**5. 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 [54-65]. These findings strongly indicate that defects in the transfer and expression of foreign genes were resolved by knowledge about two critical factors required for reprodu‐ cibility 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) [91,92,103,105-107,113,114]; 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 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 [101-103,106], 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 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.
