Seaweed protoplasts: status, biotechnological perspectives and needs

Abstract

Protoplasts are living plant cells without cell walls which offer a unique uniform single cell system that facilitates several aspects of modern biotechnology, including genetic transformation and metabolic engineering. Extraction of cell wall lytic enzymes from different phycophages and microbial sources has greatly improved protoplast isolation and their yield from a number of anatomically more complex species of brown and red seaweeds which earlier remained recalcitrant. Recently, recombinant cell wall lytic enzymes were also produced and evaluated with native ones for their potential abilities in producing viable protoplasts from Laminaria. Reliable procedures are now available to isolate and culture protoplasts from diverse groups of seaweeds. To date, there are 89 species belonging to 36 genera of green, red and brown seaweeds from which successful protoplast isolation and regeneration has been reported. Of the total species studied for protoplasts, most belonged to Rhodophyta with 41 species (13 genera) followed by Chlorophyta and Phaeophyta with 24 species each belonging to 5 and 18 genera, respectively. Regeneration of protoplast-to-plant system is available for a large number of species, with extensive literature relating to their culture methods and morphogenesis. In the context of plant genetic manipulation, somatic hybridization by protoplast fusion has been accomplished in a number of economically important species with various levels of success. Protoplasts have also been used for studying foreign gene expression in Porphyra and Ulva. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, bio-chemical synthesis of cell walls etc. This article briefly reviews the status of various developments in seaweed protoplasts research and their potentials in genetic improvement of seaweeds, along with needs that must to be fulfilled for effective realization of the objectives envisaged for protoplast research.

Expanding the Symbiodinium (Dinophyceae, Suessiales) Toolkit Through Protoplast Technology

“…In summary, we report the first generation of Symbiodinium protoplasts and the first observations of protoplast fusion in any dinoflagellate. Protoplasts have led to major breakthroughs in the biological understanding and genetic enhancement of a vast range of organisms (Bravo and Evans 2011;Carlson 1973;Davey et al 2005;Gietz and Woods 2001;Hopwood 1981;Reddy et al 2007), thus we anticipate our study may also significantly expand the experimental scope of Symbiodinium studies. Considering the recent calls for heightened focus on genetic modification of dinoflagellates (Murray et al 2016) and genetic enhancement of Symbiodinium to sustain the health of coral reefs (van Oppen et al 2015), Symbiodinium protoplast generation and fusion are valuable and timely technologies ready to be utilized.…”

“…In the mid-1900s, introduction of enzymes to digest cells walls (Bachmann and Bonner 1959;Cocking 1960;Eddy and Williamson 1957;Weibull and Bergstr€ om 1958) led to high protoplast viability and yields (Davey et al 2005). Protoplasts have since unlocked major research areas of bacteria, fungi, plants, and algae-notably genetic modification strategies (Carlson 1973;Davey et al 2005;Gietz and Woods 2001;Hopwood 1981;Reddy et al 2007) that have lead to economically valuable agricultural improvements (Bajaj 2012;Bravo and Evans 2011;Wang et al 2013). Absence of the cell wall enables alternative methods for intracellular delivery of foreign DNA into cells such as polyethylene glycol (PEG)-and liposome-mediated transformation that can improve genetic transformation efficiency in certain species (Caboche 1990;Hansen and Wright 1999;Mathur and Koncz 1998;Rakoczy-Trojanowska 2002) and intracellular delivery of RNA-protein complexes for CRISPR-Cas9 genome editing (Woo et al 2015).…”

Cultivar Development of Kelps for Commercial Cultivation—Past Lessons and Future Prospects

“…Chromosome doubling can also be achieved artificially by treatment with chemicals that inhibit spindle formation and nuclear and cell division (e.g., colchicine, oryzaline, trifluralin) (Dhooghe et al, 2011). Moreover, triploid macroalgae, as well as hybrids between species that cannot hybridize by natural means, may be obtained by protoplast fusion (somatic hybridization), followed by plant regeneration (Reddy et al, 2008;Charrier et al, 2015). For recurrent selectionbased development of triploid cultivars, it would be necessary to conduct the genetic improvement before inducing chromosome doubling, while subsequent propagation of the material prior to cultivation would have to be carried out through some form of asexual reproduction.…”

“…The use of random mutagenesis, transgenesis, and precise gene editing in algae was recently reviewed by Gan and Maggs (2017). Protocols for protoplast isolation, culture, and regeneration into plants exist for many species including kelps (Reddy et al, 2008). Random mutations can be induced by physical or chemical mutagens or occur spontaneously and be revealed as somaclonal variation in protoplast cultures and during other natural or manipulated mechanisms of regeneration, such as parthenogenesis and tissue culture (Charrier et al, 2015).…”

Detection of Epigenetic Variations in the Protoplast-Derived Germlings of Ulva reticulata Using Methylation Sensitive Amplification Polymorphism (MSAP)

“…There have been numerous studies on the isolation and regeneration of protoplasts from a wide variety of seaweeds ranging from morphologically simple leafy thallus to anatomically complex thallus (see review Reddy et al 2008). Unlike higher plants, seaweed protoplasts regenerate and differentiate into a full thallus without any amendments of phytohormones to culture medium.…”