Chapter 13 Current Advances in Seaweed Transformation

Title Current Advances in Seaweed Transformation Author(s) Mikami, Koji Citation Edited by Gandhi Rádis Baptista, An Integrated View of the Molecular Recognition and Toxinology : From Analytical Procedures to Biomedical Applications. Intech, 2013. Issue Date 2013-06-01 Doc URL http://hdl.handle.net/2115/56886 Rights(URL) http://creativecommons.org/licenses/by/3.0/ Type bookchapter File Information InTech-seaweed transformation final.pdf


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][5][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-mation mediated by A. tumefaciens has become the most commonly used method to transmit DNA fragment into higher plants [12].
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 protoplasts, 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 phycoerythrin, n-3 polyunsaturated fatty acids, porphyran, ager and carrageenan from red algae, and fucoxantine, fucoidan and alginate from brown algae [19][20][21][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][26][27][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.Establishing 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 transformation 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 summarizing pioneer and recent studies related to seaweed genetic transformation.

Pioneer studies for transient transformation
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 CaMV35S-GUS gene by pioneers for initial trials of seaweed transformation.

Recent improvement of the transient transformation system in Porphyra
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][38][39][40][41].As a result, the transient transformation system in red seaweeds has recently been improved by resolving this problem.

Optimization of codon usage in the reporter gene
Inefficient expression of foreign genes in the green alga Chlamydomonas reinhardtii is often due to the incompatibility of the codon usage in the gene's coding regions [49][50][51].Expressed sequence tag (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 means the GC content reaches a high of 65.2% [52].Since bacterial GUS and lacZ reporter genes have AT-rich codons, the incompatibility of codon usage, which generally inhibits the effective use of transfer RNA by rarely used codons in the host cells, thus decreasing the efficiency of the translation [53], might be responsible for the poor translation efficiency of foreign genes in P. yezoensis cells.It is therefore possible that modification of codon usage in the GUS gene would enable the efficient expression of this gene in P. yezoensis cells.
Accordingly, the codon usage of the GUS reporter gene was adjusted to that in the nuclear genes of P. yezoensis by introducing silent mutations [48], by which unfavorable or rare codons in the GUS reporter gene were exchanged for favorable ones without affecting amino acid sequences.The resultant artificially codon-optimized GUS gene was designated PyGUS, and its GC content was increased from 52.3% to 66.6% [48].When the PyGUS gene directed by the CaMV 35S promoter was introduced into P. yezoensis gametophytic cells by particle bombardment, low but significant expression of the PyGUS gene was observed by histochemical detection and GUS activity test, indicating enhancement of the expression level of the GUS reporter gene [29,30,48].Optimization of the codon usage of the reporter gene is therefore one of the important factors for successful expression in P. yezoensis cells [29,30,48].

Employment of endogenous strong promoters
The CaMV 35S promoter has very low activity in cells of green microalgae such as Dunaliella salina [54], Chlorella kessleri [55] and Chlorella vulgaris [56] and no activity in C. reinhardtii cells [57][58][59].Thus, a low level of PyGUS expression under the direction of the CaMV 35S promoter is likely to be caused by the low activity of this promoter in P. yezoensis cells.A hint to overcoming this problem was that employment of strong endogenous promoters such as the β-Tub, RbcS2 and Hsp70 promoters results in the efficient expression of foreign genes in microalgae [60][61][62][63][64][65].Therefore, it is likely that efficient expression of the PyGUS reporter gene in P. yezoensis cells is caused by the recruitment of endogenous strong promoters.
By comparison with steady-state expression levels by reverse transcription-polymerase chain reaction (PCR), we found two genes strongly expressed in P. yezoensis: genes encoding glyceraldehyde-3-phosphate dehydrogenase (PyGAPDH) and actin 1 (PyAct1) [29].When the PyGUS gene fused with the 5' upstream regions of these genes were introduced into gametophytic cells by particle bombardment, cells expressing the reporter gene and GUS enzymatic activity were dramatically increased [48,66].These results indicate that employment of endogenous strong promoters is another important factor necessary for high-level expression of the reporter gene in P. yezoensis cells.In addition, the original GUS gene was not activated by PyGAPDH or PyAct1 promoter [29,30,48], demonstrating that the PyGUS gene and endogenous strong promoter have a synergistic effect on the efficiency of the expression in P. yezoensis cells (Figure 1A).Therefore, the combination of endogenous strong promoters with codon optimized reporter genes is critical for successful transient transformation in Porphyra species [29,30].The established procedure of transient transformation is schematically represented in Figure 2.

Application of the transient transformation for using fluorescent proteins
The GUS reporter gene is usually used to monitor gene expression in planta; however, visualization of the reporter products requires cell killing.Reporters that function in living cells have also been established to date with fluorescent proteins used most commonly.The green fluorescent protein (GFP) has the advantage over other reporters for monitoring subcellular localization of proteins in living cells, because its fluorescence can be visualized without additional substrates or cofactors [67].At present, there are GFP variants with non-overlapping emission spectra such as cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and red fluorescent protein, which allows multicolor imaging in cells [68,69].
Until recently, there was no report about the successful expression of fluorescent proteins in seaweeds.However, based on an efficient transient transformation system in P. yezoensis, fluorescent reporter systems have recently been established in P. yezoensis [29,30,70,71].The humanized fluorescent protein genes, AmCFP, ZsGFP, and ZsYFP (Clontech) and the plant-adapted GFP(S65T) [72], the GC contents of which are as high as 63.7%, 62.8%, 61.9% and 61.4%, respectively, were strongly expressed in gametophytic cells under the direction of the PyAct1 promoter using the particle bombardment method [71] (see Figure 1B).
The analysis of subcellular localization of cellular molecules was available using humanized and plant-adapted fluorescent reporters.The first successful attempt at achieving this process was to monitor the plasma membrane localization of phosphoinositides in P. yezoensis [70].Phosphoinositides (PIs), whose inositol ring has hydroxyl groups at positions D3, D4 and D5 for phosphorylation, constitute a family of structurally related lipids, PtdIns-monophosphates [PtdIns3P, PtdIns4P and PtdIns5P], PtdIns-bisphosphates [PtdIns(3,4)P 2 , PtdIns(3,5)P 2 and PtdIns(4,5)P 2 ] and PtdIns-trisphosphate [PtdIns(3,4,5)P 3 ], all of which are detectable in plants except for PtdIns(3,4,5)P 3 [73,74].Although the PIs are a minority among membrane phospholipids, they play important roles in regulating multiple processes of development and cell responses to environmental stimuli in land plants and green algae [74,75].Recently, Li et al. [76,77] demonstrated that PIs are involved in the establishment of cell polarity in P. yezoensis monospores.The Pleckstrin homology (PH) domain, a PI-binding module, each part of which has individual substrate specificity, is usually used to monitor PIs in vivo by fusion with a fluorescent protein [78][79][80].For instance, the PH domains from human phospholipase Cδ1 (PLCδ1) are employed for the detection of PtdIns(4,5)P 2 [81], whereas that from the v-akt murine thymoma viral oncogene homolog 1 (Akt1) has dual specificity in the detection of both PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 [82].Because of this substrate specificity, we were able to visualize PtdIns(3,4)P 2 and PtdIns(4,5)P 2 at the plasma membrane with humanized AmCFP and ZsGFP fused to the PH domains from PLCδ1 and Akt1 via the direction of the PyAct1 promoter [70].Moreover, subcellular localization of transcription factors was also visualized in P. yezoensis.When complete open reading frames (ORFs) of transcription elongation factor 1 (PyElf1) and multiprotein bridging factor 1 (PyMBF1) from P. yezoensis were fused to AmCFP or ZsGFP, nuclear localization of these fusion proteins was observed in gametophytic cells, which was confirmed by overlapping of fluorescent signals with SYBR Gold staining of the nucleus [71] With the successfull visualization of subcellular localization of cellular molecules, the transient transformation system developed in P. yezoensis appearst to be powerful tool to analyze functions of genes and cellular components [29,30].

Applicability of the P. yezoensis transient transformation system in other red seaweeds
As described above, both the adjustment of codon usage of the reporter gene according to algal preference and the employment of the strong endogenous promoters are important for providing highly efficient and reproducible expression of the reporter gene in P. yezoensis.In addition to Bangiophyceae like Porphyra species, Florideophyceae are also known, including a number of industrially important species such as Gracilaria and Gelidium as sources of agar and Chondrus and Kappaphycus as sources of carrageenan.Thus, the establishment of a genetic manipulation system for both Bangiophyceae and Florideophyceae other than P. yezoensis is awaited.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 analysis of the GAPDH gene from a Florideophycean alga Chondrus crispus showed a high GC content (approximately 60%) in the coding region [83,84], which is consistent with the codon preference in P. yezoensis.Since efficient expression of the GAPDH-PyGUS gene has recently been confirmed in P. tenera [85], the applicability of the P. yezoensis transient gene expression system in other red seaweeds is expected.Indeed, using the PyGUS and sGFP(S65T) reporter genes under the direction of the PyAct1 promoter, efficient expression of PyGUS and sGFP(S65T) genes was observed in Bangiophyceae including P. tenera, P. okamurae, P. psedolinearis and Bangia fuscopurpurea, although the expression efficiency varied among species [86].Thus, the transient transformation system developed in P. yezoensis is widely applicable in Bangiophycean red algae [29,30,86].
No expression of the reporter genes was seen in Florideophyceae [29,30,86].Since the availability of the P. yezoensis promoter is responsible for this deficiency in gene expression, it is important to employ the 5' upstream region of the suitable endogenous gene from Florideophycean algae.Alternatively, it is possible that the efficiency of plasmid transfer by bombardment parameters is reduced by the cell wall and thus the size of the gold particles, target distance, acceleration pressure and/or amount of DNA per bombardment should be adjusted.
Taken together, PyGUS and sGFP(S65T) genes act synergistically with the PyAct1 promoter as a heterologous promoter for transient transformation in Bangiophycean algae.Recently, the same synergistic effect was found in P. tenera; that is, Son et al. [85] clearly indicated that the heat shock protein 70 (PtHSP70) promoter from P. tenera can activate the PyGUS gene in gametophytic cells of this alga.Moreover, the PtHSP70-PyGUS gene was expressed in P. yezoensis, P. okamurae, P. psedolinearis and B. fuscopurpurea [85,87].These findings are consistent with the importance of two critical factors for transient transformation in red seaweeds, adjustment of the codon usage in reporter genes and employment of a strong endogenous promoter.
The other important message gleaned from this experimental data is the efficient heterologous activation of PyGAPDH and PtHSP70 promoters in P. tenera and P. yezoensis, respectively [85,87].For the genetic transformation, the target site for recombination is usually determined by the DNA sequence of genes desired for disruption or modification.Thus, it is better to exclude a possibility of homologous recombination at the DNA region corresponding to the promoter sequence used for expression of the reporter gene that is usually sandwiched between two different DNA sequences from the objective gene or its flanking regions.To avoid incorrect recombination at the promoter region, it is critical to employ heterologous promoters, whose sequence has low homology to the genome sequence of the host, to direct the expression of reporter genes.It is therefore possible that PyGAPDH and PtHSP70 promoters are useful for genetic transformation in P. tenera and P. yezoensis, respectively.The number of promoters acting for heterologous reporter gene expression in red algae must be increased to develop a sophisticated system for red algal genetic transformation.

Towards genetic transformation in red seaweeds
The successful genetic transformation in red alga has been established only in unicellular algae [20,88].The first report described chloroplast transformation in the unicellular red alga Porphyridium sp.through integration of the gene encoding AHAS(W492S) into the chloroplast genome by homologous recombination, resulting in sulfometuron methyl (SMM) resistance at a high frequency in SMM-resistant colonies [89].The next report was of stable nuclear transformation in the unicellular red alga Cyanidioschyzon merolae, for which the uracil auxotrophic mutant lacking the URA5.3 gene was used for the genetic background to isolate mutants with uracil prototrophic by employing the wild-type URA5.3gene fragment as a selection maker [90].
Table 1 shows preliminary experiments with red seaweeds.The first was by Cheney et al. [26], who introduced the CaMV 35S-GUS and CaMV 35S-GFP genes in P. yezoensis genome via an Agrobacterium-mediated transformation system.In addition, they transformed P. yezoensis with a bacterial nitroreductase gene via an Agrobacterium-mediated method [28] and P. leucosticte monospores with an unknown gene by electroporation [27].However, these reports appeared on conference abstracts and thus details of experimental procedures are unknown.In related work, the genetic transformation of Gracilaria species was recently reported [91,92], in which integration of the SV40-lacZ gene was checked by PCR using genomic DNAs prepared from particle-bombarded seaweeds; however, selection of transformed cells was not performed.Taken together, these preliminary experiments are not enough to conclude the establishment of genetic transformation in red seaweeds, meaning that the genetic transformation system has not yet been fully established in red macroalgae.
As mentioned above, procedures of integration and targeting of foreign genes into the genome and selection of transformed cells must be developed for establishing the genetic transformation system, although other requirements such as an efficient gene transfer system and an efficient expression system for foreign genes have been resolved by developing the transient transformation system in Bangiophyceae [29,30].Regarding the unresolved points, knowledge about the selection of transformed cells is now accumulating.Selection marker genes are required to distinguish between transformed cells and nontransformed cells, since successful integration of a foreign gene into the host genome usually occur in only a small percentage of transfected cells.These genes confer new traits to any transformed target strain of a certain species, thus enabling the transformed cells to survive on medium containing the selective agent, where non-transformed cells die.Genes with resistance to the aminoglycoside antibiotics, which bind to ribosomal subunits and inhibit protein synthesis in bacteria, eukaryotic plastids and mitochondria [93], are generally used as selection markers.For example, the antibiotics hygromycin and geneticin (G418) are frequently used as selection agents with the hygromycin phosphotransferase (hptII) gene to inactivate hygromycin via an ATP-dependent phosphorylation [94] and the neomycin phosphotransferase II (nptII) gene to detoxify neomycin, G418 and paromomycin [93], respectively.In the green alga Chlamydomonas reinhardtii, the hygromycin phosphotransferase (aph7") gene from Streptomyces hygroscopicus and the aminoglycoside phosphotransferase aphVIII (aphH) gene from S. rimosus had been reported as selectable marker genes for hygromycin and paromomycin, respectively, with similarity in the codon usage [95][96][97].The aphH gene from S. rimosus is also applicable to the multicellular green alga Volvox carteri as a paromomycin-resistance gene [97,98].In the diatom Phaeodactylum tricornutum, the expressed chloramphenicol acetyltransferase gene (CAT) detoxifies chloramphenicol [99], and the nptII gene confers resistance to the aminoglycoside antibiotic G418 [64].Likewise, the nptII gene gives resistance to the antibiotic G418 in the diatoms Navicula saprophila and Cyclotella cryptica [100].However, it is unknown what kinds of antibiotics-based selection marker genes are available for red seaweeds, since red algae usually have strong resistance to antibiotics.
Recently, the sensitivity of P. yezoensis gametophytes to ampicillin, kanamycin, hygromycin, geneticin (G418), chloramphenicol and paromomycin was investigated, and lethal effects of these antibiotics on gametophytes were observed at more than 2.0 mg mL -1 of hygromycin, chloramphenicol and paromomycin and 1.0 mg mL -1 of G418, whereas P. yezoensis gametophytes were highly resistant to ampicillin and kanamycin [101].Although these concentrations are in fact very high in comparison with the cases for the red alga Griffithsia japonica and the green alga C. reinhardtii that were highly sensitive to 50 μg mL -1 and 1.0 μg mL -1 of hygromycin [96,102], these four antibiotics and corresponding resistance genes are suitable for the selection of genetically transformed cells from P. yezoensis gametophytes.According to these findings, it is necessary to confirm whether P. yezoensis gametophytes will obtain antibiotic tolerance by introducing plasmid constructs containing the antibiotic-resistance genes mentioned above.In this case, optimization of codon usage and the employment of strong endogenous promoter are expected for functional expression of the antibiotic resistance genes, according to the knowledge from the transient transformation system [29,30].Such efforts could effectively contribute to the establishment of the genetic transformation system in red seaweeds in the near future.

Transformation in brown seaweeds
According to Qin et al. [103], trials of genetic engineering in brown seaweeds have been started by transient expression of the GUS reporter gene under direction of the CaMV 35S promoter by particle bombardment in Laminaria japonica and Undaria pinnatifida, which were first performed in 1994 by them.Descriptions of related experiments were published later [104,105].Qin et al. then focused on the establishment of genetic transformation in brown seaweeds and provided successful reports of genetic transformation in L. japonica [103,106].Genetic transformation was performed by particle bombardment only and expression of a reporter gene was driven by the SV40 promoter that is usually used for gene expression in mammalian cells (Table 2).This promoter represented non-tissue and -cell specificity for expression of the E. coli lacZ reporter gene [105].Promoters from maize ubiquitin, algal adenine-methyl transfer enzyme and diatom fucoxanthin chlorophyll a/c-binding protein (FCP) genes are also useful for transient expression of the GUS gene, and the FCP promoter is also employable for the genetic transformation [107].Interestingly, there has been no successful genetic transformation using the CaMV 35S promoter, although this promoter is active in the transient transformation [103].
Despite the reports of successful genetic transformation, there was no experiment using antibiotics-based selection of transformants in brown seaweeds.Although the susceptibility of brown seaweeds to antibiotics has not been well studied, it was reported that L. japonica was sensitive to chloramphenicol and hygromycin, but not to ampicillin, streptomycin, kanamycin, neomycin or G418 [103,106].Since hygromycin is more effective than chloramphenicol [103,106], it is necessary to confirm the utility of the SV40-hptII gene for the selection of transformants to fully establish the genetic transformation system in kelp.To date, stably transformed microalgae have been employed to produce recombinant antibodies, vaccines or bio-hydrogen as well as to analyze the gene functions targeted for engineering [108][109][110][111]. Based on the success in genetic transformation, L. japonica is now proposed as a marine bioreactor in combination with the SV40 promoter [112].Indeed, the integration of human hepatitis B surface antigen (HBsAg) and recombinant human tissue-type plasminogen (rt-PA) genes into the L. japonica genome resulted in the efficient expression of these genes under the direction of the SV40 promoter [113,114].Therefore, L. japonica promises to be useful as the bioreactor for vaccine and other medical agents, although it is necessary to continually check the safety and value of its use by oral application.

Species
There is no competitor against the Chinease group in the field of using brown algal genetic transformation at present [103,106,115], meaning there is currently no way to confirm the replicability of the experiments.It is necessary to re-examine the effective use of the non-plant SV40 promoter and bacterial lacZ gene in brown algal genetic transformation, which is also important for the evaluation of genetic transformation in red seaweeds Gracilaria species, for which the SV40-lacZ gene was used such as transgene, as described above [91,92].

Transformation in green seaweeds
The first successfull genetic transformation in green algae was reported in the unicellular green alga Chlamydomonas reinhardtii for which the particle bombardment and glass-bead abrasion techniques were employed [116,117].The availability of electoroporation was then confirmed in C. reinhardtii and Chlorella saccharophila [118,119].These methods produce physical cellular damage, allowing DNA to be introduced into the cells.Moreover, particle bombardment was confirmed to be useful for a diverse range of species, including transient transformation in the unicellular Haematococcus pluvialis [120] and genetic transformation in the multicellular Volvox carteri and Gonium pectoral [97,[120][121][122].Agrobacterium-mediated transformation was also reported in H. pluvialis [123].Thus, all methods employed in land green plants are applicable for green microalgae [88] (see Table 3).
In contrast, there is no report about genetic transformation in green seaweeds (Table 3).To date, only two examples of transient transformation have been reported in green seaweeds, Ulva lactura by electroporation and U. pertusa by particle bombardment [124,125].As shown in Table 3, some of the experiments with micro-and macro-green algae used the promoter of the CaMV 35S gene and the coding region of the E. coli GUS gene.Although functionality of the CaMV 35S promoter and bacterial GUS coding region is the same in land green plants, the expression of the GUS reporter gene seems to be very low in the green seaweed U. lactuca [124].
In fact, codon-optimization is critical for the expression of reporters like the GFP gene and antibiotic-resistance genes in C. reinhardtii [47,90,115,126].Moreover, the HSP70A promoter was employed to increase the expression level of the reporter genes [47,115] If the rbsS-EGFP gene is useful as a reporter gene for genetic transformation in green seaweeds, the remaining problems to be settled are methods for foreign gene integration into the genome and selection of transformed cells, which is the same as the situation with red seaweeds.Reddy et al. [24] commented on the antibiotic sensitivity of green seaweeds, indicating the considerable resistance of protoplast from Ulva and Monostroma to hygromycin and kanamycin.
Insensitivity to hygromycin is inconsistent with the case for red and brown seaweeds [101][102][103]106].It is therefore necessary to check the sensitivity of green seaweed cells to other antibiotics to identify the genes employable for selection of transformed cells, which could stimulate the development of the genetic transformation system in green seaweeds.

Conclusion
It is nearly 20 years since the first transient transformation of a red seaweed with a circular expression plasmid [25], and many efforts have been made to develop a system for transient and stable expression of foreign genes in many kinds of seaweeds; however, a seaweed transformation system has still not been developed.The main problem is the employment of the CaMV 35S-GUS gene in the pioneer attempts at system development as shown in Tables 1, 2 and 3.This problem was recently resolved through the development of an efficient transient transformation system in P. yezoensis [29,30].It is clear that the CaMV 35S promoter and the GUS gene are not active in seaweed cells [48], which is supported by knowledge from green microalgae [54][55][56][57][58][59][60][61][62][63][64][65].These findings strongly indicate that defects in the transfer and expression of foreign genes were resolved by knowledge about two critical factors required for reproducibility and efficiency of transient gene expression, namely, the optimization of codon usage of coding regions and the employment of endogenous strong promoters [29,30].However, these significant improvements are not enough to allow the establishment of a genetic transformation system in seaweeds.
At present, genetic transformation is reported in red and brown seaweeds using the SV40 promoter (Tables 1 and 2) [91,92,103,[105][106][107]113,114]; however, isolation of transgenic clone lines produced from distinct single transformed cells, which is the final goal of the genetic transformation of seaweeds as a tool, has not been reported, and seaweed genetic transformation is thus not fully developed.Therefore, the next step is to develop the gene targeting system via integration of a foreign gene into the genome and the system for selection of transformed cells.Since candidates of antibiotic agents for selection of transformed algal cells were mentioned recently [101][102][103]106], it is necessary to confirm the possibility of stable integration of a plasmid or a DNA fragment containing the selection maker gene into the seaweed genome.Once a positive result is obtained, it could lead us to establish the gene targeting method via the homologous recombination using an appropriate antibiotics resistance gene, if possible, with the heterologous promoter.To this end, we must reevaluate the availability of the methods for gene transfer such as electroporation and Agrobacteriumu infection.
Due to the problems with efficient genetic transformation systems, the molecular biological studies of seaweeds are currently progressing more slowly than are the studies of land green plants.Since a genetic transformation system would allow us to perform genetic analysis of gene function via inactivation and knock-down of gene expression by RNAi and antisense RNA supression, its establishment will enhance both our biological understanding and genetical engineering for the sustainable production of seaweeds and also for the use of seaweeds as bioreactors.

Figure 1 .
Figure 1.Efficient expression of PyGUS and fluorescent proteins by the transeint transformation with circular expression plasmids in P. yezoensis gametophytic cells.(A) Expression of the codon-optimized PyGUS reporter gene under the direction of the actin 1 (PyAct1) promoter.Blue histochemically stained cells are PyGUS expression cells.Scale bar corresponds to 100 μm.(B) Expression of humanized AmCFP and plant-adapted sGFP(S65T).Gametophytic cells transiently transformed with expression plasminds containng AmCFP or sGFP(S65T) gene under the control of the PyAct1 promoter.Left and right panels show bright field and fluorescence images, respectively.Scale bar corresponds to 5 μm.

Figure 2 .
Figure 2. The established procedure of transeient transformation in P. yezoensis.A circular expression plasmid is bombarded into P. yezoensis gametophytic cells using the Bio-Rad PDS-1000/He after coating of gold particles with the plasmid.Expression of the reporter gene is observed after cultivation of the bombareded gametophyte under dark for two days; for PyGUS reporter gene, histochemical staining with X-gluc solution and fluorometric analysis of enzymatic activity are performed; for fluorescent reporter genes, bombarded sanples are examined with fluorescent microscopy.

Table 1 .
Transformation in red seaweeds.

Table 2 .
Transformation in brown seaweeds.

Table 3 .
[125]refore, it is possible that changes in codon usage in the reporter gene and promoter region could result in increased reporter gene expression in transient transformation of green seaweeds.Recently, the Rubisco small subunit (rbsS) promoter was used for expression of the EGFP reporter gene in transient transformation of U. pertusa by particle bombardment[125]; however, it is still unclear whether the rbsS promoters and the EGFP gene work well in cells in comparison with the CaMV 35S promoter and codon-optimized EGFP gene.Transformation in green algae.