**1. Introduction**

Frederick Griffith reported the discovery of transformation in 1928 [1]. Since a harmless strain of *Streptococcus pneumoniae* was altered to a virulent one by exposure to heat-killed virulent strains in mice, Griffice hypothesized that there was a transforming principle in the heat-killed strain. It took sixteen years to indentify the nature of the transforming principle as a DNA fragment released from virulent strains and integrated into the genome of a harmless strain [2]. Such an uptake and incorporation of DNA by bacteria was named transformation. Remarkably, an epoch-making technology in the form of artificial transformation protocol for the model bacterium *Escherichia coli* was established by Mandel and Higa in 1970 [3], which stimulated the development of artificial genetic transformation systems in yeasts, animals and plants. In plants, genetic transformation is a powerful tool for elucidating the functions and regulatory mechanisms of genes involved in various physiological events, and special attention has been paid to plant improvements affecting food security, human health, the environment and conservation of biodiversity. For instance, researchers have focused on the creation of organisms that efficiently produce biofuels and medically functional materials or carry stress tolerance in the face of uncertain environmental conditions [4-6].

Although the first success in the creation of transgenic mouse was carried out by injecting the rat growth hormone gene into a mouse embryo in 1982 [7], the protocol for artificial genetic transformation in plants was established earlier than that in animals. Following the discovery of the soil plant pathogen *Agrobacterium tumefaciens*, which is responsible for producing plant tumors, in 1907 [8], it was found that the tumor-inducing agent is the Ti plasmid containing T-DNA, a particular DNA segment containing tumor-producing genes that are transferred into the nuclear genome of infected cells [9]. By replacing tumor-producing genes by a gene of interest within the T-DNA region, infection of *A. tumefaciens* carrying a modified Ti plasmid results in insertion of a DNA fragment containing the desired genes into the genomes of plants by genetic recombination. Since the report of this protocol in the early 1980s [10,11], transfor‐

© 2013 Mikami; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Mikami; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

mation mediated by *A. tumefaciens* has become the most commonly used method to transmit DNA fragment into higher plants [12].

[29,30], I focus here on the current progress in red algal transient transformation with sum‐

Current Advances in Seaweed Transformation

http://dx.doi.org/10.5772/52978

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As far as I know, Donald P. Cheney is the pioneer in researching red algal transformation. He and his colleague performed transient transformation of the red alga *Kappaphycus alvarezii* using particle bombardment [25], which was the first report about the transient transformation of seaweeds (Table 1). In this case, the *Escherichia coli uidA* gene encoding β-glucuronidase (GUS) was expressed as a reporter under direction of the cauliflower mosaic virus (CaMV) 35S promoter (*CaMV 35S-GUS* gene). Since the GUS expression can be visualized as a blue color following treatment with X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) and also be quantified by fluorometric analysis [31,32], this reporter gene is widely used in land green plants having no background of the GUS activity [33,34]. In addition, the *CaMV 35S* promoter is heterologously used in land green plants as a strong constitutive and non-tissue-specific transcriptional regulator [35,36]. Therefore, it is a natural choice for the selection of the

To date, studies have been mainly focused on *Porphyra* species because of their economical values. As shown in Table 1, expression of the *CaMV 35S-GUS* gene was previously observed in *P. miniata*, *P. tenera* and *P. yezoensis*[37-42], all of which were performed by electoroporation using protoplasts. Kuang et al. [38] also tested the particle bombardment of the *CaMV 35S-GUS* gene in *P. yezoensis* and got positive results. Moreover, the availability of mammaliantype simian virus 40 (*SV40*) promoter was reported to express the *E. coli lacZ* reporter gene, encoding β-galactosidase cleaving colorless substrate X-gal (5-bromo-4-chloro-3-indolyl-βgalactopyranoside) to produce a blue insoluble product [43], in *P. haitanensis*, *Gracilaria*

As noted above, pioneer experiments of red algal transient transformation were performed using plant viral *CaMV 35S RNA* and animal viral *SV40* promoters in combination with *GUS* and *lacZ* reporter genes (Table 1). The *CaMV 35S* and *SV40* promoters are typical eukaryotic class II promoters with a TATA box and thus are generally employed to drive transgenes in dicot plant and animal cells, respectively [46,47]. However, we have found that the TATA box is not usually found in the core promoters of *P. yezoensis* genes (unpublished observation), and we thus proposed that there were differences in the promoter structure and transcriptional regulation of protein-coding genes between red algae and dicot plants. Indeed, we recently observed quite low activity of the *CaMV 35S* promoter and the *GUS* reporter gene in *P. yezoensis* gametophytec cells [29,30,48]. These observations are completely opposite from the results in previous reports using the *CaMV 35S* promoter [25,37-41]. As a result, the transient transformation system in red seaweeds has recently been improved by resolving this problem.

marizing pioneer and recent studies related to seaweed genetic transformation.

*CaMV35S-GUS* gene by pioneers for initial trials of seaweed transformation.

*chagii* and *K. alvarezii* by electroporation or particle bombardment [44,45].

**2.2. Recent improvement of the transient transformation system in** *Porphyra*

**2. Transformation in red seaweeds**

**2.1. Pioneer studies for transient transformation**

Since not all plant cells are susceptible to infection by *A. tumefaciens*, other methods were developed and are available in plants. Particle bombardment [13], which is also referred to as microprojectile bombardment, particle gun or biolistics, makes use of DNA-coated gold particles, which enables the transient and stable transformation of almost any type of cell, regardless of rigidity of the cell wall, and is thus extensively used for land plants. For proto‐ plasts, electroporation is well employed, for which a high-voltage electrical pulse temporarily disturbs the phospholipid bilayer of the plasma membrane, allowing cells to take up plasmid DNAs [14,15]. In addition, the polyethylene glycol (PEG)-mediated transformation system is also thought to affect the plasma membrane and induce the uptake of DNAs into cells [15,16] and is almost exclusively applied with the moss *Physcomitrella patens* and liverwort *Marchantia polymorpha* [17,18]. Therefore, several kinds of genetic transformation methods are now available in land green plants.

Seaweeds are photosynthetic macroalgae, the majority of which live in the sea, and are usually divided into green, red and brown algae. Traditionally, all classes of seaweeds are known as human foods especially in Asian countries; for instance, red algae are known as Nori and brown algae are called Konbu and Wakame in Japan. In addition, red and brown algae are utilized as the sources of industrially and medically valuable compounds such as phycoery‐ thrin, n-3 polyunsaturated fatty acids, porphyran, ager and carrageenan from red algae, and fucoxantine, fucoidan and alginate from brown algae [19-22]. Thus, to make new strains carrying advantageous characteristics benefiting industry and medicine, researchers have worked hard since the early 1990s to establish methods of genetic transformation in seaweeds [20,23,24]. However, the process is very difficult, and most of the early studies were reported in conference abstracts without the accompanying manuscript publication [25-28]. This situation has hampered us from gaining an understanding of gene functions in various physiological regulations and also a utilization of seaweeds in biotechnological applications.

Transformation can be divided into genetic (stable) and transient transformations under the control of the genes introduced into cells. In genetic transformation, genes introduced by genetic recombination are maintained in the genome through generations of cells, whereas in transient transformation, rapid loss of introduced foreign genes is usually observed. Estab‐ lishing the genetic transformation system requires four basal techniques: an efficient gene transfer system, an efficient expression system for foreign genes, an integration and targeting system to deliver the foreign gene into the genome, and a selection system for transformed cells. It is notable that the transient transformation system is completed by the first two of the four required systems. In this respect, the development of an efficient and reproducible transient transformation system is the most critical step to establishing a genetic transforma‐ tion system in seaweeds.

The current progress in establishing of both transient and genetic transformation systems in macroalgae is reviewed here. Although high-quality review articles for algal transformation have been published previously [20,23,24], I believe addressing the recent activity in seaweed transformation provides valuable information for seaweed molecular biologists and breeding scientists. Since considerable technical improvement was recently made in red seaweeds [29,30], I focus here on the current progress in red algal transient transformation with sum‐ marizing pioneer and recent studies related to seaweed genetic transformation.
