**4. 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* [97,120-122]. *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 *GU*S 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]. 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 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.


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

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

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

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

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* [97,120-122]. *Agrobacterium*-mediated transformation was also reported in *H. pluvialis* [123]. Thus, all methods employed in land green plants are applicable

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 *GU*S 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]. 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

which the *SV40-lacZ* gene was used such as transgene, as described above [91,92].

continually check the safety and value of its use by oral application.

**4. Transformation in green seaweeds**

Applications

334

for green microalgae [88] (see Table 3).

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.

Insensitivity to hygromycin is inconsistent with the case for red and brown seaweeds [101-103,106]. 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.

**Author details**

Address all correspondence to: komikami@fish.hokudai.ac.jp

Experimental Medicine 1944;79(2) 137–158.

United States of America 1982;79(7) 2268-7222.

of crown gall tumorigenesis. Cell 1977;11(2) 263-271.

Molecular Biology 1970;53(1) 159–162.

Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate, Japan

[1] Griffith, F. The significance of pneumococcal types. Journal of Hygiene 1928;27(2)

Current Advances in Seaweed Transformation

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

337

[2] 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

[3] Mandel M, Higa A. Calcium-dependent bacteriophage DNA infection. Journal of

[4] Griesbeck C, Kobl I, Heitzer M. *Chlamydomonas reinhardtii*. A protein expression sys‐ tem for pharmaceutical and biotechnological proteins. Molecular Biotechnology

[5] Torney F, Moeller L, Scarpa A, Wang K. Genetic engineering approaches to improve bioethanol production from maize. Current Opinion in Biotechnology 2007;18(3)

[6] Bhatnagar-Mathur P, Vadez V, Sharma KK. Transgenic approaches for abiotic stress tolerance in plants: retrospect and prospects. Plant Cell Reports 2008;27(3) 411-424.

[7] Doehmer J, Barinaga M, Vale W, Rosenfeld MG, Verma IM, Evans RM. Introduction of rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector: ex‐ pression and regulation. Proceedings of the National Academy of Sciences of the

[8] Smith EF, Townsend CO. A plant tumor of bacterial origin. Science 1907;25(643)

[9] Chilton MD, Drummond MH, Merio DJ, Sciaky D, Montoya AL, Gordon MP, Nester EW. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis

Koji Mikami\*

**References**

113–159.

2006;34(2) 213-223.

193-199.

671-673.
