-Charpentier, C., Pizarro-Mora, F., Estrades, A. and Ahmad, P. 2015. Plant transcriptomics and responses

- 56 - to environmental stress: an overview. J. Genet. 94 , of the U.S.– Japan Conference, Hakone, pp. 63-75. 525-537. 30. Keum, Y. S. 2010. Sphacelariales, Cutleriales, . 16. Hwang, B., Lee, J. H. and Bang, D. 2018. Single-cell In: Sook, S. (ed), Algal flora of Korea. RNA sequencing technologies and bioinformatics Heterokontophyta: Phaeophyceae: , , pipelines. Exp. Mol. Med 50 , 96. , Sphacelariales, Cutleriales, Ralfisales, https://doi.org/10.1038/s12276-018-0071-8 Laminariales, vol. 2, National Institute of Biological 17. Shulse, C. N, Cole, B.J, Ciobanu, D., Lin, J., Yoshinaga, Resources, Incheon, pp. 19-69. Y., Gouran, M., Turco, G. M., Zhu, Y., O’Malley, R. 31. Keum, Y. S., Oak, J. H., Draisma, S. G. A., Prud'homme C., Brady, S. M. and Dickel, D. E. 2019. High-throughput van Reine, W. F. and Lee, I. K. 2005. Taxonomic single-cell transcriptome profiling of plant cell types. Cell reappraisal of Sphacelaria rigidula and S. fusca Rep. 27 , 2241-2247. (Sphacelariales, Phaeophyceae) based on morphology and 18. Reddy, C. R. K., Gupta, M. K., Mantri, V. A. and molecular data with special reference to S. didichotoma . Bhavanath, J. 2008. Seaweed protoplast: status, Algae 20 , 1-13. biotechnological perspectives and need . J. Appl. Phycol. 32. Bustamante, D. E., Won, B. Y. and Cho, T. O. 2016. 20 , 619-632. The conspecificity of Pterosiphonia spinifera and P. 19. Efroni, I. and Birnbaum, K. D. 2016. The potential of arenosa (Rhodomelaceae, Ceramiales) inferred from single-cell profiling in plants. Genome Biol. 17 , 65. morphological and molecular analyses. Algae 31 , https://doi.org/10.1186/s13059-016-0931-2 . 105-115. 20. Burris, K P., Dlugosz, E. M., Collins, A. G., Stewart, 33. Destombe, C. and Douglas, S. E. 1991. Rubisco spacer C. N. and Lenaghan, S. C. 2016. Development of a rapid, sequence divergence in the rhodophyte alga Gracilaria low-cost protoplast transfection system for switchgrass verrucosa and closely related species. Curr Genet. 19 , (Panicum virgatum L.). Plant Cell Rep. 35 , 693-704. 395-398. 21. Fujimura, T., Kawai, T., Kajiwara, T., Ishida, Y. 1994. 34. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, Volatile components in protoplast isolated from the J., Zhang, Z., Miller, W. and Lipman, D. J. 1997. Gapped marine brown alga Dictyopteris prolifera (Dictyotales). BLAST and PSI-BLAST: a new generation of protein Plant Tissue Cult. Lett. 11 , 34-39. database search programs. Nucleic Acids Res. 25 , 22. Fisher, D. D and Gibor, A. 1987. Production of 3389-3402. protoplasts from the brown alga, Sargassum muticum 35. Benet, H., Gall, E. Ar, Asensi, A. and Kloareg, B. 1997. (Yendo) Fensholt (Phaeophyta). Phycologia 26 , 488-495. Protoplast regeneration from gametophytes and 23. Chen, C. S and Shyu, J. F. 1995. Isolation of protoplast sporophytes of some species in the order Laminariales from four species of brown algae. Bot. Bull. Acad. Sinica (Phaeophyceae). Protoplasma . 199 , 39-48. 35 , 95-104. 36. Coelho, S. M., Scornet, D., Rousvoal, S., Peters, N., 24. Matsumura, W. 1998. Efficient isolation and culture of Dartevelle, L., Peters, A. F. and Cock, J. M. 2012. viable protoplasts from Laminaria longissima Miyabe Isolation and regeneration of protoplast from Ectocarpus . (Phaeophyceae). Bull. Fac. Fish. Hokkaido Univ. 49 , Cold Spring Harb. Protoc 2012 , 361-364. doi: 85-90. 10.1101/pdb.prot067959 25. Avila-Peltroche, J., Won, B. Y. and Cho, T. O. 2019. 37. Abràmoff, M. D., Magalhães, P. J. and Ram, S. J. 2004. Protoplast isolation and regeneration from Hecatonema Image processing with ImageJ . Biophoton Int. 11 , 36-42. terminale (Ectocarpales, Phaeophyceae) using a simple 38. Venables, W. N. and Ripley, B. D. 2002. Modern mixture of commercial enzymes. J. Appl. Phycol. 31 , Applied Statistics with S. 4 th edition. Springer, NewYork. 1873-1881. http://www.stats.ox.ac.uk/pub/MASS4. 26. Ducreux, G. and Kloareg, B. 1988. Plant regeneration 39. Ferrari, S. L. P. and Cribari-Neto, F. 2004. Beta from protoplast of Sphacelaria (Phaeophyceae). Planta regression for modelling rates and proportions. J. App. 174 , 25-29. Stat. 31 , 799-815. 27. Rusig, A-M., Le Guyader, H. and Ducreux, G. 1994. 40. Cribari-Neto, F and Zeileis, A. 2010. Beta regression Dedifferentiation and microtubule reorganization in the in R. J. Stat. Softw. 34 , 1-24. apical cell protoplast of Sphacelaria (Phaeophyceae). 41. Sellke, T., Bayarri, M. J. and Berger, J. O. 2001. Protoplasma 179 , 83-94 Calibration of p values for testing precise null hypotheses. 28. Cocking, E. C. 1972. Plant cell protoplasts – isolation Am. Stat. 55 , 62-71. and development. Annu. Rev. Plant Physiol. 23 , 29-50. 42. Lee, S. R., Oak, J. H., Keum, Y. S., Lee, J. A. and 29. Provasoli, L. 1968. Media and prospects for the Chung, I. K. 2011. Utility of rbc S gene as a novel target cultivation of marine algae. In: Watanabe, A. and Hattori, DNA region for brown algal molecular systematics. A. (eds), Cultures and collections of algae. Proceedings Phycol. Res. 59 , 34-31.

- 57 - 43. Bhojwani, S. S. and Razdan, M. K. 1996. Plant Tissue 49. Kloareg, B., Polne-Fuller, M. and Gibor, A. 1989. Mass Culture: Theory and Practice, a Revised Edition. Elsevier, production of viable protoplasts from Macrocystis Amsterdam, p. 338. pyrifera . Plant Sci. 62 , 105-112. 44. Cronshaw, J., Myers, A. and Preston, R. D. 1958. A 50. Huang, L., Zhou, J., Li, X., Peng, Q., Lu, H. and Du, chemical and physical investigation of the cell walls of Y. 2013. Characterization of a new alginate lyase from some marine algae. Biochim. Biophys. Acta. 27 , 89-103. newly isolated Flavobacterium sp. S20. J. Ind. Microbiol. 45. Kloareg, B. and Quatrano, R. S. 1988. Structure of the Biotechnol. 40 , 113-122. cell walls of marine algae and ecophysiological functions 51. Xiaoke, H., Xiaolu, J. and Huashi, G. 2003. Isolation of the matrix polysaccharides. Oceanogr. Mar. Biol. of protoplast from Undaria pinnatifida by alginate lyase Annu. Rev. 26 , 259-315. digestion. J. Ocean. Univ. Qingdao. 2, 58-61. 46. Salmeán, A. A., Duffieux, D., Harholt, J., Qin, F., 52. Burns, A.R., Oliveira, L. and Bisalputra, T. 1984. A Michel, G., Czjzek, M., Willats, W. G. T and Hervé, C. cytochemical study of cell wall differentiation during bud 2017. Insoluble (1 →3), (1 →4)- β-D-glucan is a initiation in the brown alga Sphacelaria furcigera . Bot. component of cell walls in brown algae (Phaeophyceae) Mar. 27 , 45-54 . and is masked by alginates in tissues. Sci. Rep. 7, 2880. 53. Collén, J., Roeder, V., Rousvoal, S., Collin, O., Kloareg, doi:10.1038/s41598-017-03081-5. B. and Boyen, C. 2006. An expressed sequence tag 47. Thibault, J.F. and Rouau, X. 1990. Studies on enzymic analysis of thallus and regenerating protoplasts of hydrolysis of polysaccharides in sugar beet pulp. Chondrus crispus (Gigartinales, Rhodophyceae). J. Carbohy. Polym. 13 , 1-16. Phycol. 42 , 104-112. 48. Butler, D. M., Ostgaard, K., Boyen, C., Evans, L. V., 54. Roeder, V., Collén, J., Rousvoal, Corre, E., Leblanc, C. Jensen, A. and Kloareg, B. 1989. Isolation conditions for and Boyen, C. 2005. Identification of stress gene high yields of protoplasts from Laminaria saccharina and transcripts in Laminaria digitata (Phaeophyceae) L. digitata (Phaeophyceae). J. Exp. Bot. 40 , 1237-1246 protoplast cultures by expressed sequence tag analysis. J. Phycol. 41 , 1227-1235.

- 58 -