Summary of nuclear, mitochondrial and plastid DNA regions used for identification in Rhodophyta .
Seaweeds are a primary source of hydrocolloids, which can be processed into various food additives, cosmetics, and pharmaceuticals. The inability of current commercial seaweed farming projects to meet industrial demands is underscored by a plethora of challenges, which include the lack of high-quality germplasm with the desired cultural characteristics. This chapter describes the current trends in commercial seaweed production and the potential technological advances in production methods and genetic selection strategies, which can be applied to raise the productivity of seaweed farms. Molecular markers have become increasingly relevant to the selection of a diverse range of wild varieties for domestication, and this augurs well for strain identification. The development of high-density linkage maps based on molecular markers offers an avenue for the implementation of molecular breeding strategies based on quantitative trait loci (QTLs). Concurrently, productivity of existing varieties can be enhanced by the analysis of exogenous factors known to affect the growth and survival of tissue-cultured seedlings. The application of photobioreactors for tissue culture is another important development, which will be digressed upon. In addition to this, quality control which focuses on the comparison of chemical and physical qualities of the tissue-cultured and conventional cultivated seaweeds will become increasingly relevant to the development of industry standards for sustainable seaweed production to fulfill the increasing demands of seaweed-related industries.
- Genetic marker
- Marker-assisted selection (MAS)
- Quantitative trait loci (QTLs)
- Tissue culture
1. Seaweeds and their economic importance
Seaweeds are marine macroalgae generally found living in oceans and coastal areas throughout the world. The classification of seaweeds has been typically based on their phenotypical features, which include pigmentation and photosynthetic properties. Since the mid-nineteenth century, seaweeds have been empirically distinguished into three main divisions based on their color: red algae (phylum: Rhodophyta) consist of about 6000 species, brown algae (phylum: Ochrophyta) consist of about 1750 species, and green algae (phylum: Chlorophyta) consist of about 1200 species . Until recently, a wide variety of seaweeds and their products have been studied for their industrial, culinary, and renewable energy applications, which include cosmetics, chemistry, paint, medicine, biofuel, etc. [2, 3]. Increasing global demand for seaweed resources and overexploitation of natural seaweeds have highlighted the need for sustainable seaweed cultivation to significantly increase captive production in mariculture systems.
Among all the seaweed-based products, hydrocolloids viz. carrageenans, agar, and alginates continue to be the principal extracts, which received commercial attention through their application in various industries [4, 5].
2. Current limitations of conventional seaweed breeding and alternative solutions
Emerging applications of hydrocolloids in food industry and other hydrocolloid-related industries have led to an enhancement of the economic values of red algae including
Current practices of seaweed cultivation are predominantly based on traditional methods, where seaweeds are exposed to environmental challenges and pathogens [12, 13]. Most of the seaweeds are cultivated using seedlings produced by vegetative propagation from cultured germplasm. Through this practice, parasites or pathogens from the harvested seaweeds may be re-introduced and subsequently reduced the productivity of the farm. The other logistical problems faced by conventional seaweed farmers include the identification of appropriate sites for farming, labor intensive tasks such as inspection, disease, and seedling losses resulting from extreme weather conditions and water quality. In order to increase the productivity, modern biotechnology via tissue culture can be considered as one of the best options to overcome the conventional breeding challenges including shortage of raw material for planting and seedlings destruction by epiphytes, subsequently facilitate the propagation of high-quality seaweeds [9, 14, 15].
3. Application of molecular markers in seaweed breeding
The establishment of an effective seaweed breeding programs is founded on the selection of strains of seaweed with desirable cultural characteristics such as high growth rates, high carrageenan content, disease resistance, and accelerated growth in response to supplemental fertilizers. Phenotypical identification methods are currently the standards by which specific seaweed strains are selected. Although invaluable, morphological characterization can be time-consuming and requires a high level of expertise to discriminate key morphological features indicative of the seaweed species. In addition to this, the physical characteristics of seaweeds tend to be variable as they are directly influenced by environmental factors . Most of these seaweeds cannot be distinguished on the basis of one or collection of specimen using morphological characters alone, and an exhaustive taxonomic study is essential before the variety can be identified. For example, high morphological plasticity within the Hawaiian
3.1. Genetic marker for identification of commercially important seaweed species
To date, the application of different genes for the genetic identification of seaweed species is widely carried out, where the targeted DNA regions are the nuclear, plastid, and mitochondrial DNA (mtDNA). Most molecular characterization targets seaweeds with economic value such as
Nuclear ribosomal regions, which include sequences of large subunit (28S or LSU), small subunit (18S or SSU), and the intergenic transcribed spacers (ITS1, ITS2), can be served as target sites for molecular markers because the ribosomal DNA (rDNA) genes contain both highly conserved and variable regions that can be used as diagnostic tools for certain organisms . The small subunit (18S) and the large subunit (28S) regions are the most used regions for marker development as they are best suited for inference at high taxonomic levels . However, ITS region is often targeted for intraspecific genetic studies in Chlorophyceae (
mtDNA has a higher mutation rate that gives rise to variation in its DNA sequence . mtDNA is usually used to analyze the phylogenetic relationships of groups within a species or individuals that are closely related . The gene map of mtDNA of the red alga,
Loci derived from the chloroplast genome (cpDNA) can be used for the identification of seaweed species due to the low frequency of structural changes and low sequence evolution rate of cpDNA . The cpDNA loci that are routinely used for seaweed identification are the ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCo) gene, specifically the large subunit of the RuBisCo (
Identification of commercially important seaweeds based on standard DNA barcodes or single marker amplification has proven to be useful as the phenotypic plasticity of the species can confound traditional taxonomic approaches. Table 1 showed the summary of nuclear, mitochondrial, and plastid DNA regions that are used for the identification of rhodophyta . Molecular markers are still valuable, despite the increasing popularity of next generation sequencing technologies, where the identity of an unknown seaweed species can be acquired based on a simple polymerase chain reaction amplification and a single sequence read (two sequence reads if both strands are sequenced). Examples of DNA markers used to identify commercially important seaweeds are given in Table 2 [46, 52–57].
|Small subunit ribosomal DNA||~1800|
|Internal transcribed spacer ribosomal DNA||~650–1100|
|Large subunit ribosomal DNA||~2700|
|Photosystem I P700 chlorophyll a apoprotein A1||~1600|
|Photosystem I P700 chlorophyll a apoprotein A2||1250|
|Photosystem II thylakoid membrane protein D1||~950|
|Plastid LSU (23S) domain V||~370|
|Ribulose-1,5-bisphosphate carboxylase large subunit||~1350|
|Cytochrome c oxidase subunit 1 DNA barcode region||~664|
|Cytochrome c oxidase subunit 1 extended fragment||~1232|
|Cytochrome oxidase subunit 2-3 intergenic spacer||~350–400|
|SSU ribosomal DNA||Forward primer|
|ITS ribosomal DNA||Forward primer|
3.2. Quantitative trait loci (QTLs) for economically important traits
Quantitative trait locus (QTL) analysis is a statistical method that links two types of information, phenotypic data (trait measurements) and genotypic data (usually molecular markers), in an attempt to explain the genetic basis of variation in complex traits . A wide range of agronomic traits in crop plants, including plant productivity and stress tolerance, are complex traits controlled by QTLs . Identification of these QTLs will facilitate the development of novel varieties of seaweeds via conventional breeding approaches as well as genetic engineering. A (QTL) is a region of DNA (the locus) that correlates with variation in a phenotype, which is designated as the “Quantitative Trait”. The QTL contains the genes that encode for the phenotype is tightly linked to the trait over successive generations. Traits of agronomic importance may be controlled by a single gene or in most cases by multiple genes. For example, if we bred a disease tolerant seaweed with a seaweed that yields a high amount of carrageenan, we can generally assume that the resultant hybrid will be a high yielding, disease tolerant variety. This is a generalized assumption based on the hypothesis that the traits are discrete (Figure 1). Markers are generally selected based on regions that flank the quantitative trait (Figure 2) and these can be validated over successive breeding cycles.
However, it should be noted that the majority of traits are not discrete. Analysis of hybrids reveals that segregating populations consist of individuals with continuous traits. In the case of seaweeds, one example of this class of QTL can be the diversity of pigmentation observed in variants of
Commercial breeders adopt a range of approaches in order to integrate diverse traits in order to produce an elite strain with all the desired agronomic traits. This approach entails the selection of wild genotypes purely on the basis of their phenotypic characters. This is an ideal strategy to adopt in the case of seaweeds, where no established elite strains exist. Marker-assisted gene pyramiding aims to produce individuals with superior economic traits according to the optimal breeding scheme, which involves selecting a series of favorite target alleles after cross of base populations and pyramiding them into a single genotype . The strategy for pyramiding is depicted in Figure 3.
An alternative approach which can be undertaken when a grower has an elite strain, which needs to be supplemented with additional traits is backcrossing with the parental genotype over successive generations. This approach (Figure 4) mitigates the likelihood of genes encoding for undesired traits from the wild type from manifesting in the hybrid phenotype.
The first step in developing a linkage map using QTLs involves information pertaining to phenotypes and their association with specific genotypes . Ideally, molecular breeding should commence with the collection of all the available phenotypes from the wild. The second step will be the identification of specific traits, which are associated with each phenotype; once this has been established, the third step will involve the elucidation of genomic information and its conversion into suitable molecular markers. This can be done using available genomic information or on the basis of expressed sequence tags (ESTs) [62–64]. Seaweeds, unlike terrestrial plants, have unusual breeding cycles  and the mechanism need to be established prior to commencing a defined breeding program. New insights into the draft genomes of
Current research work on QTL mapping in seaweeds has focused on commercially cultivated species, which are
3.3. Marker-assisted selection (MAS)
Marker-assisted selection is defined as an indirect selection method of an individual with desired traits in a breeding program based on DNA markers . The important of MAS in a seaweed breeding program is to obtain basic genetic knowledge of the chosen commercially important seaweed. Some desired seaweed traits, such as crop yield or phycocolloid content, may be controlled by one gene or a group of genes. Therefore, it is beneficial to develop markers for a range of commercially important seaweed species to provide the foundation needed for MAS in the seaweed breeding program .
In seaweed farming, specifically for the phycocolloid industry, the desired traits of seaweed would be disease resistance, suitable carrageenan content, high productivity, and yield. These desired characteristic or traits can be genetically simple, where only one gene is involved. However, most economically important crops tend to have traits that are genetically complex, where it is controlled by many genes (QTL) and the environment . For example, Babu et al.  had detected a total of 47 QTLs for drought resistance traits from various plant water stress indicators to increase production and yield of rice in rainfed agriculture ecosystems. To date, there are no reports in the literature of the application of MAS in seaweed breeding program. Recently, Maili et al.  had successfully developed eight out of 112 single loci DNA markers to discriminate between varieties of
4. Mass propagation of seaweed seedlings via tissue culture
Repeated vegetative propagation applied in conventional seaweed cultivation was found decreasing genetic variability of seaweeds and subsequently contribute to the decreased in growth rates and yields and increased susceptibility to diseases . Micro-propagation via tissue culture technology has been proposed as an alternative method compared to conventional breeding to resolve the seedling shortage problem and increase the productivity of seaweed raw materials. Micro-propagation is a versatile tool to produce high number of uniform specimens from selected strains with desirable characteristics and increase seed stock production in shorter period of time . However, challenges including lack of optimized protocols to obtain axenic cultures and regeneration of explants have limited the widespread use of tissue culture technology in commercial seaweed production. The efficacy of seaweed tissue culture is depends on the effective manipulation of endogenous (age, source, developmental stage, and physiological state of explants) and exogenous (media composition, light, salinity, pH, and temperature) factors [80, 81]. Current researches have been strategized to improve the culture conditions for mass production of high-quality laboratory seedlings to enhance the overall productivity of seaweed cultivation [3, 9, 14, 15].
4.1. Preparation of axenic cultures
Explants have to be sterilized in order to obtain the axenic cultures for mass propagation in tissue culture . Seaweed samples collected from the wild are associated with a significant level of biological contamination, which is likely to be commensal or symbiotic; therefore, it is necessary to surface sterilize the explants with general disinfectants as well as targeted antibiotics prior to cultivation. Povidone iodine and alcohol are common disinfectants used for surface sterilization as they have a localized activity compared to the narrow spectrum antibiotics with their functionality limited to specific classes of microbes . Surface sterilization of seaweeds is difficult as they lack of thick protective surface, and therefore, sodium hypochlorite and similar agents can easily damage the tissues especially newly regenerated thallus . Prolonged exposure of explants to excessive disinfectants (e.g., more than 5 min in 2% betadine and more than 72 h in 5% antibiotic mixture) was reported causing patches of damaged surface on thallus and explants .
4.2. Media composition
Culture media commonly used for rapid propagation of rhodophyte are reported to be Provasoli’s enriched seawater (PES) , seawater supplemented with von Stosch (VS) solution , and seawater enriched with half strength “f medium” (F/2) . The selection of culture media for seaweed propagation is highly dependent on the nutrient level, ambient water, and cultured species. The optimized culture medium for economically important
4.3. Plant growth regulators
The addition of plant growth regulators and their role in seaweed tissue culture have been extensively reviewed [92–94]. Cellular competence to plant hormones in cultivated seaweeds is significant only if the cells possess ability to perceive, transduce, and respond to the hormonal signal . The common plant hormones used in seaweed tissue culture are auxins (indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid), cytokinins (benzyadenine, isopentenyladenine, kinetin), and gibberellins (gibberellic acid). The presence of phytoregulators in tissue culture medium is known to be able to stimulate tissue elongation and contributes to the overall plant growth . Generally, auxins are used to increase protein synthesis, induce morphogenesis, and elicit changes in genetic expression of explants , while cytokinins are used to stimulate cell division, enhance metabolic activities, and affect cell differentiation in seaweed tissue cultures . Combination of auxins (α-naphthalenacetic acid and phenylacetic acid) and cytokinins (N6-(2-isopentenyl) adenine and 6-benzylaminopurine) has been reported to induce the highest callus growth in
4.4. Organic fertilizers and biostimulant
The organic requirements for axenic seaweed culture are remained unclear although additions of organic complexes (coconut milk, yeast, and algal extracts) to increase the growth rates of seaweed tissue have been reported . Three commercially available formulated fertilizers and biostimulant in global market for seaweed cultivation are Acadian marine plant extract powder (AMPEP), Gofar600 (GF), and natural seaweed extract (NSE). AMPEP is extracted from
5. Exogenous factors affecting seaweed tissue culture
Studies on optimizing the growth of economically important seaweeds, especially
5.1. Light intensity
Light source is one of the most important parameters to be optimized in seaweed cultivation. The intensity, wavelength, and spectral quality of light, all influence the photosynthetic productivity of algae. Different strains or varieties of seaweed may exhibit different optimum growth range and tolerance to different light resources.
5.2. Aeration activity
Aeration is an ideal method of energy transfer; whereby, atmospheric carbon dioxide is diffused into the culture medium. Continuous aeration is an important process to provide enough carbon dioxide for carbon fixation in seaweed metabolism. Meanwhile, carbon source can be provided in the organic form such as glycerol yet the addition of glycerol in the culture medium was found to reduce the morphogenetic capacity and totipotency of the explants . Continuous aeration (at 30.0 L h−1) was found to significantly enhance the growth of
Salinity is reported to be one of the factors affecting the growth [15, 80] and exerting strong influences on the photosynthetic capacity [117, 118] of the cultured seaweeds. Prolonged exposure to low salinity may induce stress that led to reduced photosynthetic efficiency, inhibited cell division, and subsequently result in stunted growth and declined in growth rate .
The ordinary seawater is slightly alkaline (pH ~8) with bicarbonate ions (HCO3 −) constituted about 91% of total dissolve inorganic carbon (DIC), followed by 8% of carbonate ions (CO3 2−), and 1% of dissolved CO2 . Alterations in seawater pH may vary the equilibrium of carbonate system and change the concentration of inorganic carbon species , subsequently affect the growth of seaweeds which depend on the supply of inorganic carbon for photosynthesis. The pH range for normal growth of most seaweed cultures was reported to be 7–9 with optimum growth in between 8.2 and 8.7 .
6. Optimal growth of seaweed micro-propagules in tissue culture and photobioreactor
In order to maximize the growth of micro-propagules and enhance the productivity of seaweed propagation, incorporation of optimized parameters in their combination in tissue culture system (Figure 5a) and application of photobioreactor with optimal growth condition (Figure 5b) are highly recommended. Maximum DGRs of
Although macroalgal tissue culture is underdeveloped relative to that of land plants, there are more than 85 species of seaweeds from which tissue culture aspects including successful callus formation, plant regeneration, somatic embryogenesis, and thallus development have been reported [92, 127]. Exploitation of seaweed organogenetic potential for the isolation of superior clones has been initiated since the late twentieth century to improve the performance of cultivated species including
7. Acclimatization of tissue-cultured seedlings prior to farming
While the studies of seaweed tissue culture and micro-propagation have been reported from various literatures, information about acclimatization and successful out-planting of tissue-cultured seedlings are still limited to date. Acclimatization to ex vitro conditions (nursery or glasshouse) is necessary to provide a buffer condition to the seaweed cultures for suitable adaptation before their exposure to the complex open sea environment . Direct planting out of tissue-cultured seaweeds without going through the acclimatization phase may cause stress and shock to the seedlings due to sudden changes in environmental conditions . Therefore, effective acclimatization process is considered to be a key element to enhance the survival rate of tissue-cultured seaweeds after they have been out-planted to the open sea.
Transferring of micro-propagated
Furthermore, acclimatization of
8. Quality assessment of tissue-cultured seaweeds
Comparison of the quality between tissue-cultured and conventional cultivated
|Carrageenan properties||Tissue cultured ||Conventional cultivated |
Gel strength (g cm-2)
Sulphate content (%)
|67.3 ± 16.4|
1280.0 ± 25.0
703.5 ± 14.1
34.2 ± 10.9
|51.5 ± 21.0|
87.8 ± 20.9
288.3 ± 19.3
7.5 ± 6.7
In terms of other chemical composition, tissue-cultured