Current Advances in Seaweed Transformation

Chapter 13 Current Advances in Seaweed Transformation Koji Mikami Additional information is available at the end of the chapter http://dx.doi.org/10.5772/52978 . Introduction Frederick Griffith reported the discovery of transformation in [ ]. 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 [ ]. 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 [ ], 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 [ - ]. 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 [ ], 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 [ ], 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 [ ]. 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 s [ , ], 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. 324 An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical Applications mation mediated by A. tumefaciens has become the most commonly used method to transmit DNA fragment into higher plants [ ]. Since not all plant cells are susceptible to infection by A. tumefaciens, other methods were developed and are available in plants. Particle bombardment [ ], 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 [ , ]. 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 [ , ] and is almost exclusively applied with the moss Physcomitrella patens and liverwort Marchantia polymorpha [ , ]. 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- polyunsaturated fatty acids, porphyran, ager and carrageenan from red algae, and fucoxantine, fucoidan and alginate from brown algae [ - ]. Thus, to make new strains carrying advantageous characteristics benefiting industry and medicine, researchers have worked hard since the early s to establish methods of genetic transformation in seaweeds [ , , ]. However, the process is very difficult, and most of the early studies were reported in conference abstracts without the accompanying manuscript publication [ - ]. 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 [ , , ], 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 Current Advances in Seaweed Transformation http://dx.doi.org/10.5772/52978 [ , ], I focus here on the current progress in red algal transient transformation with sum‐ marizing pioneer and recent studies related to seaweed genetic transformation. . Transformation in red seaweeds . . 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 [ ], which was the first report about the transient transformation of seaweeds Table . 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 S promoter CaMV S-GUS gene . Since the GUS expression can be visualized as a blue color following treatment with X-gluc -bromo- -chloro- -indolyl-β-D-glucuronide and also be quantified by fluorometric analysis [ , ], this reporter gene is widely used in land green plants having no background of the GUS activity [ , ]. In addition, the CaMV S promoter is heterologously used in land green plants as a strong constitutive and non-tissue-specific transcriptional regulator [ , ]. Therefore, it is a natural choice for the selection of the CaMV S-GUS gene by pioneers for initial trials of seaweed transformation. To date, studies have been mainly focused on Porphyra species because of their economical values. As shown in Table , expression of the CaMV S-GUS gene was previously observed in P. miniata, P. tenera and P. yezoensis [ - ], all of which were performed by electoroporation using protoplasts. Kuang et al. [ ] also tested the particle bombardment of the CaMV SGUS gene in P. yezoensis and got positive results. Moreover, the availability of mammaliantype simian virus SV promoter was reported to express the E. coli lacZ reporter gene, encoding β-galactosidase cleaving colorless substrate X-gal -bromo- -chloro- -indolyl-βgalactopyranoside to produce a blue insoluble product [ ], in P. haitanensis, Gracilaria chagii and K. alvarezii by electroporation or particle bombardment [ , ]. . . 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 S RNA and animal viral SV promoters in combination with GUS and lacZ reporter genes Table . The CaMV S and SV 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 [ , ]. 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 S promoter and the GUS reporter gene in P. yezoensis gametophytec cells [ , , ]. These observations are completely opposite from the results in previous reports using the CaMV S promoter [ , - ]. As a result, the transient transformation system in red seaweeds has recently been improved by resolving this problem. 325 326 An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical Applications Species Status of Gene transfer method Promoter Marker or Reporter Ref. expression Kappaphycus alvarezii transient particle bombardment CaMV 35S GUS [25] Porphyra miniata transient electroporation CaMV 35S GUS [37] Porphyra yezoensis transient Electroporation CaMV 35S GUS [38] particle bombardment Porphyra tenera transient electroporation CaMV 35S GUS [39] Porphyra yezoensis transient electroporation rbcS GUS [40] Porphyra yezoensis transient electroporation CaMV 35S GUS [41] Porphyra yezoensis transient electroporation CaMV 35S GUS [42] lacZ [44] β-tubulin Gracilaria changii transient particle bombardment SV40 Porphyra haitanensis transient Porphyra yezoensis SV40 CAT [128] transient electriporation SV40 CAT, GUS [129] Porphyra yezoensis transient electroporation Rubisco GUS, sGFP(S65T) [130] Porphyra yezoensis transient particle bombardment CaMV 35S PyGUS [48] PyGAPDH Porphyra yezoensis transient particle bombardment PyAct1 PyGUS [66] Porphyra yezoensis transient particle bombardment PyAct1 AmCFP [70] Porphyra yezoensis transient particle bombardment PyAct1 AmCFP, ZsGFP, [71] ZsYFP, sGFP(S65T) Porphyra tenera transient particle bombardment Porphyra yezoensis Porphyra species* PtHSP70 transient particle bombardment PyAct1 Bangia fuscopurpurea Porphyra species* PyGUS [85] PyGUS [86] PyGAPDH sGFP(S65T) transient particle bombardment PtHSP70 PyGUS [87] stable Agrobacterium-mediated CaMV 35S GUS [26] Bangia fuscopurpurea Porphyra yezoensis gene transfer Porphyra leucostica stable ekectroporation CaMV 35S lacZ [27] Porphyra yezoensis stable Agrobacterium-mediated (unknown) (unknown) [28] gene transfer Kappaphycus alvarezii stable particle bombardment SV40 lacZ [45] Porphyra haitanensis stable glass bead agitation SV40 lacZ [131] EGFP Gracilaria changii stable particle bombardment SV40 lacZ [91] Gracilaria gracilis stable particle bombardment SV40 lacZ [92] *Porphyra species used are P. yezoensis, P. tenera, P. okamurae, P. onoi, P. variegate and P. pseudolinearis. Table 1. Transformation in red seaweeds. Current Advances in Seaweed Transformation http://dx.doi.org/10.5772/52978 . . . 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 [ - ]. 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 . % [ ]. 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 [ ], 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 [ ], 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 . % to . % [ ]. When the PyGUS gene directed by the CaMV S promoter was introduced into P. yezoensis gametophytic cells by particle bombard‐ ment, 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 [ , , ]. Optimization of the codon usage of the reporter gene is therefore one of the important factors for successful expression in P. yezoensis cells [ , , ]. . . . Employment of endogenous strong promoters The CaMV S promoter has very low activity in cells of green microalgae such as Dunaliella salina [ ], Chlorella kessleri [ ] and Chlorella vulgaris [ ] and no activity in C. reinhardtii cells [ - ]. Thus, a low level of PyGUS expression under the direction of the CaMV S 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, RbcS and Hsp promoters results in the efficient expression of foreign genes in microalgae [ - ]. 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- -phosphate dehydrogenase PyGAPDH and actin PyAct [ ]. When the PyGUS gene fused with the ’ upstream regions of these genes were introduced into gameto‐ phytic cells by particle bombardment, cells expressing the reporter gene and GUS enzymatic activity were dramatically increased [ , ]. 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 PyAct promoter [ , , ], demonstrating that the PyGUS gene and endog‐ enous strong promoter have a synergistic effect on the efficiency of the expression in P. yezoensis cells Figure A . Therefore, the combination of endogenous strong promoters with 327 328 An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical Applications codon optimized reporter genes is critical for successful transient transformation in Porphyra species [ , ]. The established procedure of transient transformation is schematically represented in Figure . . . . 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 liv‐ ing cells have also been established to date with fluorescent proteins used most common‐ ly. 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 [ ]. 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 [ , ]. Until recently, there was no report about the successful expression of fluorescent pro‐ teins in seaweeds. However, based on an efficient transient transformation system in P. yezoensis, fluorescent reporter systems have recently been established in P. yezoensis [ , , , ]. The humanized fluorescent protein genes, AmCFP, ZsGFP, and ZsYFP Clontech and the plant-adapted GFP S T [ ], the GC contents of which are as high as . %, . %, . % and . %, respectively, were strongly expressed in gametophytic cells under the direction of the PyAct promoter using the particle bombardment method [ ] see Figure B . 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 [ ]. Phosphoinositides PIs , whose inositol ring has hydroxyl groups at positions D , D and D for phosphorylation, constitute a family of structurally related lipids, PtdIns-monophosphates [PtdIns P, PtdIns P and PtdIns P], PtdIns-bisphosphates [PtdIns , P , PtdIns , P and PtdIns , P ] and PtdIns-trisphosphate [PtdIns , , P ], all of which are detectable in plants except for PtdIns , , P [ , ]. Although the PIs are a minority among membrane phos‐ pholipids, they play important roles in regulating multiple processes of development and cell responses to environmental stimuli in land plants and green algae [ , ]. Recently, Li et al. [ , ] 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 [ - ]. For instance, the PH domains from human phospholipase Cδ PLCδ are employed for the detection of PtdIns , P [ ], whereas that from the v-akt murine thymoma viral oncogene homolog Akt has dual specificity in the detection of both PtdIns , P and PtdIns , , P [ ]. Because of this substrate specificity, we were able to visualize PtdIns , P and PtdIns , P at the plasma membrane with humanized AmCFP and ZsGFP fused to the PH domains from PLCδ and Akt via the direction of the PyAct promoter [ ]. Current Advances in Seaweed Transformation http://dx.doi.org/10.5772/52978 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 direc‐ tion 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. The established procedure of transeient transformation in P. yezoensis. A circular expression plasmid is bom‐ barded 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. 329 330 An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical Applications Moreover, subcellular localization of transcription factors was also visualized in P. ye‐ zoensis. When complete open reading frames ORFs of transcription elongation factor PyElf and multiprotein bridging factor PyMBF from P. yezoensis were fused to AmCFP or ZsGFP, nuclear localization of these fusion proteins was observed in gameto‐ phytic cells, which was confirmed by overlapping of fluorescent signals with SYBR Gold staining of the nucleus [ ] 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 [ , ]. . . . 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 impor‐ tant 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 Geli‐ dium as sources of agar and Chondrus and Kappaphycus as sources of carrageenan. Thus, the establishment of a genetic manipulation system for both Bangiophyceae and Florideo‐ phyceae 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 con‐ tent approximately % in the coding region [ , ], which is consistent with the codon preference in P. yezoensis. Since efficient expression of the GAPDH-PyGUS gene has re‐ cently been confirmed in P. tenera [ ], the applicability of the P. yezoensis transient gene expression system in other red seaweeds is expected. Indeed, using the PyGUS and sGFP S T reporter genes under the direction of the PyAct promoter, efficient expres‐ sion of PyGUS and sGFP S T genes was observed in Bangiophyceae including P. ten‐ era, P. okamurae, P. psedolinearis and Bangia fuscopurpurea, although the expression efficiency varied among species [ ]. Thus, the transient transformation system devel‐ oped in P. yezoensis is widely applicable in Bangiophycean red algae [ , , ]. No expression of the reporter genes was seen in Florideophyceae [ , , ]. Since the availa‐ bility of the P. yezoensis promoter is responsible for this deficiency in gene expression, it is important to employ the ’ upstream region of the suitable endogenous gene from Florideo‐ phycean algae. Alternatively, it is possible that the efficiency of plasmid transfer by bombard‐ ment 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 S T genes act synergistically with the PyAct 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. [ ] clearly indicated that the heat shock protein PtHSP promoter from P. tenera can activate the PyGUS gene in gametophytic cells of this alga. Moreover, the PtHSP -PyGUS gene was expressed in P. yezoensis, P. okamurae, P. psedolinearis and B. fuscopurpurea [ , ]. These findings are consistent Current Advances in Seaweed Transformation http://dx.doi.org/10.5772/52978 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 PtHSP promoters in P. tenera and P. yezoensis, respectively [ , ]. 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 PtHSP 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 [ , ]. The first report described chloroplast transformation in the unicellular red alga Porphyridium sp. through integration of the gene encoding AHAS W S into the chloroplast genome by homologous recombination, resulting in sulfometuron methyl SMM resistance at a high frequency in SMM-resistant colonies [ ]. The next report was of stable nuclear transformation in the unicellular red alga Cyanidioschyzon merolae, for which the uracil auxotrophic mutant lacking the URA . gene was used for the genetic background to isolate mutants with uracil prototrophic by employing the wild-type URA . gene fragment as a selection maker [ ]. Table shows preliminary experiments with red seaweeds. The first was by Cheney et al. [ ], who introduced the CaMV S-GUS and CaMV S-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 [ ] and P. leucosticte monospores with an unknown gene by electroporation [ ]. 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 [ , ], in which integration of the SV -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 331 332 An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical Applications system and an efficient expression system for foreign genes have been resolved by devel‐ oping the transient transformation system in Bangiophyceae [ , ]. Regarding the unre‐ solved 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 [ ], are generally used as selection markers. For example, the antibiotics hygromycin and geneticin G are frequently used as selection agents with the hygromycin phos‐ photransferase hptII gene to inactivate hygromycin via an ATP-dependent phosphoryla‐ tion [ ] and the neomycin phosphotransferase II nptII gene to detoxify neomycin, G and paromomycin [ ], respectively. In the green alga Chlamydomonas reinhardtii, the hy‐ gromycin phosphotransferase aph ” gene from Streptomyces hygroscopicus and the ami‐ noglycoside phosphotransferase aphVIII aphH gene from S. rimosus had been reported as selectable marker genes for hygromycin and paromomycin, respectively, with similari‐ ty in the codon usage [ - ]. The aphH gene from S. rimosus is also applicable to the multicellular green alga Volvox carteri as a paromomycin-resistance gene [ , ]. In the diatom Phaeodactylum tricornutum, the expressed chloramphenicol acetyltransferase gene CAT detoxifies chloramphenicol [ ], and the nptII gene confers resistance to the amino‐ glycoside antibiotic G [ ]. Likewise, the nptII gene gives resistance to the antibiotic G in the diatoms Navicula saprophila and Cyclotella cryptica [ ]. However, it is un‐ known what kinds of antibiotics-based selection marker genes are available for red sea‐ weeds, since red algae usually have strong resistance to antibiotics. Recently, the sensitivity of P. yezoensis gametophytes to ampicillin, kanamycin, hygromycin, geneticin G , chloramphenicol and paromomycin was investigated, and lethal effects of these antibiotics on gametophytes were observed at more than . mg mL- of hygromycin, chloramphenicol and paromomycin and . mg mL- of G , whereas P. yezoensis gameto‐ phytes were highly resistant to ampicillin and kanamycin [ ]. 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 μg mL- and . μg mL- of hygromycin [ , ], 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 [ , ]. Such efforts could effectively contribute to the establishment of the genetic transformation system in red seaweeds in the near future. Current Advances in Seaweed Transformation http://dx.doi.org/10.5772/52978 . Transformation in brown seaweeds According to Qin et al. [ ], trials of genetic engineering in brown seaweeds have been started by transient expression of the GUS reporter gene under direction of the CaMV S promoter by particle bombardment in Laminaria japonica and Undaria pinnatifida, which were first performed in by them. Descriptions of related experiments were published later [ , ]. Qin et al. then focused on the establishment of genetic transformation in brown seaweeds and provided successful reports of genetic transformation in L. japonica [ , ]. Genetic transformation was performed by particle bombardment only and ex‐ pression of a reporter gene was driven by the SV promoter that is usually used for gene expression in mammalian cells Table . This promoter represented non-tissue and -cell specificity for expression of the E. coli lacZ reporter gene [ ]. Promoters from maize ubiq‐ uitin, algal adenine-methyl transfer enzyme and diatom fucoxanthin chlorophyll a/c-bind‐ ing protein FCP genes are also useful for transient expression of the GUS gene, and the FCP promoter is also employable for the genetic transformation [ ]. Interestingly, there has been no successful genetic transformation using the CaMV S promoter, although this promoter is active in the transient transformation [ ]. 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 G [ , ]. Since hygromycin is more effective than chloramphenicol [ , ], it is necessary to confirm the utility of the SV -hptII gene for the selection of transformants to fully establish the genetic transformation system in kelp. Species Status of Gene transfer method Promoter expression Marker or Ref. Reporter Laminaria japonica transient particle bombardment CaMV 35S GUS [103] Laminaria japonica stable particle bombardment SV40 GUS [105] Laminaria japonica transient particle bombardment CaMV 35S, UBI, GUS [107] AMT Laminaria japonica stable particle bombardment FCP GUS [107] Laminaria japonica stable particle bombardment SV40 HBsAg [113] Laminaria japonica stable particle bombardment SV40 Rt-PA [114] Laminaria japonica stable particle bombardment SV40 bar [114] Undaria pinnatifida transient particle bombardment CaMV 35S GUS [103] Undaria pinnatifida transient particle bombardment SV40 GUS [104] Table 2. Transformation in brown seaweeds. 333 334 An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical Applications To date, stably transformed microalgae have been employed to produce recombinant anti‐ bodies, vaccines or bio-hydrogen as well as to analyze the gene functions targeted for engi‐ neering [ ]. Based on the success in genetic transformation, L. japonica is now proposed as a marine bioreactor in combination with the SV promoter [ ]. Indeed, the integration of human hepatitis B surface antigen HBsAg and recombinant human tissue-type plasmi‐ nogen rt-PA genes into the L. japonica genome resulted in the efficient expression of these genes under the direction of the SV promoter [ , ]. 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. There is no competitor against the Chinease group in the field of using brown algal genetic transformation at present [ , , ], 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 SV 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 SV -lacZ gene was used such as transgene, as described above [ , ]. . 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 [ , ]. The availability of electoroporation was then confirmed in C. reinhardtii and Chlorella saccharophila [ , ]. 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 [ ] and genetic transformation in the multicellular Volvox carteri and Gonium pectoral [ , ]. Agrobacterium-mediated transformation was also reported in H. pluvialis [ ]. Thus, all methods employed in land green plants are applicable for green microalgae [ ] see Table . In contrast, there is no report about genetic transformation in green seaweeds Table . To date, only two examples of transient transformation have been reported in green seaweeds, Ulva lactura by electroporation and U. pertusa by particle bombardment [ , ]. As shown in Table , some of the experiments with micro- and macro-green algae used the promoter of the CaMV S gene and the coding region of the E. coli GUS gene. Although functionality of the CaMV S 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 [ ]. In fact, codon-optimization is critical for the expression of reporters like the GFP gene and antibiotic-resistance genes in C. reinhardtii [ , , , ]. Moreover, the HSP A promoter was employed to increase the expression level of the reporter genes [ , ]. Therefore, 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 Current Advances in Seaweed Transformation http://dx.doi.org/10.5772/52978 in transient transformation of U. pertusa by particle bombardment [ ] however, it is still unclear whether the rbsS promoters and the EGFP gene work well in cells in comparison with the CaMV S promoter and codon-optimized EGFP gene. Species Status of Gene transfer method Promoter expression Marker or Ref. Reporter Microalga Chlamidominas stable particle bombardment stable glass bead agitation [116] reinhardtii Chlamidominas Nitrate reductase reinhardtii Chlamidominas Nitrate [117] reductase stable electroporation CaMV 35S CAT [118] stable glass bead agitation rbcS2 aphVIII [95] stable glass bead agitation β2-tubulin Aph7” [96] Chlorella saccharophila transient electroporation CaMV 35S GUS [119] Haematococcus pluvialis transient particle bombardment SV40 lacZ [120] Haematococcus pluvialis stable Agrobacterium-mediated CaMV 35S GUS,GFP, [123] reinhardtii Chlamidominas reinhardtii Chlamidominas reinhardtii gene transfer Volvox Carteri stable Volvox Carteri stable Volvox Carteri stable hptII particle bombardment β2-tubulin arylsulfatase [121] aphVIII [98] aphH [97] aphVIII [122] particle bombardment Hsp70A-rbcS2 glass bead agitation fusion particle bombardment β-tubulin, Hsp70A Gonium pectoral stable particle bombardment VcHsp70A Ulva lactuca transient electroporation CaMV 35S GUS [124] Ulva pertusa transient particle bombardment UprbcS EGFP [125] Seaweed Table 3. Transformation in green algae. 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. [ ] commented on the antibiotic sensitivity of green seaweeds, indicating the consider‐ able resistance of protoplast from Ulva and Monostroma to hygromycin and kanamycin. 335 336 An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical Applications Insensitivity to hygromycin is inconsistent with the case for red and brown seaweeds [ , ]. 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 years since the first transient transformation of a red seaweed with a circular expression plasmid [ ], 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 S-GUS gene in the pioneer attempts at system development as shown in Tables , and . This problem was recently resolved through the development of an efficient transient transformation system in P. yezoensis [ , ]. It is clear that the CaMV S promoter and the GUS gene are not active in seaweed cells [ ], which is supported by knowledge from green microalgae [ - ]. These findings strongly indicate that defects in the transfer and expression of foreign genes were resolved by knowledge about two critical factors required for reprodu‐ cibility and efficiency of transient gene expression, namely, the optimization of codon usage of coding regions and the employment of endogenous strong promoters [ , ]. 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 SV promoter Tables and [ , , , , , ] 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 transfor‐ mation 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 [ , ], 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 resist‐ ance 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. Current Advances in Seaweed Transformation http://dx.doi.org/10.5772/52978 Author details Koji Mikami* Address all correspondence to [email protected] Faculty of Fisheries Sciences, Hokkaido University, - - Minato, Hakodate, Japan References [ ] Griffith, F. The significance of pneumococcal types. Journal of Hygiene – . [ ] Avery OT, MacLeod CM, MaCarty M. Studies on the chemical nature of the sub‐ stance inducing transformation of Pneumococcal types induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus Type III. Journal of Experimental Medicine – . [ ] Mandel M, Higa A. 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