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Transient Transformation of Red Algal Cells: Breakthrough Toward Genetic Transformation of Marine Crop Porphyra Species

Written By

Koji Mikami, Ryo Hirata, Megumu Takahashi, Toshiki Uji and Naotsune Saga

Submitted: 11 November 2010 Published: 06 September 2011

DOI: 10.5772/20982

From the Edited Volume

Genetic Transformation

Edited by María Alvarez

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1. Introduction

Genetic transformation is a powerful tool not only for elucidating the functions and regulatory mechanisms of genes involved in various physiological events but also for establishing organisms that efficiently produce biofuels and medically functional materials or carry stress tolerance in face of uncertain environmental conditions (Griesbeck et al., 2006; Torney et al., 2007; Bhatnagar-Mathur et al., 2008). Eukaryotic algae classified into microalgae and macroalgae (seaweeds) are highly diverse photosynthetic plants that are utilized as human food and animal feed as well as sources of valuable compounds such as fatty acids, pigments, vitamins and polysaccharides (Hallmann, 2007; Sugawara et al, 2011). Because of their importance in ecology and industry, algae are now considered promising organisms for economical and industrial applications and are thus a target of genetic transformation (Walker et al., 2005; Hallmann, 2007; Blouin et al., 2011). To date, genetic transformation has succeeded in microalgae; thus, stably transformed microalgae are now employed to produce recombinant antibodies, vaccines, or bio-hydrogen as well as to analyze the gene functions targeted for engineering (Sun et al., 2003; Zorin et al., 2009; Specht et al., 2010; Wu et al., 2010). However, it has proven difficult to establish transgenic macroalgae, which has hampered understanding their gene functions in various physiological regulations and also their utilization in biotechnological applications.

The red macroalga Porphyra yezoensis is the most popular sea crop in Japan with economical and pharmaceutical importance as the source of foods such as “nori” and pharmacological regents such as the sulfated polysaccharide “porphyran”, which has anti-tumor and anti-allergic activities (Noda et al., 1990; Zemke-White & Ohno, 1999; Ishihara et al., 2005). Recently, non-beneficial climate change due to global warming has decreased the quality and yield of P. yezoensis at algal farms by enhancing discoloration and red rot disease caused by fungal infection (Kakinuma et al., 2008; Park et al., 2000). Although breeding of P. yezoensis by traditional selection and crossing methods has progressed to obtain strains showing high growth rates and economically valuable characteristics, these methods have limitations in terms of the isolation of strains carrying heat-stress tolerance or disease resistance. Development of a molecular breeding method based on genetic transformation is expected to resolve this problem using knowledge about gene functions in P. yezoensis.

Despite the difficulty of genetic transformation, genetic information about P. yezoensis is now accumulating by a collection of ESTs from leafy gametophytes and filamentous sporophytes (Nikaido et al., 2000; Asamizu et al., 2003) and also by ongoing full-length cDNA and whole genome analyses. Genetic information provides a sophisticated way to isolate P. yezoensis genes of interest; however, utilization of this knowledge – in both basic biological research and molecular breeding of P. yezoensis – is hindered by the lack of a genetic transformation system.

Given this situation, it is clear that the development of a genetic transformation system for P. yezoensis is critical for sustainable production as well as the enhancement of biological study. In our laboratory much effort has been expended on the establishment of a genetic transformation system; as a result we have identified several factors preventing the establishment of a genetic transformation system in macroalgae: for example, the lack of efficient methods for foreign gene transfer and expression systems and the absence of valid selectable markers for isolation of genetically transformed cells. Thus, it is considered worthwhile to develop a transient gene expression system, because some of the above inhibiting factors, such as defects in the transfer and expression of foreign genes, could be removed by using non-integrated plasmids containing genes for expression. Since we have recently succeeded in developing an efficient transient gene expression system in Bangiophyceae including Porphyra species, the establishment and successful application of this system in a molecular biological study are summarized here.


2. Development of a transient gene expression system in P. yezoensis cells

2.1. Problems in previously published experiments

Expression of foreign genes in macroalgae has already been attempted using bacterial lacZ (β-galactosidase) and uidA (β-glucronidase, GUS) genes under direction by promoters of the cauliflower mosaic virus 35S RNA (CaMV 35S) and simian virus 40 (SV40) genes (Kübler et al., 1994; Kuang et al., 1998; Huang et al., 1996; Gan et al., 2003; Jiang et al., 2003). 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 (Kang & An, 2005; Funabashi et al., 2010). However, we have observed a quite low activity of the CaMV 35S promoter in P. yezoensis cells as shown in Figs. 1 and 2 (Fukuda et al., 2008), which is strongly supported by the fact that the TATA box is not found in the core promoters of P. yezoensis genes (unpublished observation), suggesting differences in the promoter structure and transcriptional regulation of protein-coding genes between red algae and dicot plants. We believe that the same could be predicted for the SV40 promoter. Thus, it seems to be necessary to reconfirm the results of reporter expression using the CaMV 35S and SV40 promoters in red algae like P. miniata, P. tenera, Kappaphycus alvarezii and Gracilaria changii in previously published literatures (Kurtzman & Cheney, 1991; Kübler et al., 1994; Okauchi & Mizukami, 1999; Cheney et al., 2001; Gan et al., 2003).

In addition, the use of the GUS reporter gene is also a problem, since we have already confirmed that the GUS reporter gene is not functional in red algal cells by histochemical analysis and enzymatic activity test (see below). This is supported by previous reports indicating background levels of the GUS enzymatic activity in red and brown macroalgal cells when the GUS reporter gene was driven by CaMV 35S and endogenous beta-tubulin promoters (Liu et al., 2003; Gong et al., 2005).

According to the above findings, previous reports of successful expression of the GUS reporter gene under the direction of the CaMV 35S and SV40 promoters are not likely to be replicated. Thus, an important step is to enable the effective expression of the GUS reporter genes in P. yezoensis cells, which should eventually lead to the establishment of a transient gene expression system via the development of procedures for introduction and expression of foreign genes as described below.

2.2. Codon-optimization of the reporter gene

Inefficient expression of foreign genes in algal cells is often due to the incompatibility of the codon usage in their coding regions, inhibiting the effective use of transfer RNA by rarely used codons in the host cells and thus decreasing the efficiency of the translation (Mayfield & Kindle, 1990). In this respect, successful development of foreign gene expression in green alga Chlamydomonas reinhardtii may be an important clue to overcoming the deficiency of the GUS reporter gene in P. yezoensis cells. For example, a low expression level of the gene encoding a green fluorescent protein (GFP) from the jellyfish Aequorea victoria, whose codon usage is rich in AT residues, was dramatically increased by adjustment of the GC content of the coding region corresponding to the codon usage in C. reinhardtii nuclear genes (Fuhrmann et al., 1999). In addition, the synthetic luciferase gene whose codon usage was adapted to that in nuclear genes from C. reinhardtii was also expressed efficiently, although the original luciferase gene had less efficiency in C. reinhardtii cells (Fuhrmann et al., 2004; Ruecker et al., 2008; Shao & Bock, 2008). In fact, EST analysis of P. yezoensis reveals that the codons in P. yezoensis nuclear genes frequently contain G and C residues especially in their third letters, by which the GC content reaches a high of 65.2% (Nikaido et al., 2000). Since bacterial GUS and lacZ reporter genes have AT-rich codons, the incompatibility of codon usage might be responsible for the poor translation efficiency of foreign genes in P. yezoensis cells. We therefore postulated that modification of codon usage in the GUS gene would enable the efficient expression of this gene in P. yezoensis cells. This possibility led us to modify the codon usage of the GUS reporter gene to that in the nuclear genes of P. yezoensis by introducing silent mutations in the GUS coding region, by which unfavorable or rare codons in the GUS reporter gene were exchanged for favorable ones without affecting amino acid sequences. In the resultant artificially codon-optimized GUS gene, designated PyGUS, the GC content was increased from 52.3% to 66.6%.

To transfer the plasmid into cells, we employed particle bombardment, also referred to as micro-projectile bombardment, particle guns, or biolistics, which are among the most popular techniques for achieving nuclear and organelle transformation not only for algae but also for plants, fungi, insects, animals and even bacteria. In this method, DNA-coated heavy-metals, usually gold or tungsten particles, are introduced into cells by helium pressure, which enables the introduction of foreign DNA regardless of whether the cell has a cell wall. This method has been successfully applied in many algal species such as C. reinhardtii (Kindle et al., 1989), Volvox carteri (Schiedlmeier et al., 1994), Dunaliella salina (Tan et al., 2005), and Phaeodactylum tricornutum (Apt et al., 1996).

When the PyGUS gene directed by the CaMV 35S promoter was introduced into P. yezoensis gametophytic cells by particle bombardment, very low but significant expression of the PyGUS gene was observed by histochemical detection and GUS activity test (Figs. 1 and 2), indicating the enhancement of the expression level of the GUS reporter gene. Optimization of the codon usage of the reporter gene is therefore one of the important factors for successful expression in P. yezoensis cells (Fukuda et al., 2008; Mikami et al., 2009; Takahashi et al., 2010; Uji et al., 2010; Hirata et al., 2011).

2.3. Employment of an endogenous strong promoter

A low level of PyGUS expression under the direction of the CaMV 35S promoter was thought to be caused by the low activity of this promoter in P. yezoensis cells. Although the CaMV 35S promoter is widely used as a heterologous promoter because of its strong, constitutive and non-tissue-specific transcriptional function in dicot plant cells (Louis et al., 2010; Wally & Punja, 2010), it is well known that this promoter has very low activity in cells of green algae such as Dunaliella salina (Tan et al., 2005), Chlorella kessleri (El-Sheekh, 1999), and Chlorella vulgaris (Chow & Tung, 1999) and no activity in C. reinhardtii cells (Day et al., 1990; Blakenship & Kindle, 1992; Lumbreras et al., 1998). However, employment of strong endogenous promoters such as the β-Tub, RbcS2 and Hsp70 promoters has achieved efficient expression of foreign genes in these algae (Davies et al., 1992; Stevens et al., 1996; Schroda et al., 2000; Walker et al., 2004). In addition, many endogenous promoters have been developed and successfully used for foreign gene expression in cells of some microalgae, such as the diatom Phaeodactylum tricornutum and the Chlorarachniophyte Lotharella amoebiformis (Zaslavskaia et al., 2000; Hirakawa et al., 2008). Therefore, it is possible that recruitment of suitable strong promoters could result in efficient expression of the PyGUS reporter gene in P. yezoensis cells.

Since we found strong expression of P. yezoensis genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin 1 (Act1) by reverse transcription-PCR (RT-PCR) (see Fig. 5), the 5’ upstream regions of these genes were isolated from the P. yezoensis genome and fused with the PyGUS gene to construct the expression plasmids pPyGAPDH-PyGUS and pPyAct1-PyGUS in addition to the pPyGAPDH-GUS and pPyAct1-GUS plasmids. When pPyGAPDH-GUS and pPyGAPDH-PyGUS were introduced into gametophytic cells by particle bombardment, cells expressing the reporter gene and GUS enzymatic activity were dramatically increased using the PyGUS gene as a reporter, although the original GUS gene was not efficiently expressed (Figs. 1 and 2). The same results were obtained using pPyAct1-PyGUS and pPyAct1-GUS plasmids (Figs. 1 and 2). Thus, the combination of endogenous strong promoters with codon optimization brings a synergistic effect on the efficiency of the expression of the reporter gene. These results indicate that employment of an endogenous strong promoter is another important factor necessary for high-level expression of the reporter gene in P. yezoensis cells (Fukuda et al., 2008).

It is noteworthy that significant expression of PyGUS under the direction of the PyAct1 and PyGAPDH promoters was also observed in sporophytic cells in P. yezoensis, when the expression plasmid was introduced using particle bombardment (Fig. 3A). However, the PyGUS enzymatic activity was much lower than those in gametophytic cells (Fig. 3B). This difference was thought to be mainly due to the difference in DNA-transferring efficiency based on the difficulty of targeting the cell for gene transfer in sporophytes of thin filamentous form by particle bombardment.

Our transient gene expression system with the above methodological imorovement is represented schematically in Fig. 4.

2.4. Evaluation of gene transfer methods

Our initial development of transient expression of the PyGUS reporter gene in P. yezoensis cells depended on the use of particle bombardment, in which the gene transfer rate was usually 103 per μg of DNA. However, it is necessary to increase the efficiency of the foreign gene expression to 106 to 107 per μg of DNA, because the establishment of a genetic

Figure 1.

Effcts of the strength of promoters and codon optimization of the reporter gene on the efficiency of the foreign gene expression in P. yezoensis gametophytic cells. The 835 bp CaMV 35S (green), 2.3 kb PyGAPDH (orange) or 3.0 kb PyAct1 (red) promoter was fused to the coding region of native (sky blue) or codon-optimized PyGUS (blue), which resulted in the construction of p35S-GUS, p35S-PyGUS, pPyGAPDH-GUS, pPyGAPDH-PyGUS, pPyAct1-GUS and pPyAct1-PyGUS. The tNOS (yellow) indicates the nos gene terminator (253 bp). Gametophytic cells of P. yezoensis were transiently transformed with each expression plasmid by particle bombardment and then examined by histochemical staining 48 h after transient transformation. Scale bar corresponds to 100 μm.

Figure 2.

Confirmation of the synergistic effects of the use of strong promoters and codon optimization on the efficiency of foreign gene expression in P. yezoensis gametophytic cells. The GUS or PyGUS enzymatic activity (dark gray) or the number of GUS- or PyGUS-expressing cells (light gray) in gametophytic cells of P. yezoensis were compared. The inner box shows a magnified figure of p35S-GUS (pBI221) and p35S-PyGUS.

Figure 3.

Availability of PyGUS for transient gene expression in sporophytic cells of P. yezoensis. (A) Sporophytic cells expressing PyGUS by transient transformation with pPyAct1–PyGUS and stained with X-gluc solution after 48 h of bombardment. Upper and lower panels show low- and high-magnification images, respectively. Scale bar corresponds to 100 μm (upper) and 10 μm (lower). (B) Comparison of GUS activity between gametophytic and sporophytic cells of P. yezoensis when pPyAct1-PyGUS or pPyGAPDH-PyGUS was introduced.

Figure 4.

Representation of the transient gene expression system established in P. yezoensis.

transformation system requires a large number of gene-transferred cells, a small portion of which are stably transformed by integration of the foreign gene into the genome. Thus, it is important to develop an efficient method of gene transfer into P. yezoensis cells toward the establishment of a stable transformation system via genetic recombination. However, our preliminary experiments with the glass beads method (Kindle, 1990; Feng et al., 2009), PEG method (Ohnuma et al., 2008) and magnetofection (Plank et al., 2003; Svingen et al., 2009) resulted in failure to introduce foreign genes into P. yezoensis. Thus, particle bombardment appears to be the only method available for transferring foreign genes into P. yezoensis cells at present, and further development of a gene transfer method to acheive very high efficient expression of the PyGUS reporter gene is required.

In fact, there have been several reports on DNA transfer into Porphyra cells using electroporation (Kübler et al., 1994) and Agrobacterium-mediated transformation (Cheney et al., 2001). Although the experiments in these reports had problems in the efficiency of the reporter gene expression based on the use of the CaMV 35S promoter and original GUS reporter gene, they are valuable as points of comparison for the increase in efficiency of gene transfer and expression by using, for example, the PyAct1-PyGUS or PyGAPDH-PyGUS reporter gene.


3. Application of the transient gene expression

3.1. Promoter analysis by PyGUS reporter system

Elucidation of the regulatory mechanisms of gene expression is crucial to understanding the molecular mechanisms governing plant stress responses, leading to enhancement of stress tolerance in plants through genetic transformation (Shinozaki & Yamaguchi-Shinozaki, 2007; Bhatnagar-Mathur et al., 2008). The transient gene expression system has been successfully applied to analyze promoters of stress-inducible genes to identify stress-responsible cis-regulatory elements in higher plants. For example, cold- and drought-responsible elements have been identified using the GUS reporter system in barley and Arabidopsis thaliana (Brown et al., 2001; Sakamoto et al., 2004). These successes are based on the quantitative correlation between promoter strength and reporter gene expression. Confirmation of this point in the transient gene expression system is indispensable for further analysis of stress-inducible genes in P. yezoensis.

To check the correlation between the promoter strength and expression level of the PyGUS reporter in P. yezoensis cells, mRNA expression levels of the actin 1 (PyAct1), GAPDH (PyGAPDH), Na+-ATPase (PyATP1A) and transcription elongation factor 1 (PyElf1) genes were compared with PyGUS enzymatic activities derived from expression plasmids of the PyGUS gene under the direction of these promoters. The result indicated a strong correlation between the mRNA levels of these genes analyzed by RT-PCR and GUS enzymatic activities via expression of the PyGUS reporter gene directed by the corresponding promoter (Fig. 5), demonstrating the applicability of our transient gene expression system with the PyGUS gene to promoter analyses of P. yezoensis genes. Identification of cis-regulatory elements by promoter analysis is now underway for stress-inducible genes using the transient PyGUS reporter expression system.

Figure 5.

Correlation of mRNA abundance and the PyGUS enzymatic activity in transient gene expression in P. yezoensis cells. (A) Comparison of mRNA abundance for PyAct1, PyATP1A, PyElf1and PyGAPDH genes in gametophytic cells. RNA samples were separately prepared from gametophytic cells transiently transformed by these genes and used for quantitative RT-PCR with gene-specific primer sets. The relative abundance of each mRNA is presented, with the abundance of the PyAct1 mRNA set as 100. (B) Comparison of the GUS enzymatic activities under the direction of different endogenous promoters. Gametophytic cells were transiently transformed with expression plasmids of the PyGUS gene under the direction of the PyAct1, PyATP1A, PyElf1and PyGAPDH promoters by particle bombardment. The GUS enzymatic activities were then examined 48 h after transient transformation. Data shown are means ± SD from three independent experiments.

3.2. Development of fluorescent protein reporter system

Since histochemical and quantitative enzymatic analyses in the PyGUS reporter system require cell killing, monitoring of the reporter expression during development or response to environmental stress in living cells is impossible to analyze. However, living cell imaging to monitor gene expression is possible using fluorescent proteins without additional substrates and cofactors (Ehrhardt, 2003). Indeed, fluorescent proteins are usually employed as useful tools to visualize protein localization in living cells in land plants (Buschmann et al., 2010; Kokkirala et al., 2010; Meng et al., 2010; Pribat et al., 2010).

We first examined the usability of fluorescent proteins as reporters in P. yezoensis cells, since the expression of fluorescent protein has not yet been demonstrated in red algal cells. Table 1 lists fluorescent protein genes such as green fluorescent protein (GFP) and its variants cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) from various organisms including the jellyfish Aequorea victoria, reef coral Zoanthus sp. and copepoda Pontellina plumata, whose codon usage is biased to be GC-rich by humanization. When these genes were expressed in P. yezoensis cells under the direction of the PyAct1 promoter, a fluorescent signal was clearly observed in every transiently transformed cell. Fig. 6A represents the expression of AmCyan and ZsGreen as examples. Interestingly, the gene encoding plant-adapted GFP(S65T) (Niwa et al., 1999) under the direction of the PyAct1 promoter also produced the GFP signal (Fig. 6A). Since the GC contents of humanized fluorescent proteins and sGFP(S65T) are as high as 60% (Table 1), a GC content of 60% appears to be sufficient for efficient expression of foreign genes in P. yezoensis cells.

Applicability of the fluorescent protein system to visualize protein localization in P. yezoensis cells has already been demonstrated. For example, as shown in Fig. 6B, plasma membrane localization of Pleckstrin homology (PH) domains from human phospholipase Cδ1 was demonstrated using AmCyan (Mikami et al., 2009) and nuclear localization of P. yezoensis transcription factor PyElf1 was also confirmed using AmCyan and ZsGreen (Uji et al., 2010). Thus, these applications will provide new opportunities for analyzing subcellular localization of various genes from P. yezoensis.

Table 1.

Properties of humanized and plant-adapted fluorescent proteins whose expression was confirmed in P. yezoensis.

Figure 6.

Successful expression of fluorescent proteins in P. yezoensis gametophytic cells. (A) Expression of humanized fluorescent proteins and plant-adapted sGFP(S65T). Gametophytic cells were transiently transformed with expression plasmids containing the AmCyan, ZsGreen or sGFP(S65T) gene under the control of the PyAct1 promoter by particle bombardment, 48 h after which cells were examined by fluorescent microscopy. Upper and lower panels show bright field and fluorescence images, respectively. The scale bar corresponds to 5 μm. (B) Visualization of subcellular localization with humanized fluorescent proteins in P. yezoensis cells. Expression plasmids pPyAct1-PLCδ1PH-AmCyan (a and b), pPyAct1-AmCyan-PyElf1 (c and d) and pPyAct1-ZsGreen-PyElf1 (e and f) were introduced into gametophytic cells of P. yezoensis by particle bombardment. Gametophytes were examined by fluorescent microscopy after 48 h of transient transformation. Plasma membrane localization of AmCyan fused with the PH domain from human phospholipase δ1 in gametophytic cells (a, bright field image; b, fluorescent image) and nuclear localization of AmCyan and ZsGreen fused with PyElf1 in gametophytic cells (c and e, bright field images; d and f, fluorescent images) are presented. The scale bar corresponds to 5 μm.

3.3. Application of PyGUS and sGFP(S65T) reporter systems in other red macroalgae

Since red macroalgae include a number of industrially important species such as Porphyra as the source of the food known as Nori, Gracilaria and Gelidium as sources of agar and Chondrus and Kappaphycus as sources of carrageenan, the establishment of a genetic manipulation system for these algae is eagerly anticipated. However, a transient gene expression system has not yet been developed in these red macroalgae other than P. yezoensis. In fact, EST analysis of P. haitanensis revealed that the GC content of the ORFs in this alga was as high as that in P. yezoensis, and the GC content at the third position of the triplets was significantly higher than those at the other two positions, which is consistent with the result reported in P. yezoensis (Fan et al., 2007). Similarly, analysis of the GAPDH gene from Chondrus crispus showed a high GC content (approximately 60%) in the cording region (Liaud et al., 1993). Thus, it is possible that the PyGUS gene could be commonly functional in red algae as a reporter of the gene expression. Indeed, applicability of the PyGUS gene in P. tenera has recently been confirmed by another laboratory (Son et al., 2011).

The applicability of the P. yezoensis transient gene expression system was examined using the PyGUS and sGFP(S65T) reporter genes under the direction of the PyAct1 promoter in the red macroalgae listed in Table 2. Good expression of PyGUS and sGFP(S65T) was observed in Bangiophyceae including genus Porphyra and Bangia, whereas no expression of the reporter genes was seen in Florideophyceae (Table 2). Thus, it was concluded that the transient gene expression system developed in P. yezoensis is widely applicable in Bangiophycean red algae (Hirata et al., 2011).

The number of reporter-expressing cells varies among species (Table 2; Hirata et al., 2011), suggesting the possibility of the improvement of the gene transfer and expression systems in each species using respective endogenous strong promoters to drive the expression of the PyGUS and the sGFP(S65T) reporter genes. Similarly, expression of these reporter genes in Florideophycean algae seems to be possible if suitable endogenous promoters can be employed. Establishment of an efficient transient gene expression system will stimulate future development of stable transformation systems in various red macroalgae, enabling more effective utilization of them for industrial purposes.

Table 2.

Comparison of the number of reporter-expressing cells among red macroalgae after transfection of the pAct1-PyGUS or pAct1-sGFP(S65T) plasmid. The data shown are means ± S.D. from three independent experiments.


4. Conclusion

Genetic transformation is indispensable both to elucidation of gene function and to molecular breeding of crop plants. Our major goal is to establish a stable transformation system to produce Porphyra species carrying biotic and abiotic stress tolerances. For this purpose, our recent development of a transient gene expression system for Bangiophycean algae, by which the lack of transfer and expression systems for foreign genes was resolved, is the first breakthrough toward the establishment of a genetic transformation method in red macroalgae. We now know that the construction of an expression plasmid requires both optimization of codon usage in coding regions of the reporter gene and recruitment of endogenous strong promoters; also that particle bombardment is the proven method of gene transfer into red algal cells. The next steps are therefore development of targeted or random integration systems of foreign genes into the genome and selection of marker genes useful for isolation of transformed cells. Resolution of these methodological problems could contribute to accelerating progress in molecular biological research on physiological regulations that will be useful for the industrial production of transgenic red macroalgae.



This work was suported in part by a grant from the Regional Innovation Cluster Program (Global Type) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan to N.S. and the Hokusui Foundation to K.M.


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Written By

Koji Mikami, Ryo Hirata, Megumu Takahashi, Toshiki Uji and Naotsune Saga

Submitted: 11 November 2010 Published: 06 September 2011