**3. Transformation in brown seaweeds**

system and an efficient expression system for foreign genes have been resolved by devel‐ oping the transient transformation system in Bangiophyceae [29,30]. 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 [93], are generally used as selection markers. For example, the antibiotics hygromycin and geneticin (G418) are frequently used as selection agents with the hygromycin phos‐ photransferase (*hptII*) gene to inactivate hygromycin via an ATP-dependent phosphoryla‐ tion [94] and the neomycin phosphotransferase II (*nptII*) gene to detoxify neomycin, G418 and paromomycin [93], respectively. In the green alga *Chlamydomonas reinhardtii,* the hy‐ gromycin phosphotransferase (*aph7"*) 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 [95-97]. 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 amino‐ glycoside 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 un‐ known what kinds of antibiotics-based selection marker genes are available for red sea‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

weeds, since red algae usually have strong resistance to antibiotics.

near future.

Applications

332

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* gameto‐ phytes 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 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 ex‐ pression 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 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 [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.


**Table 2.** Transformation in brown seaweeds.

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 [108-111]. 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 plasmi‐ nogen (*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.

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

**Gene transfer method Promoter Marker or**

stable particle bombardment [116]

stable electroporation CaMV 35S CAT [118]

stable glass bead agitation rbcS2 aphVIII [95]

stable glass bead agitation β2-tubulin Aph7" [96]

stable glass bead agitation Nitrate reductase Nitrate

*Chlorella saccharophila* transient electroporation CaMV 35S GUS [119] *Haematococcus pluvialis* transient particle bombardment SV40 lacZ [120]

*Volvox Carteri* stable particle bombardment β2-tubulin arylsulfatase [121]

*Gonium pectoral* stable particle bombardment VcHsp70A aphVIII [122]

*Ulva lactuca* transient electroporation CaMV 35S GUS [124] *Ulva pertusa* transient particle bombardment UprbcS EGFP [125]

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 consider‐ able resistance of protoplast from *Ulva* and *Monostroma* to hygromycin and kanamycin.

glass bead agitation

gene transfer

**Reporter**

Current Advances in Seaweed Transformation

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

reductase

CaMV 35S GUS,GFP,

Hsp70A-rbcS2 fusion

Hsp70A

hptII

aphVIII [98]

aphH [97]

**Ref.**

335

[117]

[123]

the *CaMV 35S* promoter and codon-optimized *EGFP* gene.

**expression**

*Haematococcus pluvialis* stable *Agrobacterium-*mediated

*Volvox Carteri* stable particle bombardment

*Volvox Carteri* stable particle bombardment β-tubulin,

**Species Status of**

**Microalga** *Chlamidominas reinhardtii*

*Chlamidominas reinhardtii*

*Chlamidominas reinhardtii*

*Chlamidominas reinhardtii*

*Chlamidominas reinhardtii*

**Seaweed**

**Table 3.** Transformation in green algae.

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].
