Seaweed micropropagation techniques and their potentials: an overview

The seaweed industry worldwide uses 7.5–8.0 million tonnes of wet seaweeds annually with a majority of it derived from cultivated farms, as the demand for seaweed based-products exceeds the supply of seaweed raw material from natural stocks. The main advantage of cultivation is that it not only obviates overexploitation of natural populations but also facilitates the selection of germplasm with desired traits. To enhance the economic prospects of seaweed cultivation, varied practices, such as simple and cost effective cultivation methods, use of select germplasm as seed stock coupled with good farm management practices, etc., are adopted. Nevertheless, in vitro cell culture techniques have also been employed as they facilitate development and propagation of genotypes of commercial importance. There are more than 85 species of seaweeds for which tissue culture aspects have been reported. Although the initial aim of these techniques focuses mostly on genetic improvement and clonal propagation of seaweeds for mariculture, recently the scope of these techniques has been extended for use in bioprocess technology for production of high value chemicals of immense importance in the pharmaceutical and nutraceutical sectors. Recently, there has been a phenomenal interest in intensifying seaweed tissue and cell culture research to maximize the add-on value of seaweed resources. This paper deals with the status of seaweed micropropagation techniques and their applications in the context of the marine biotech industry. Further, it also provides an analysis of the problems to be resolved for removing the barriers that are impeding the true realization of potentials offered by these techniques for sustainable development and utilization of seaweed resources.     

Seaweed protoplasts: status,biotechnological perspectives and needs

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.     

Evaluation of properties of several cheese-ripening fungi for potential biotechnological applications

Strains of Penicillium camemberti and Penicillium roqueforti were tested for properties that could be important for future biotechnological applications of these fungi. Penicillium camemberti CECT 2267 and P. roqueforti NRRL 849 showed the most promising performances in terms of growth, protoplast production, and protoplast regeneration abilities. Transformation of these strains with a plasmid containing gene encoding phleomycin resistance showed that they also have a high transformation frequency. In addition, both strains showed low extracellular proteolytic activity. Thus, these strains have all the characteristics to make them suitable for future genetic improvement, recombinant protein production, and other potential biotechnological applications.     

Barley scutellum protoplasts: Isolation, culture and plant regeneration

Many applications of cereal protoplast culture systems are still limited by the difficulties of regeneration from suspension cells which are the usual protoplast source. The objective of the present study therefore was to investigate the conditions for the development of a culture system for protoplasts capable of plant regeneration isolated directly from immmature scutella of barley. The procedure developed involves a two-stage pre-culture of scutellar tissue, followed by vacuum infiltration with cell wall degrading enzymes and the culture of alginate-embedded protoplasts. The pre-culture of the scutella and the co-cultivation of protoplasts with nurse cells were the most important factors for the success of the culture system, but several other parameters affecting protoplast yield, viability and sustained division were identified, including the developmental stage of the embryo, the use of cold conditioning periods during pre-culture, the composition of the pre-culture and protoplast culture medium, and the embedding matrix. Protoplasts isolated from scutellar tissues of barley cvs Dissa, Clipper, Derkado and Puffin were capable of sustained division in culture. Macroscopic protoplast-derived tissues were obtained in all cultivars, except ev. Puffin, and fertile plants were regenerated from cvs Dissa and Clipper 3–4 months after protoplast isolation. The procedure described provides a novel approach for the isolation of totipotent protoplasts in barley which avoids the need for suspension cultures.     

Biochemical characteristics of cellulose and a green alga degradation by Gilvimarinus japonicas 12-2T,and its application potential for seaweed saccharification

ABSTRACT

Cellulose is one of the major constituents of seaweeds, but reports of mechanisms in microbial seaweed degradation in marine environment are limited, in contrast to the multitude of reports for lignocellulose degradation in terrestrial environment. We studied the biochemical characteristics for marine cellulolytic bacterium Gilvimarinus japonicas 12-2^T in seaweed degradation. The bacterial strain was found to degrade green and red algae, but not brown algae. It was shown that the bacterial strain employs various polysaccharide hydrolases (endocellulase, agarase, carrageenanase, xylanase, and laminarinase) to degrade seaweed polysaccharides. Electrophoretic analysis and peptide sequencing showed that the major protein bands on the electrophoresis gel were homologous to known glucanases and glycoside hydrolases. A seaweed hydrolysate harvested from the bacterial culture was found useful as a substrate for yeasts to produce ethanol. These findings will provide insights into possible seaweed decomposition mechanisms of Gilvimarinus, and its biotechnological potential for ethanol production from inedible seaweeds.     

Plant tissue culture. Biotechnology. Milestones

Summary The progress in the development of the technologies of plant tissue and cell culture over the past four decades has been remarkable. This article covers my personal reflections on the various topics and is based on my involvement in the field during that period. There are three fundamental technologies which constitute most of what is referred to as plant in vitro technologies or tissue culture. The origin and some of the key persons involved in the development of each of these procedures will be discussed. The technology that is most common is growing plant tissue on gel-solidified nutrient media. That technology is being used in the most vital procedures, namely the regeneration of plants from cultured cells. The culture of plant cells in liquid suspension was developed very shortly after that, and has become a very effective technology for plant regeneration by somatic embryogenesis. The method of meristem culture arose out of a need for developing plants that were virus-free. In many species the technique is now being used to produce virus-free crop plants. Another important technology is the culture of anthers and microspores for producing haploid and homozygous plants. Included with plant tissue culture is the development of the plant protoplast and cell fusion technologies for the production of new plant hybrids. The final aspect of the development concerns the integration of tissue culture with molecular genetics, which has developed into the rapidly expanding field of biotechnology.     

Plant cell and biotechnology studies in <Emphasis Type="Italic">Linum usitatissimum</Emphasis> – a review

The species Linum usitatissimum (flax/linseed) has been the focus of a great deal of both basic and applied research effort in plant cell and biotechnology studies in recent years. In this review we consider applications of the techniques of plant biotechnology in this species under several distinct headings. Plant cell and tissue regeneration strategies and applications are discussed, and the applications of the techniques of somatic embryogenesis, protoplast isolation, culture and fusion and cell suspension cultures in this species are described. A major area of study is the use of anther and microspore culture where clear advantages to breeding programmes could be applied. In addition, embryo and ovary culture studies have resulted in significant findings. The more recent technologies of gene transfer and expression by genetic transformation are reviewed, and a section on strategies for improvements in technological quality is also included. Finally we propose conclusions and future prospects for this ancient, but still highly relevant crop.     

Differential response of varying salinity and temperature on zoospore induction, regeneration and daily growth rate in Ulva fasciata (Chlorophyta, Ulvales)

Seaweed cultivation is imperative to augment increasing industrial demand. Ulva fasciata Delile is a potential seaweed for cultivation with applications in food industries. There is a renewed interest in large-scale aquaculture of this species in India due to its envisaged demand in snack food products. In the present study, we have successfully demonstrated the possibility of inducing zoospores in vegetative tissue, effective regeneration and improved growth in this seaweed by manipulating salinity (from 15 to 30 psu) and temperature (from 15 to 35°C). The optimum salinity and temperature requirement for zoospores induction were found to be 15 psu and 25°C, respectively. The quadriflagellate zoospores showed negative phototaxis and the settlement and germination pattern similar to several other green seaweeds. The optimum regeneration (78.53?±?10.05%) was recorded at 25°C and 30 psu salinity. The maximum daily growth rate (16.1?±?0.28%) was at 25°C and 30 psu salinity which corresponded to the field conditions. This method could be further refined at nursery culture to achieve artificial seeding essential for the success of commercial cultivation of this seaweed.     

Algicidal activity of the seaweed Corallina pilulifera against red tide microalgae

Extracts of seaweeds from the coast of Korea have been tested in vitro for algicidal activity against the growth of the toxic microalga Cochlodinium polykrikoides. Blooms of C. polykrikoides and the ensuing mass mortalities of farmed fish and shellfish are an escalating and worrisome trend. Cell growth of C. polykrikoides was inhibited by the addition to the culture medium of several seaweed extracts. Inhibition of growth resulted from methanol-soluble extracts of the seaweeds Corallina pilulifera, Ulva pertusa, Ishige foliacea and Endarachne binghamiae. Growth inhibition also resulted from the water-soluble extract of C. pilulifera. Powder and dry tissue from the seaweed C. pilulifera also inhibited cell growth of C. polykrikoides. The active algicidal products of C. pilulifera showed stable activity when boiled, exposed to light, or when treated under alkaline condition. Corallina pilulifera had no regional and seasonal variations in this algicidal activity. A powder of the seaweed C. pilulifera, the most potent species, showed algicidal activity against several red tide microalgae, especially C. polykrikoides, Gymnodiniummikimotoi, G. sanguineum, Heterosigma akashiwo, Prorocentrum triestinum and Pyraminonas sp. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro biotechnological approaches on <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andrews: advancements and opportunities

In vitro biotechnological advancement of Vanilla plays a major role in germplasm conservation, genetic engineering, accelerated clonal multiplication and production of disease-free plants with enviable aromatic properties. Several attempts have been taken place for the establishment of efficient in vitro protocol for Vanilla in the past few decades. Optimization of various conditions during different phases of micropropagation, for instance development of in vitro aseptic cultures, multiple shoot regeneration, rooting and acclimatization of the plantlets are discussed in this review. In addition to basic micropropagation techniques, various other in vitro biotechnological applications such as clonal fidelity assessment, genetic transformation, synthetic seed technology and cryopreservation are also highlighted. Apart from the existing data, applied aspects like embryo rescue, mutation breeding, genetic engineering, protoplast fusion, somaclonal variation, in vitro enhancement of vanillin production through cell suspension culture, hairy root culture or bioreactors and cryopreservation need to be investigated further. Overall, the current review gives a synopsis on progress and prospect of in vitro culture of Vanilla.     

Factors influencing plant regeneration from protoplasts isolated from long-term cell suspension culture of recalcitrant Indica rice cultivar IR36

Summary Factors influencing successful establishment of embryogenic cell-suspension cultures and plant regeneration from longterm cell suspension-derived protoplasts of the recalcitrant Indica rice cultivar IR36 were studied. The factors included cell and protoplast culture medium, protoplast culture procedure, the source of nurse cells, and the regeneration procedure. Embryogenic cell suspension cultures could only be established from mature seed-derived callus of IR36 in AA-based medium (Müller and Grafe, 1978). Protoplast-derived colonies could be obtained only using the filter-membrane nurse-culture procedure when Lolium multiflorum suspension cells served as nurse, rather than wild rice (Oryza ridleyi) and Japonica rice (Oryza sativa cv. Taipei 309) cells. The utilization of a two-step regeneration procedure led to regeneration of fertile plants from protoplasts isolated from 2-yr-old cell suspensions of IR36, one of the most important but recalcitrant rice cultivars.     

Protoplast isolation and regeneration from <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariales,Rhodophyta)

The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol. The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL^-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained.     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Cell culture in autologous fibrin scaffolds for applications in tissue engineering

In tissue engineering techniques, three-dimensional scaffolds are needed to adjust and guide cell growth and to allow tissue regeneration. The scaffold must be biocompatible, biodegradable and must benefit the interactions between cells and biomaterial. Some natural biomaterials such as fibrin provide a structure similar to the native extracellular matrix containing the cells. Fibrin was first used as a sealant based on pools of commercial fibrinogen. However, the high risk of viral transmission of these pools led to the development of techniques of viral inactivation and elimination and the use of autologous fibrins. In recent decades, fibrin has been used as a release system and three-dimensional scaffold for cell culture. Fibrin scaffolds have been widely used for the culture of different types of cells, and have found several applications in tissue engineering. The structure and development of scaffolds is a key point for cell culture because scaffolds of autologous fibrin offer an important alternative due to their low fibrinogen concentrations, which are more suitable for cell growth.     

Protoplast regeneration from gametophytes and sporophytes of some species in the order Laminariales (Phaeophyceae)

Summary Protoplasts were isolated from sporophytes and from gametophyte cultures of several species in the order Laminariales. For each example, the isolation and culture procedures were investigated systematically, to identify conditions leading to plant regeneration. After dedifferentiation through a filamentous stage, protoplasts isolated from adultLaminaria saccharina sporophytes regenerated polystichous bladelets. In contrast, cells isolated fromLaminaria digitata sporophytes proved recalcitrant in culture, except when the donor plants were undifferentiated sporelings. The most critical factors for protoplast development were the origin of explants, the osmoticum used for cell isolation, cultivation in plain seawater, and the absence of stress during the first two weeks of culture. We also found that protoplast isolation from the sporophytes of members of the Laminariales results in the release of hydrogen peroxide, up to 5–120 μM final concentration in the macerating medium, a characteristic which may be related to protoplast recalcitrance. Protoplasts isolated from the gametophytic phase readily regenerated into normal gametophytes, capable of gametogenesis and producing sporophytes by fertilization.     

Some effects of plant growth regulators on tissue cultures of the marine red alga Agardhiella subulata (Gigartinales,Rhodophyta)

We examined whether auxins and cytokinins, either singly or in combination, stimulate cell division in tissue cultures of a red seaweed. Our experimental model consisted of filamentous and callus-like growths that developed from cross-sectional discs cut from young branches of Agardhiella subulata. Plant growth regulators were added to the medium to give combinations of an auxin with a cytokinin over a range of concentrations (1 µg L^–1 –10 mg L^–1). Several mixtures of auxins and cytokinins, as well as some single auxins, cytokinins and phenolics, stimulated cell division and growth in the tissue cultures beyond that of controls. The treatments that were effective included: phenylacetic acid/zeatin; phenylacetic acid/6-benzylaminopurine; -naphthaleneacetic acid/zeatin; 2,4,5-trichlorophenoxyacetic acid/6-benzylaminopurine; and indoleacetic acid/kinetin. High concentrations of cytokinins (i.e. 10 mg L^–1) inhibited the regeneration of plants in some of the cell cultures. These results provide further evidence that growth regulators can be used for the tissue culture of seaweeds and for the study of developmental phenomena in these plants.     

Production of clonal planting materials from Gracilaria changii and Kappaphycus alvarezii through tissue culture and culture of G. changii explants in airlift photobioreactors

Global demand for seaweed resources has increased due to their emergent use as sources of biopharmaceuticals, nutraceuticals and biofuels. These high-valued products make possible the use of micropropagation techniques that may be more costly than conventional mariculture. This study reports the successful tissue culture of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva and Gracilaria changii (B. Xia & Abbott) Abbott, Zhang and Xia. Callus induction of K. alvarezii was successfully developed following an explant sterilisation protocol. Callus formation and regeneration of K. alvarezii was observed in solidified Provasoli’s enriched seawater medium. Different culture conditions such as agar concentration, growth hormones, nutrients, irradiance and enrichment media were investigated to determine the suitable conditions for explant culture of G. changii. Proliferations of adventitious shoots were induced under the most suitable culture conditions. G. changii explants were successfully cultured in airlift photo-bioreactors, with no decrease in the carbohydrate content in the G. changii explants. This micropropagation technique can provide a useful alternative system for seedling production of economically important seaweeds.     

VARIATION IN ANATOMICAL AND MATERIAL PROPERTIES EXPLAINS DIFFERENCES IN HYDRODYNAMIC PERFORMANCES OF FOLIOSE RED MACROALGAE (RHODOPHYTA)1

Over the last two decades, many studies on functional morphology have suggested that material properties of seaweed tissues may influence their fitness. Because hydrodynamic forces are likely the largest source of mortality for seaweeds in high wave energy environments, tissues with material properties that behave favorably in these environments are likely to be selected for. However, it is very difficult to disentangle the effects of materials properties on seaweed performance because size, shape, and habitat also influence mechanical and hydrodynamic performance. In this study, anatomical and material properties of 16 species of foliose red macroalgae were determined, and their effects on hydrodynamic performance were measured in laboratory experiments holding size and shape constant. We determined that increased blade thickness (primarily caused by the addition of medullary tissue) results in higher flexural stiffness (EI), which inhibits the seaweed’s ability to reconfigure in flowing water and thereby increases drag. However, this increase is concurrent with an increase in the force required to break tissue, possibly offsetting any risk of failure. Additionally, while increased nonpigmented medullary cells may pose a higher metabolic cost to the seaweed, decreased reconfiguration causes thicker tissues to expose more photosynthetic surface area incident to ambient light in flowing water, potentially ameliorating the metabolic cost of producing these cells. Material properties can result in differential performance of morphologically similar species. Future studies on ecomechanics of seaweeds in wave‐swept coastal habitats should consider the interaction of multiple trade‐offs.     

Microtubule organization in the apical cell of Sphacelaria (Phaeophyceae) and its related protoplast

The growth of the filamentous brown alga Sphacelaria depends on a large, strongly polarized, apical cell. The protoplast derived from this cell can be distinguished in a heterogeneous suspension by cytological markers, so it is possible to study development of the cytoskeleton during protoplast isolation and the first steps of regeneration. In the initial cell, microtubules show an asymmetric distribution along the axis; they are mainly located at the distal part around the physodes. After protoplast isolation, this polarity initially seems to be maintained; subsequently, the microtubules radiate from the two centrioles and spread out to the plasmalemma. This experimental model is suitable for investigating the development of the polarity of the initial cell, and the sequence of the first morphogenetic events leading to protoplast regeneration.     

The effects of reactive nitrogen and oxygen species on the regeneration and growth of cucumber cells from isolated protoplasts

The present study investigated changes in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in isolated mesophyll protoplasts and cell cultures of the cucumber Cucumis sativus cv. Marketer. Although only a minor increase in the level of nitrogen oxide (NO) was observed during the first 7 days of culture following protoplast isolation, a substantial accumulation of ROS was detected. Compounds known to modulate endogenous ROS and RNS levels were employed to study their role in cucumber protoplast regeneration and growth. Supplementing the culture medium with the NO donors S-nitrosoglutathione and sodium nitroprusside and the ROS scavenger ascorbate significantly increased protoplast viability and cell density. In contrast, cell density was significantly decreased following the addition of catalase to the medium. Scavenging of ROS and RNS induced the formation of cucumber microcalli, thus suggesting a differential role of NO in the maintenance of cell viability and in the control of cell division. Our findings confirm the crucial role of controlled ROS and RNS production in both protoplast regeneration and cellular growth and differentiation.