Production Of Protoplasts Research Articles

Improved Protoplast Production Protocol for Fungal Transformations Mediated by CRISPR/Cas9 in Botrytis cinerea Non-Sporulating Isolates.

Botrytis cinerea is a necrotrophic fungus that causes considerable economic losses in commercial crops. Fungi of the genus Botrytis exhibit great morphological and genetic variability, ranging from non-sporogenic and non-infective isolates to highly virulent sporogenic ones. There is growing interest in the different isolates in terms of their methodological applications aimed at gaining a deeper understanding of the biology of these fungal species for more efficient control of the infections they cause. This article describes an improvement in the protoplast production protocol from non-sporogenic isolates, resulting in viable protoplasts with regenerating capacity. The method improvements consist of a two-day incubation period with mycelium plugs and orbital shaking. Special mention is made of our preference for the VinoTaste Pro enzyme in the KC buffer as a replacement for Glucanex, as it enhances the efficacy of protoplast isolation in B459 and B371 isolates. The methodology described here has proven to be very useful for biotechnological applications such as genetic transformations mediated by the CRISPR/Cas9 tool.

Optimization of protoplast isolation and subsequent regeneration from the economically important brown alga Ecklonia cava using response surface methodology

Ecklonia cava is an edible kelp species distributed in Korea and Japan with diverse uses in ecological restoration, traditional medicine, and the pharmaceutical and abalone industries. These features make E. cava a good candidate for cellular biotechnology research, which is often dependent on protoplasts. To date, however, there have been no studies that have focused on the isolation of protoplast from E. cava. In this study, we sought to optimize the yield of protoplasts from cultured young sporophytes of E. cava using commercial enzymes, with a particular focus on identifying the key conditions for achieving high protoplast yield, including enzyme concentration, chelation pre-treatment, growth, temperature, incubation time, pH, and osmolarity. Protoplast production conditions were modeled using response surface methodology (RSM) via Box–Behnken design (BBD), and the practical utility of these conditions was experimentally verified. We found that an enzyme composition of 1.67 % cellulase RS and 4 U mL−1 alginate lyase without chelation pre-treatment, at 1570 mOsm L−1 H2O and pH 6 produced the highest protoplast yield of 4.45 ± 2.16 × 107 protoplasts g−1 fresh weight when incubated at 20 °C for 4 h using young (2-week-old) cultures. Our data revealed a strong positive correlation between growth rate and protoplast yield, thereby indicating that growth rate could serve as an index for determining the optimal timing of protoplast isolation from cultured material. The optimized protocol resulted in protoplasts capable of cell wall formation, division, and regeneration. These findings indicate the efficacy of commercial enzymes in protoplast isolation and that BBD-mediated RSM is a useful approach for the production of viable protoplasts from kelp sporophytes.

Protoplasts Isolation from Sardinian Germoplasm of Saffron (Crocus sativus L.)

Genetic studies of saffron indicate low heritability and strong environmental effects for most of the traits examined, suggesting local variability that has been selected over time. In vitro culture can be of support for genetic improvement programs, multiplication, or production of secondary metabolites. This is particularly relevant for saffron due to its triploidy. In the present work, the in vitro culture of Sardinian genotypes of saffron, using protoplasts, is described. Protoplasts were obtained from young leaves using a KAO enzyme mixture and cultured on KM8P substrate. The use of Evans et al. enzymes did not result in the production of protoplasts. The results presented in this work should be considered as the initial phase in the in vitro production of secondary metabolites obtained from local saffron genotypes.

Generation of protoplasts provides a powerful experimental research tool for biological and pathogenicity studies of Pythium insidiosum.

Pythiosis is a high-mortality infectious condition in humans and animals. The etiologic agent is Pythium insidiosum. Patients present with an ocular, vascular, cutaneous/subcutaneous, or gastrointestinal infection. Antifungal medication often fails to fight against P. insidiosum. The effective treatment is limited to radical surgery, resulting in organ loss. Fatal outcomes are observed in advanced cases. Pythiosis needs to be studied to discover novel methods for disease control. Genome data of P. insidiosum is publicly available. However, information on P. insidiosum biology and pathogenicity is still limited due to the lack of a cost-effective animal model and molecular tools. We aimed to develop a high-efficiency protocol for generating P. insidiosum protoplast, and used it to set up an animal model, in vitro drug susceptibility assay, and DNA transformation for this pathogen. P. insidiosum protoplast was successfully generated to establish a feasible pythiosis model in embryonic chicken eggs and an efficient in vitro drug susceptibility assay. DNA transformation is a critical method for gene manipulation necessary for functional genetic studies in pathogens. Attempts to establish a DNA transformation method for P. insidiosum using protoplast were partly successful. Significant work needs to be done for genetically engineering a more robust selection marker to generate stable transformants at increased efficiency. This study is the first to report an efficient P. insidiosum protoplast production for clinical and research applications. Such advances are crucial to speeding up the pathogen's biology and pathogenicity exploration.

Enhanced antimicrobial activity of lactic acid bacteria through genome shuffling and genetic variability among shuffled strains.

In this investigation, lactic acid bacteria (LAB) isolated from milk were tested for their antibacterial properties and improved the antimicrobial activity of these isolates using genome shuffling. A total of sixty-one isolates were found in eleven samples, which were then tested using the agar diffusion method for their antibacterial activity against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa. Thirty-one strains exhibited antibacterial activity against at least one of the tested pathogens, with an inhibitory zone's diameter varying between 15.0 and 24.0mm. Two isolates that showed the highest antimicrobial activity were identified as Lactobacillus plantarum CIP 103151 and Lactobacillus plantarum JCM 1149 according to 16S rRNA analysis. In the present study, applying genome shuffling approach significantly enhanced the antibacterial activity of L. plantarum. The initial populations were obtained via ultraviolet irradiation and were treated using the protoplast fusion method. The ideal condition for the production of protoplasts was 15mg/ml of lysozyme and 10μg/ml of mutanolysin. After two rounds of fusion, ten recombinants exhibited a significant increase in the inhibition zones versus S. aureus, S. typhimurium, P. aeruginosa, and E. coli, reaching up to 1.34, 1.31, 1.37, and 1.37-fold increase in inhibitory zone respectively. Random Amplified Polymorphic DNA results showed clear differences in DNA banding patterns among the wild strain of L. plantarum CIP 103151 and the three selected shuffled strains using primers 1283 & OPA09. On the other hand, no change was obtained using primers OPD03 neither among the wild strain and the three recombinant strains nor among the three shuffled strains.

Protoplast Preparation for Algal Single-Cell Omics Sequencing.

Single-cell sequencing (SCS) is an evolutionary technique for conducting life science research, providing the highest genome-sale throughput and single-cell resolution and unprecedented capabilities in addressing mechanistic and operational questions. Unfortunately, the current SCS pipeline cannot be directly applied to algal research as algal cells have cell walls, which makes RNA extraction hard for the current SCS platforms. Fortunately, effective methods are available for producing algal protoplasts (cells without cell walls), which can be directly fed into current SCS pipelines. In this review, we first summarize the cell wall structure and chemical composition of algal cell walls, particularly in Chlorophyta, then summarize the advances made in preparing algal protoplasts using physical, chemical, and biological methods, followed by specific cases of algal protoplast production in some commonly used eukaryotic algae. This review provides a timely primer to those interested in applying SCS in eukaryotic algal research.

Identification of the main proteins secreted byKluyveromyces marxianusand their possible roles in antagonistic activity against fungi

Lytic enzymes secreted by Kluyveromyces marxianus can lyse Saccharomyces cerevisiae cells. Their ability to hydrolyze yeast cell walls can be used in biotechnological applications, such as the production of glucans and protoplasts, as well as a biological control agent against plant pathogenic fungi. Herein, 27 proteins secreted by K. marxianus were identified by mass spectrometry analyses. Importantly, 14 out of the 27 proteins were classified as hydrolases. Indeed, the enzyme extract secreted by K. marxianus caused damage to S. cerevisiae cells and reduced yeast cell viability. Moreover, K marxianus inhibited spore germination and mycelial growth of the phytopathogenic fungus Botrytis cinerea in simultaneous cocultivation assays. We suggest that this inhibition may be partially related to the yeast's ability to secrete lytic enzymes. Consistent with the in vitro antagonistic tests, K. marxianus was able to protect strawberry fruits inoculated with B. cinerea. Therefore, these findings suggest that K. marxianus possesses potential as a biocontrol agent against strawberry gray mold during the postharvest stage and may also have potential against other phytopathogenic fungi by means of its lytic enzymatic arsenal.

An improved protocol for protoplast production, culture, and whole plant regeneration of the commercial brown seaweed Undaria pinnatifida

Protoplasts (i.e., living plant cells devoid of cell wall) represent an important biotechnological tool with utility for the conduction of in vitro studies and crop improvement in economic cultivars. Undaria pinnatifida is an annual brown kelp commonly used in Asian cuisine and several industries because of its biological properties. It is one of the major seaweed cultivars used worldwide. This study establishes a simple and effective protocol for protoplast isolation, culture, and whole plant regeneration of U. pinnatifida . The results showed that high protoplast yields (2–4 × 10 7 protoplast g −1 of fresh weight) were obtained using the commercially available enzymes cellulase “Onozuka” RS (1 %) and alginate lyase (4 U mL −1 ) without any pre-treatment and with a short incubation time (2–4 h). The isolation protocol described herein presented with more effective results with meristematic explants and was superior to the protocols reported for U. pinnatifida and other brown algae. Additionally, the enzymatic mixture could be reused up to six times. Whole plant regeneration was accomplished after 3 months in culture at 20 °C using two culture media, and a mixture of antibiotics (penicillin G, streptomycin, and chloramphenicol) was added once at the beginning of the culture. Our regeneration protocol was less laborious and more cost-effective, exhibiting final plating efficiency higher than previously reported and demonstrating the potential to be used to produce up to 3 × 10 4 sporophytes g −1 of explant. The established protocol can be easily used for genetic manipulation, protoplast fusion, or micropropagation, thus enabling the expansion of the use of protoplasts in algae. • Single-step production of protoplasts from the edible seaweed Undaria pinnatifida using commercial enzymes • The reusability of the enzyme shows that the process is economically viable. • Antibiotics and temperature are key factors for protoplast survival, division and normal regeneration. • Regeneration protocol is simpler and more effective than previously reported.

Physcomitrium patens Protoplasting and Protoplast Transfection.

Protoplast production with the moss Physcomitrium (Physcomitrella) patens has a long and successful history. As a tool, it has not only been the base of reverse genetic studies covering research fields as diverse as development, metabolism, or gene network regulation but also allowed its development as a bioengineering platform for protein production. We present here a standardized protocol for protoplast production from Physcomitrium (Physcomitrella) patens protonemata. Additionally, we detail procedures for their transfection, their plating for optimal regeneration, and three alternative selection approaches. To improve the consistency of protoplast regeneration, we describe a new option for protoplast embedding. The use of an alginate matrix to regenerate moss protoplast alleviates the use of warm agarized medium. Thus, it optimizes transformed protoplast survival without any morphological detrimental effect or impact on transfection efficiency.

Protoplast isolation from Dictyopteris pacifica and Scytosiphon lomentaria, using a simple commercial enzyme preparation

BackgroundProtoplasts (i.e., naked plant cells) can be used for in vitro manipulations and genetic improvement in cultivars with economic value. During the last decade, protoplast research in economic brown algae has been scarce, and it is usually hampered by the use of non-commercial enzymes or crude extracts for isolating protoplasts. Dictyopteris pacifica is part of a brown algal genus well known by its wide chemical diversity and biological properties. Scytosiphon lomentaria is an edible brown seaweed with antioxidant, antitumor, and antiviral properties. So far, there are no protoplast isolation protocols using commercial enzymes for these two economic brown algae. In this study, we obtained protoplasts from cultured samples of D. pacifica and S. lomentaria using commercially available enzymes. Additionally, we investigated the effects of Driselase inclusion and Ca-chelation pre-treatment on protoplast yields in order to optimize the conditions for protoplast preparations. ResultsProtoplasts were isolated from Dictyopteris pacifica and Scytosiphon lomentaria using the commercially available Cellulase Onozuka RS (1%) and Alginate lyase (4 U mL−1), and short incubation time (4 h). Driselase did not show significant effects on protoplast production in both species. Ca-chelation pre-treatment only increased the number of protoplasts in D. pacifica. Under optimal conditions, the protoplast yields from D. pacifica and S. lomentaria were 4.83 ± 2.08 and 74.64 ± 32.49 × 106 protoplasts g−1 fresh weight, respectively. The values obtained for S. lomentaria were 2–3 orders of magnitude higher than previously reported. ConclusionsOur results show that high protoplast yields can be obtained from D. pacifica and S. lomentaria using a simple mixture of commercial enzymes (Cellulase RS and Alginate lyase) and short incubation time (4 h). This work also represents the first report of protoplast isolation in D. pacifica. The method proposed here can help to expand protoplast technology in more brown algal species.

Qu-2, a robust poplar suspension cell line for molecular biology

Populus spp. have long been used as model woody plant species for molecular biology research. However, tissues of poplar are often recalcitrant to experimental procedures for molecular studies. We generated a hormone autotrophic poplar suspension cell line from a hybrid of Populus alba × P. berolinensis ‘Yinzhong’, named Qu-2. Qu-2 cells are suitable as a model biological system for studying woody plants. Qu-2 cells have many advantages over suspension cell lines derived so far from any other woody plants. Qu-2 cells are very easy to cultivate and can grow on several common plant culture media without the addition of any plant hormone. They show exceptionally high growth rates, reaching an approximately 150-fold increase in biomass after one week of culturing. Another important unique characteristic of Qu-2 cells is that they can be cryopreserved and readily reactivated. Qu-2 cells are suitable for molecular manipulations such as protoplast production, transient transformation, and RNA-seq analysis. Therefore, Qu-2 cells have the great potential to be an excellent model cell line in tree molecular biological research, ranging from physiology to gene function. The Qu-2 cells will be made available to the plant community for research.

Subcellular Localization of the D27 Protein in Sugarcane (Saccharum spp. Hybrids) Using an Optimized Protoplast Isolation, Purification, and Transient Gene Expression Protocol

The transient gene expression of protoplasts in plant is a considerable tool for gene functional research that has been widely used in gene analysis and functional characterization. Therefore, the objectives of this study were to develop a protocol for the isolation and purification of sugarcane protoplasts (Saccharum spp. hybrids), conduct transient PEG-mediated protoplast transfection with D27, and localize the D27 protein in sugarcane protoplasts. Total yield and viability of protoplasts were optimized for enzyme combination, mannitol concentration, and duration and temperature of enzymatic hydrolysis. High production of intact protoplasts (10.94 × 106 protoplasts g−1 FW) and a survival rate of > 80.0% was achieved through enzymatic hydrolysis at constant temperature of 28 °C, 60–70 rpm min−1 for 8 h in a solution containing 2.0% cellulase R-10, 0.5% macerozyme R-10, 0.6% pectolyase Y-23, 20 mM 2-(N-morpholine) ethanesulfonic acid (MES), 20 mM KCl, and 400 mM mannitol (pH 5.7). Using GFP as the reporter gene, the protoplasts were transformed most efficiently with 25% PEG 4000 for 25 min and the ScD27 protein was localized in the chloroplasts. The localization of ScD27 protein in sugarcane protoplast demonstrated that the newly developed protocol was functionally effective. This optimized sugarcane protoplast isolation, purification, and transient expression protocol lays a foundation for future molecular biology research in sugarcane.

Optimization of protoplast isolation from the gametophytes of brown alga Undaria pinnatifida using response surface methodology

The microscopic gametophytic phase of the commercial brown alga, Undaria pinnatifida, can be used for several applications, including the production of bioactive compounds, aquaculture and as germplasm bank. Therefore, gametophytes are good candidates for cellular biotechnology techniques, many of which rely on protoplasts (“naked” living plant cells). This study reports on the optimization of protoplast yield from male and female gametophytes of U. pinnatifida using different mixtures of commercial enzymes and chelation pre-treatment. Key conditions for achieving the highest protoplast yield, such as enzyme combinations, chelation pre-treatment, growth, temperature, incubation time, pH and osmolarity, were investigated. Protoplast isolation conditions were modelled by using response surface methodology (RSM) via Box-Behnken design (BBD) and subsequently experimentally verified in its predictability of protoplast production. The enzyme composition with 1% cellulase RS, 2 U mL−1 alginate lyase and 1% driselase with chelation pre-treatment, at 2481–2591 mOsm L−1 H2O and adjusted to pH 5.8–6.1, produced the highest protoplast yields of 3.12 ± 0.51 × 106 protoplasts g−1 fresh weight for male gametophyte and 2.11 ± 0.08 × 106 for female gametophyte when incubated at 20 °C for 4–7 h using cultures at mid or early exponential phase, respectively. These conditions also gave high amounts of protoplasts from other strains of Korea. Our results show the effectiveness of commercial enzymes combined with chelation pre-treatment in protoplast isolation and RSM with BBD is a useful method for rapidly producing the higher yields of protoplasts from brown alga.

High-Throughput Screening for Engineered Nanoparticles That Enhance Photosynthesis Using Mesophyll Protoplasts.

Certain engineered nanoparticles (NPs) have unique properties that have exhibited significant potential for promoting photosynthesis and enhancing crop productivity. Understanding the fundamental interactions between NPs and plants is crucial for the sustainable development of nanoenabled agriculture. Leaf mesophyll protoplasts, which maintain similar physiological response and cellular activity as intact plants, were selected as a model system to study the impact of NPs on photosynthesis. The mesophyll protoplasts isolated from spinach were cultivated with different NMs (Fe, Mn3O4, SiO2, Ag, and MoS2) dosing at 50 mg/L for 2 h under illumination. The potential maximum quantum yield and adenosine triphosphate (ATP) production of mesophyll protoplasts were significantly increased by Mn3O4 and Fe NPs (23% and 43%, respectively), and were decreased by Ag and MoS2 NPs. The mechanism for the photosynthetic enhancement by Mn3O4 and Fe is to increase the photocurrent and electron transfer rate, as revealed by photoelectrochemical measurement. GC-MS based single cell type metabolomics reveal that NPs (Fe and MoS2) altered the metabolic profiles of mesophyll cells during 2 h of illumination period. Separately, the effect of NPs exposure on photosynthesis and biomass were also conducted at the whole plant level. A strong correlation was observed with protoplast data; plant biomass was significantly increased by Mn3O4 exposure (57%) but was decreased (24%) by treatment of Ag NPs. The use of mesophyll protoplasts can be a fast and reliable tool for screening NPs to enhance photosynthesis for potential nanofertilizer use. Importantly, inclusion of a metabolic analysis can provide mechanistic toxicity data to ensure the development "safer-by-design" nanoenabled platforms.

Эффективность транзиентной экспрессии в протопластах разных сортов картофеля

Production of mesophilic protoplasts in potato (Solanum tuberosum L.) for transient gene expression is a mandatory technological stage in testing efficiency of new genetic constructs and CRISPR/Cas genome editing. In this study, isolation of protoplasts was carried out from leaves of potato plants of nine Russian cultivars grown in vitro for 6-7 weeks in vessels with foil caps, which prevented gas exchange with the environment. We first ever demonstrated the possibility of obtaining 2.4∙106 to 4.6∙106 viable protoplasts from one gram of aseptic plant leaves depending on the used potato cultivar. We established that the level of transfection of protoplasts with the genetic construct pHBT-sGFP-NosT amounts from 10 to 49% depending on the cultivar, which is sufficient for subsequent successful analysis of the efficiency of genetic constructs and potato genome editing. potato, Solanum tuberosum, protoplasts, plasmid DNA, transfection, transient expression, gfp The work was financially supported by the Comprehensive Research Program «Development of Potato Breeding and Seed Production».

Effect of 2,4-dichlorophenoxyacetic on in Vitro Callus Induction Using Leaf of Rocoto (Capsicum Pubescens Ruiz & Pav.) Cv. Serrano

The rocoto (Capsicum pubescen Ruiz & Pav.) is a plant of the genus Capsicum with center of origen in Perú, Used in national cuasina and of great valué for its pharmaceutical and medicinal propretores, relatad to capsaicin. In the present work a methodology was developed to determine the effect of 2,4-dichlorophenoxyacetic on in vitro callus induction from leaves in rocoto cv. Serrano. Were used seedlings rocoto cv. Serrano germínate in vitro, and subsequently the youngest leaves were selected, which were sectioned and placed in the different culture media. Five treatments with different concentrations of 2,4-D were evaluated. The best results were obtained with treatments T4 and T5 for the callus induction in 21 days allowed 72% callus proliferation. This study, a pioneer for the species, opens expectations for the potential use of callus of rocotos in other applications such as the production of protoplasts, somatic hybrids, somatic embryos and their use in polyploidization.

Development and validation of a novel and robust cell culture system in soybean (Glycine max (L.) Merr.) for promoter screening.

A novel soybean cell culture was developed, establishing a reliable and rapid promoter assay to enable high-throughput automated screening in soybean protoplasts relevant to shoot tissues in whole plants. Transient reporter gene assays can be valuable to rapidly estimate expression characteristics of heterologous promoters. The challenge for maximizing the value of such screens is to combine relevant cells or tissues with methods that can be scaled for high-throughput screening, especially for crop-rather than model species. We developed a robust and novel soybean cell suspension culture derived from leaf-derived callus for protoplast production: a platform for promoter screening. The protoplasts were transfected with promoter-reporter constructs, of which were chosen and validated against known promoter expression profiles from tissue-derived protoplasts (leaves, stems, and immature cotyledons) and gene expression data from plants. The cell culture reliably produced 2.82 ± 0.94 × 108 protoplasts/g fresh culture mass with a transfection efficiency of 31.06 ± 7.69% at 48h post-incubation. The promoter-reporter gene DNA expression levels of transfected cell culture-derived protoplasts were most similar to that of leaf- and stem-derived protoplasts (correlation coefficient of 0.99 and 0.96, respectively) harboring the same constructs. Cell culture expression was also significantly correlated to endogenous promoter-gene expression in leaf tissues as measured by qRT-PCR (correlation coefficient of 0.80). Using the manual protocols that produced these results, we performed early stage experiments to automate protoplast transformation on a robotic system. After optimizing the protocol, we achieved up to 29% transformation efficiency using our robotic system. We conclude that the soybean cell culture-to-protoplast transformation screen is amenable to automate promoter and gene screens in soybean that could be used to accelerate discoveries relevant for crop improvement. Key features of the system include low-cost, facile protoplast isolation, and transformation for soybean shoot tissue-relevant molecular screening.

Characterization of Pleurotus djamor neohaplonts recovered by production of protoplasts and chemical dedikaryotization.

Production of hybrid strains is accomplished by mating monosporic isolates or neohaplonts, obtained either by chemical dedikaryotization or by production of protoplast. However, differences in growth rate among recovered neohaplonts have been reported. The presence of phenotypic and genetic changes among the neohaplonts recovered either by chemical dedikaryotization or by production of protoplast, was evaluated by measuring growth and morphology, and by molecular characterization using six ISSR markers to identify polymorphisms. Neohaplonts recovered by both methods presented variation in growth rate depending on their compatibility type and recovery method. Using ISSR markers, 59.2% polymorphism was established. Neohaplonts recovered by both monokaryotization procedures presented differences in growth rate and polymorphism.

An Automated Protoplast Transformation System.

Efficient plant protoplast production from cell suspension cultures, leaf, and stem tissue allows for single-cell plant biology. Since protoplasts do not have cell walls, they can be readily transformed to enable rapid assessment of regulatory elements, synthetic constructs, gene expression, and more recently genome-editing tools and approaches. Historically, enzymatic cell wall digestion has been both expensive and laborious. Protoplast production, transformation, and analysis of fluorescence have recently been automated using an integrated robotic system. Here we describe its use for bulk protoplast isolation, counting, transformation, and analysis at very low cost for high-throughput experiments.

Protoplast isolation and regeneration from Hecatonema terminale (Ectocarpales, Phaeophyceae) using a simple mixture of commercial enzymes

Protoplast systems are essential for genome-editing and gene silencing technologies. In brown algae, protoplast isolation has been hampered by protocols that use non-commercial enzymes or crude extracts. This study is the first to report the production of protoplasts from cell-filament suspension cultures of the brown alga, Hecatonema terminale (Kutzing) Kylin, using different mixtures of commercial enzymes and chelation pre-treatment. In this study, mixture A (cellulase RS and alginate lyase) with chelation pre-treatment produced the highest number of protoplasts (3.52 ± 0.23 × 105 protoplasts g−1 FW). Chelation pre-treatment showed high effects on all kinds of enzyme mixtures. The effects of these different mixtures were examined by two-way ANOVA. We also investigate the optimal protoplast density and regeneration medium for protoplast regeneration. Of 16 combinations for regeneration media, RM1 with lowest initial protoplast density (2.4 × 103 protoplasts mL−1) showed the highest value (74%) of final plating efficiency (FPE) after 13 days of culture. The well-defined heterotrichous thalli with phaeophycean hairs were clearly distinguished after 22 days of culture.