**2. Transformation in red seaweeds**

mation mediated by *A. tumefaciens* has become the most commonly used method to transmit

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

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 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 [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

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-3 polyunsaturated fatty acids, porphyran, ager and carrageenan from red algae, and fucoxantine, fucoidan and alginate from brown algae [19-22]. 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 [25-28]. 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. 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‐

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

DNA fragment into higher plants [12].

Applications

324

available in land green plants.

tion system in seaweeds.

### **2.1. 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.

To date, studies have been mainly focused on *Porphyra* species because of their economical values. As shown in Table 1, expression of the *CaMV 35S-GUS* gene was previously observed in *P. miniata*, *P. tenera* and *P. yezoensis*[37-42], all of which were performed by electoroporation using protoplasts. Kuang et al. [38] also tested the particle bombardment of the *CaMV 35S-GUS* gene in *P. yezoensis* and got positive results. Moreover, the availability of mammaliantype simian virus 40 (*SV40*) promoter was reported to express the *E. coli lacZ* reporter gene, encoding β-galactosidase cleaving colorless substrate X-gal (5-bromo-4-chloro-3-indolyl-βgalactopyranoside) to produce a blue insoluble product [43], in *P. haitanensis*, *Gracilaria chagii* and *K. alvarezii* by electroporation or particle bombardment [44,45].

### **2.2. 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 [25,37-41]. As a result, the transient transformation system in red seaweeds has recently been improved by resolving this problem.

*2.2.1. Optimization of codon usage in the reporter gene*

enable the efficient expression of this gene in *P. yezoensis* cells.

*2.2.2. Employment of endogenous strong promoters*

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 [49-51]. 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

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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 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 [29,30,48]. Optimization of the codon usage of the reporter gene is therefore one

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 [57-59]. 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 [60-65]. Therefore, it is likely that efficient expression of the *PyGUS* reporter gene

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 gameto‐ phytic 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 endog‐ enous 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

of the important factors for successful expression in *P. yezoensis* cells [29,30,48].

in *P. yezoensis* cells is caused by the recruitment of endogenous strong promoters.

*\*Porphyra species* used are *P. yezoensis, P. tenera, P. okamurae, P. onoi, P. variegate* and *P. pseudolinearis.*

**Table 1.** Transformation in red seaweeds.

### *2.2.1. Optimization of codon usage in the reporter gene*

**Species Status of**

Applications

326

Porphyra tenera Porphyra yezoensis

Porphyra species\* Bangia fuscopurpurea

Porphyra species\* Bangia fuscopurpurea **expression**

*Porphyra yezoensis* transient electroporation CaMV 35S

*Porphyra yezoensis* transient particle bombardment CaMV 35S

*Porphyra yezoensis* stable *Agrobacterium-*mediated

*Porphyra yezoensis* stable *Agrobacterium-*mediated

**Table 1.** Transformation in red seaweeds.

*Porphyra yezoensis* transient Electroporation

**Gene transfer method Promoter Marker or Reporter Ref.**

β-tubulin

PyGAPDH

PyGAPDH

transient particle bombardment PtHSP70 PyGUS [87]

CaMV 35S GUS [38]

GUS [42]

PyGUS [48]

[71]

[86]

[131]

ZsYFP, sGFP(S65T)

sGFP(S65T)

CaMV 35S GUS [26]

(unknown) (unknown) [28]

EGFP

PyGUS [85]

*Kappaphycus alvarezii* transient particle bombardment CaMV 35S GUS [25] *Porphyra miniata* transient electroporation CaMV 35S GUS [37]

*Porphyra tenera* transient electroporation CaMV 35S GUS [39] *Porphyra yezoensis* transient electroporation rbcS GUS [40] *Porphyra yezoensis* transient electroporation CaMV 35S GUS [41]

*Gracilaria changii* transient particle bombardment SV40 lacZ [44] *Porphyra haitanensis* transient SV40 CAT [128] *Porphyra yezoensis* transient electriporation SV40 CAT, GUS [129] *Porphyra yezoensis* transient electroporation Rubisco GUS, sGFP(S65T) [130]

*Porphyra yezoensis* transient particle bombardment PyAct1 PyGUS [66] *Porphyra yezoensis* transient particle bombardment PyAct1 AmCFP [70]

*Porphyra yezoensis* transient particle bombardment PyAct1 AmCFP, ZsGFP,

gene transfer

gene transfer

*\*Porphyra species* used are *P. yezoensis, P. tenera, P. okamurae, P. onoi, P. variegate* and *P. pseudolinearis.*

*Porphyra haitanensis* stable glass bead agitation SV40 lacZ

*Porphyra leucostica* stable ekectroporation CaMV 35S lacZ [27]

*Kappaphycus alvarezii* stable particle bombardment SV40 lacZ [45]

*Gracilaria changii* stable particle bombardment SV40 lacZ [91] *Gracilaria gracilis* stable particle bombardment SV40 lacZ [92]

transient particle bombardment PtHSP70

transient particle bombardment PyAct1 PyGUS

particle bombardment

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

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 [49-51]. 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 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 [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].

#### *2.2.2. 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 [57-59]. 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 [60-65]. 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 gameto‐ phytic 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 endog‐ enous 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.

### *2.2.3. 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 [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].

**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

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**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.

panels show bright field and fluorescence images, respectively. Scale bar corresponds to 5 μm.

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* [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)P2, PtdIns(3,5)P2 and PtdIns(4,5)P2] and PtdIns-trisphosphate [PtdIns(3,4,5)P3], all of which are detectable in plants except for PtdIns(3,4,5)P3 [73,74]. 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 [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 [78-80]. For instance, the PH domains from human phospholipase Cδ1 (PLCδ1) are employed for the detection of PtdIns(4,5)P2 [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)P2 and PtdIns(3,4,5)P3 [82]. Because of this substrate specificity, we were able to visualize PtdIns(3,4)P2 and PtdIns(4,5)P2 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].

codon optimized reporter genes is critical for successful transient transformation in *Porphyra* species [29,30]. The established procedure of transient transformation is schematically

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

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 [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

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* [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

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)P2, PtdIns(3,5)P2 and PtdIns(4,5)P2] and PtdIns-trisphosphate [PtdIns(3,4,5)P3], all of which are detectable in plants except for PtdIns(3,4,5)P3 [73,74]. 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 [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 [78-80]. For instance, the PH domains from human phospholipase Cδ1 (PLCδ1) are employed for the detection of PtdIns(4,5)P2 [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)P2 and PtdIns(3,4,5)P3 [82]. Because of this substrate specificity, we were able to visualize PtdIns(3,4)P2 and PtdIns(4,5)P2 at the plasma membrane with humanized AmCFP and ZsGFP fused to the PH domains from PLCδ1 and Akt1 via the direction of the *PyAct1*

*2.2.3. Application of the transient transformation for using fluorescent proteins*

represented in Figure 2.

Applications

328

imaging in cells [68,69].

[71] (see Figure 1B).

promoter [70].

**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.

Moreover, subcellular localization of transcription factors was also visualized in *P. ye‐ zoensis*. 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 gameto‐ phytic cells, which was confirmed by overlapping of fluorescent signals with SYBR Gold staining of the nucleus [71]

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

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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

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 *URA5.3* gene fragment as a

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

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

sophisticated system for red algal genetic transformation.

**2.3. Towards genetic transformation in red seaweeds**

has not yet been fully established in red macroalgae.

promoter.

selection maker [90].

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].

### *2.2.4. 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 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 re‐ cently 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 expres‐ sion of *PyGUS* and *sGFP(S65T)* genes was observed in Bangiophyceae including *P. ten‐ era, P. okamurae, P. psedolinearis* and *Bangia fuscopurpurea*, although the expression efficiency varied among species [86]. Thus, the transient transformation system devel‐ oped 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 availa‐ bility 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 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(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.

### **2.3. Towards genetic transformation in red seaweeds**

Moreover, subcellular localization of transcription factors was also visualized in *P. ye‐ zoensis*. 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 gameto‐ phytic cells, which was confirmed by overlapping of fluorescent signals with SYBR Gold

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

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

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 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 re‐ cently 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 expres‐ sion of *PyGUS* and *sGFP(S65T)* genes was observed in Bangiophyceae including *P. ten‐ era, P. okamurae, P. psedolinearis* and *Bangia fuscopurpurea*, although the expression efficiency varied among species [86]. Thus, the transient transformation system devel‐

*2.2.4. Applicability of the P. yezoensis transient transformation system in other red seaweeds*

oped 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 availa‐ bility 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 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(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

staining of the nucleus [71]

Applications

330

functions of genes and cellular components [29,30].

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 *URA5.3* gene 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 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‐ weeds, since red algae usually have strong resistance to antibiotics.

**3. Transformation in brown seaweeds**

promoter is active in the transient transformation [103].

**expression**

**Species Status of**

**Table 2.** 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 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

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

*Laminaria japonica* transient particle bombardment CaMV 35S GUS [103] *Laminaria japonica* stable particle bombardment SV40 GUS [105]

*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]

**Gene transfer method Promoter Marker or**

AMT

**Reporter**

Current Advances in Seaweed Transformation

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

333

GUS [107]

**Ref.**

transformants to fully establish the genetic transformation system in kelp.

*Laminaria japonica* transient particle bombardment CaMV 35S, UBI,

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 near future.


**1. Introduction**

As is known, high-strength concrete currently includes concrete with a compressive strength of 60–100 MPa and higher at the age of 28 days [1,2]. With the use of classic technology, obtaining high-strength concrete is possible by ensuring the high quality of raw materials, low values of the water-cement ratio, and sufficient compaction of the concrete mixture. In practice, these requirements are achieved by reducing the water content of the concrete mixture when using stiff mixtures, or by introducing plasticizing additives, increasing the activity and specific surface of cement, introducing hardening accelerators, reducing water consumption, and optimizing the grain composition of aggregates with high physical and mechanical properties [1–6].

The technology of high-strength concrete of the new generation involves the introduction of superplasticizer additives with a high (20–40%) water-reducing effect and dispersed active mineral fillers into the composition of concrete mixtures. These components provide both important individual effects, namely achieving extremely low values of the watercement ratio while maintaining ease of installation, an increase in the volume of hydration products, the degree of cement hydration, and a significant synergistic effect. With the simultaneous introduction of superplasticizer additives and a dispersed active filler, the rheological potential of the plasticizing additive is fully realized on the one hand, whereas on the other hand, in the conditions of a limited water-cement ratio, the positive effect of the dispersed active filler on the structure formation of cement stone and concrete is manifested [7–10].

In hydration systems, the physical interaction of cement particles and the filler is significantly affected by the so-called "compressed conditions" [11], which are characterized by a sharp increase in the concentration of the solid phase and the transition of part of the volume water into film water. At the same time, the change in free surface energy

**Citation:** Dvorkin, L.; Zhitkovsky, V.; Marchuk, V.; Makarenko, R. High-Strength Concrete Using Ash and Slag Cements. *Mater. Proc.* **2023**, *13*, 16. https://doi.org/10.3390/ materproc2023013016

Academic Editors: Katarzyna Mróz, Tomasz Tracz, Tomasz Zdeb and Izabela Hager

Published: 14 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

between the solid and liquid phases contributes to the intensive formation of crystal nuclei of hydration products, which is in accordance with the Gibbs–Volmer theory [12]. With the optimal concentration and dispersion of the active additive, a fine-grained structure of the binder is formed, which has a positive effect on the technical properties of the artificial stone.

The structuring effect of dispersed additives in concrete increases when the physicochemical activity of their surface increases. Methods of mechanical activation of additives include grinding them with cement in the presence of plasticizers and, if necessary, other chemical admixtures. This method of activation is the basis for obtaining composite cement with low water consumption [13].

An important indicator of the quality of concrete as a structural material is the specific consumption of cement per unit of concrete strength. In traditional concretes, this indicator is on average close to 10 kg/MPa, in new generation concretes it decreases two or more times [7–9].

The best active fillers for high-strength concrete, as demonstrated by many studies and by practice, are microsilica additives [14,15]. At the same time, the use of microsilica is complicated due to the need for its granulation or briquetting for transportation and dosing, as well as to significant fluctuations in its composition and properties.

As regards active fillers for high-strength concrete, the use of dispersed siliceous and aluminosilicous materials based on local raw materials and industrial waste is a practical interest [14,16]. Among such materials, the most common is coal fly ash and granulated blast furnace slag. The main task of our work was to figure out the technological parameters for obtaining high-strength concrete using composite ash and slag-containing cement and to research their complex structure and technical properties.


**3. Discussion**

In this study, the roots of *S. baicalensis* seedlings treated with white LED light contained the highest levels of baicalin, baicalein, and wogonin and lower levels of most sugars than the other plant parts, suggesting the need for energy to enhance the biosynthesis of phenolic compounds, including the three described here. These results agree with previous studies showing that sugar concentrations for anthocyanin accumulation were lower in purple kohlrabi than in green kohlrabi [14] and that a fungal elicitor allowed for the more rapid depletion of sugar pools to promote alkaloid biosynthesis in cell cultures of *Papaver somniferum* [15].

Numerous previous studies have reported that LED illumination can enhance secondary metabolite production in vegetables and medicinal plants. White LED illumination has been shown to increase the accumulation of phenolics in *Agastache rugosa* seedlings [16], carotenoids in *Fagopyrum tataricum* sprouts [17], and glucosinolates in *Brassica juncea* sprouts [11], compared with other colored LED lights, consistent with the findings of this study. Blue LED light has been reported to increase accumulations of phenolics in *Brassica napus* [18] and *Glycine max* sprouts [19], and red LED light has been shown to enhance both phenolic compounds in the leaves of *Myrtus communis* in vitro [20] and carotenoid production in the outer peel layer of citrus fruit [21].

The metabolic networks of glutamine, glutamate, aspartate, and asparagine are involved in various nitrogen-related processes, including nitrogen assimilation by plants, metabolism into amino acids and other nitrogen-containing compounds, transport between source and sink, stress-associated metabolism, and carbon-nitrogen partitioning. Glutamine is derived from ammonium assimilation and can be converted into glutamic acid with α-ketoglutarate, a TCA cycle intermediate. This glutamic acid is further metabolized into aspartic acid, which is converted to asparagine. The first three compounds can be used for the synthesis of proteinogenic and non-proteinogenic amino acids, amides, and other nitrogenous compounds. Asparagine is a prominent nitrogen transport agent as well as a proteinogenic amino acid [22–24].

In this study, glutamate, glutamine, asparagine, and aspartate showed decreasing concentration levels in the roots, leaves, and stems of *S. baicalensis* seedlings treated with different LED lights, with a related reduction of their derivatives. This result was supported by the positive correlations between these four compounds and their derivatives. Furthermore, since shikimate and phenylalanine, which are derived from the shikimate pathway, were negatively correlated with most phenolic compounds, the biosynthesis of these compounds, including phenolic acids and flavones must have been assisted by intermediates or precursors. These findings corroborate a previous study reporting that the internal pool of phenylalanine was lower in purple kohlrabi, which contained a high amount of phenolic compounds, reflecting a precursor supply to produce phenolic acids and anthocyanins [14].

Artificial LED source is important to regulate the lighting systems in a plant factory to produce high-quality plant materials. Therefore, this study suggests that *S. baicalensis* seedlings, containing a high number of health-beneficial compounds, can be produced under LED lights in limited space since *S. baicalensis* was generally cultivated in the field and indicates that the optimal light was white LED for flavone accumulation in *S. baicalensis* seedlings.


**3. Domestication of diadromous and marine fish species**

#### **3.1. The Atlantic salmon (***Salmo salar***)**

The farming of the Atlantic salmon started in the early nineteenth century in the United Kingdom in order to rebuild river populations for angling [9]. Nevertheless, this is only at the end of the 1960s that the farming in sea cages was used for the first time in Norway [57]. From the year 1970, the first ever family-based breeding program of the Atlantic salmon was initiated in Norway in a brand new research institute entitled AKVAFORSK [57, 58]. Even though the production of the Atlantic salmon was only 100 tons in Norway at this time, the equivalent of about 3.6 million US\$ was covered for two-thirds by the Norwegian government and the rest by industry (not working in the aquaculture field) and nongovernmental organizations [57]. In parallel, research programs were started to develop new dry pellet feed adapted to the Atlantic salmon, which were available from 1982 [57]. After about 40 years of farming, the time to produce a standard market-sized 4 kg fish has been halved, and while 3 kg of dry matter (in moist feed) was necessary to produce 1 kg of salmon in the beginning, this has also been reduced to 1.15 kg (dry pellets) [37]. Other traits were progressively added to the selection index such as age at sexual maturity, disease resistance, stress resistance, and quality of the flesh [10, 37, 57, 58]. From the early stages of the selective breeding programs, eggs and juveniles were sold to the industry resulting in that close to 100% of the production of the Atlantic salmon in Norway and in the rest of the world are now based on improved stocks [10, 57, 58]. Farmed salmon is regarded as one of the most domesticated fish species farmed for food, and one Norwegian strain has been exposed to ≥12 generations of domestication [59, 60].

produce the Atlantic salmon, among which the four leading countries are Norway, Chile, the United Kingdom, and Canada [19, 47, 62]. In Norway and Chile (where this species was introduced), the farming of the Atlantic salmon has an enormous economic importance [63, 64]. In Norway, the farming of Atlantic salmon is the third largest industry after petroleum and mining [47]. In Chile, the exports of the Atlantic salmon represent about two-thirds of the total Chilean fisheries exports and became the third most important export commodity after copper and wood products [47]. Most part of the production (on-growing) is now performed in sea cages [19, 61], with hatcheries working with either flow-through or recirculating aqua-

Fish Domestication: An Overview

79

http://dx.doi.org/10.5772/intechopen.79628

The Atlantic salmon has evolved in few decades from a luxury item, which was consumed only at specific periods in the year (particularly at Christmas), to convenience products [9]. He has become in 2014 the ninth most produced aquatic products globally, with more than 2.3 million tons. The success of its farming in Norway (and in Chile) is mainly due to the presence of numerous suitable sites for its production, a dynamic research and industry as well as government support [9]. Besides, the Atlantic salmon displays several features (high growth in cages, flesh strongly appreciated by consumers, etc.) that contribute to its farming success [9]. In parallel, the capture by fisheries has strongly decreased from 118,000 tons in 1980 to 73,000 tons in 1990 to less than 40,000 ton in 2000 [47]. Today, almost 100% of the Atlantic salmon is

The traditional farming of the European seabass in the Mediterranean Sea consisted of collecting juveniles in the wild and releasing them in semi-artificial coastal lagoons, such as "Vallicoltura" in Italy [66, 67]. Within these lagoons, seabass was reared extensively [68]. Nevertheless, from the year 1960, in the face of strong competition for wild juveniles between the on-growers and the decrease of natural resources [66], the first rearing trials were initiated in France and Italy [69]. In the middle of the 1980s, the development of reliable methods of reproduction techniques and husbandry methods allowed higher survival [68], and their diffusion rapidly led to the development of a true industry in several countries along the Mediterranean Sea [66, 68]. In the 1990s, the first breeding programs were initiated, first in France and Israel, then in Greece, Spain, and Italy, which allow obtaining the eighth generation of selection in the oldest program [67]. However, a large proportion of the broodstock used today is still coming from wild breeders or first-generation individuals

The aquaculture production of seabass was almost inexistent in 1950 (**Figure 7**). With the control of the life cycle in captivity, the aquaculture production increased exponentially from the middle of 1980 to reach 134,711 tons in 2010 (**Figure 7**). In the same period of time, the capture by fisheries increased from 4460 tons in 1980 to 10,853 tons in 2010 [9]. Consequently, 9 of 10 seabasses consumed in the world are now farmed. The main producers are all located around the Mediterranean Sea: Turkey, Greece, Egypt, Spain, and Italy [9]. Most of the production (on-growing) is realized in sea cages, followed by tanks and lagoon [68].

culture systems [65].

coming from farming [47].

[66, 69, 70].

**3.2. The European seabass (***Dicentrarchus labrax***)**

In parallel to its domestication, the production was multiplied by 5000 (**Figure 6**) from 294 tons in 1970 to more than 1.4 million tons in 2010 [19, 61]. A dozen of countries currently

**Figure 6.** Global aquaculture production from 1950 to 2016 of the Atlantic salmon *Salmo salar* (data from the FAO database).

produce the Atlantic salmon, among which the four leading countries are Norway, Chile, the United Kingdom, and Canada [19, 47, 62]. In Norway and Chile (where this species was introduced), the farming of the Atlantic salmon has an enormous economic importance [63, 64]. In Norway, the farming of Atlantic salmon is the third largest industry after petroleum and mining [47]. In Chile, the exports of the Atlantic salmon represent about two-thirds of the total Chilean fisheries exports and became the third most important export commodity after copper and wood products [47]. Most part of the production (on-growing) is now performed in sea cages [19, 61], with hatcheries working with either flow-through or recirculating aquaculture systems [65].

The Atlantic salmon has evolved in few decades from a luxury item, which was consumed only at specific periods in the year (particularly at Christmas), to convenience products [9]. He has become in 2014 the ninth most produced aquatic products globally, with more than 2.3 million tons. The success of its farming in Norway (and in Chile) is mainly due to the presence of numerous suitable sites for its production, a dynamic research and industry as well as government support [9]. Besides, the Atlantic salmon displays several features (high growth in cages, flesh strongly appreciated by consumers, etc.) that contribute to its farming success [9]. In parallel, the capture by fisheries has strongly decreased from 118,000 tons in 1980 to 73,000 tons in 1990 to less than 40,000 ton in 2000 [47]. Today, almost 100% of the Atlantic salmon is coming from farming [47].

#### **3.2. The European seabass (***Dicentrarchus labrax***)**

**3. Domestication of diadromous and marine fish species**

The farming of the Atlantic salmon started in the early nineteenth century in the United Kingdom in order to rebuild river populations for angling [9]. Nevertheless, this is only at the end of the 1960s that the farming in sea cages was used for the first time in Norway [57]. From the year 1970, the first ever family-based breeding program of the Atlantic salmon was initiated in Norway in a brand new research institute entitled AKVAFORSK [57, 58]. Even though the production of the Atlantic salmon was only 100 tons in Norway at this time, the equivalent of about 3.6 million US\$ was covered for two-thirds by the Norwegian government and the rest by industry (not working in the aquaculture field) and nongovernmental organizations [57]. In parallel, research programs were started to develop new dry pellet feed adapted to the Atlantic salmon, which were available from 1982 [57]. After about 40 years of farming, the time to produce a standard market-sized 4 kg fish has been halved, and while 3 kg of dry matter (in moist feed) was necessary to produce 1 kg of salmon in the beginning, this has also been reduced to 1.15 kg (dry pellets) [37]. Other traits were progressively added to the selection index such as age at sexual maturity, disease resistance, stress resistance, and quality of the flesh [10, 37, 57, 58]. From the early stages of the selective breeding programs, eggs and juveniles were sold to the industry resulting in that close to 100% of the production of the Atlantic salmon in Norway and in the rest of the world are now based on improved stocks [10, 57, 58]. Farmed salmon is regarded as one of the most domesticated fish species farmed for food, and

one Norwegian strain has been exposed to ≥12 generations of domestication [59, 60].

In parallel to its domestication, the production was multiplied by 5000 (**Figure 6**) from 294 tons in 1970 to more than 1.4 million tons in 2010 [19, 61]. A dozen of countries currently

**Figure 6.** Global aquaculture production from 1950 to 2016 of the Atlantic salmon *Salmo salar* (data from the FAO database).

**3.1. The Atlantic salmon (***Salmo salar***)**

78 Animal Domestication

The traditional farming of the European seabass in the Mediterranean Sea consisted of collecting juveniles in the wild and releasing them in semi-artificial coastal lagoons, such as "Vallicoltura" in Italy [66, 67]. Within these lagoons, seabass was reared extensively [68]. Nevertheless, from the year 1960, in the face of strong competition for wild juveniles between the on-growers and the decrease of natural resources [66], the first rearing trials were initiated in France and Italy [69]. In the middle of the 1980s, the development of reliable methods of reproduction techniques and husbandry methods allowed higher survival [68], and their diffusion rapidly led to the development of a true industry in several countries along the Mediterranean Sea [66, 68]. In the 1990s, the first breeding programs were initiated, first in France and Israel, then in Greece, Spain, and Italy, which allow obtaining the eighth generation of selection in the oldest program [67]. However, a large proportion of the broodstock used today is still coming from wild breeders or first-generation individuals [66, 69, 70].

The aquaculture production of seabass was almost inexistent in 1950 (**Figure 7**). With the control of the life cycle in captivity, the aquaculture production increased exponentially from the middle of 1980 to reach 134,711 tons in 2010 (**Figure 7**). In the same period of time, the capture by fisheries increased from 4460 tons in 1980 to 10,853 tons in 2010 [9]. Consequently, 9 of 10 seabasses consumed in the world are now farmed. The main producers are all located around the Mediterranean Sea: Turkey, Greece, Egypt, Spain, and Italy [9]. Most of the production (on-growing) is realized in sea cages, followed by tanks and lagoon [68].

industry is entirely based on the stocking of wild-caught specimens, which are reared in cages

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Despite the lack of control of the full life cycle in captivity, the aquaculture production has evolved very quickly from the year 1990 to reach 4080 tons in 2010 (**Figure 8**). More than 10 countries around the Mediterranean Sea currently produce this species, that is, around 60–70 farms [72 - 74]. The leading producers are Croatia, Malta, Turkey, Spain, and Tunisia [77, 78]. The first kind of production, which dates back to the 1980s, is based on the capture of breeders, whose weight varies between 40 and 400 kg, during the migration season, most often close to the spawning areas [73]. Those large individuals are then transported at low speed (1–1.5 knots) over distances ranging from few to 100 km, which might sometimes take several weeks, before being transferred to very large sea cages [73, 79]. Within those very large rearing cages, which may reach 50–60 m of diameter (sometimes even larger than 100 m) and 20–35 m deep, fish are fattened during several months, time required to rebuild muscle fat content that confers to the high-quality flesh researched on the Japanese market of sushi and sashimi [71, 73, 76]. During the middle of 1990s, a second type of production was initiated and consists of capturing immature individuals (8–20 kg in body weight), which are then reared during about 2–3 years in smaller sea cages than for breeders (50–60 m of diameter for about 20 m deep) to get fish of 30–50 kg [73, 76]. This type of production is mainly developed in the Adriatic Sea, particularly in Croatia [71, 76]. In all rearing systems, tunas are most often feed small forage fishes, among which sardinella (*Sardinella aurita*), pilchard (*Sardina pilchardus*), herring (*Clupea harengus*), mackerel (*Scomber scombrus*), chub mackerel (*Scomber japonicas*), horse mackerel (*Trachurus* sp.), bogue (*Boops boops*), and some cephalopods [71, 73, 75, 78, 79]. The feed conversion rates, which are estimated on a wet feed/wet tuna biomass, are most often high and vary between 10 and 20:1 [73, 78]. In other words, it requires 20 kg of forage fish to make 1 kg of tuna [73]. These feed conversion rates may be even beyond 40:1 for large specimens [73]. Two close relative species to the Atlantic bluefin tuna are also currently produced in other geographic areas using similar methods, namely the Northern Pacific bluefin tuna (*Thunnus orientalis*) in Mexico [78, 79] and the Southern bluefin tuna (*Thunnus maccoyii*) in Australia [80]. In 2002, Japanese scientists were able to obtain eggs and larvae of *T. orientalis* in an artificial setting; yet, there is not a broad knowledge of how to culture tuna in captive conditions, and much research is

during a period varying from few months to 2 or 3 years [72–76].

needed to consistently control the entire life cycle in captivity [78].

wild stocks [72, 73, 80].

**4. Conclusions**

In conclusion, the aquaculture production of Atlantic bluefin tuna has strongly increased within the past two decades, mainly driven by the Japanese market [78], its high commercial value associated with its high growth (30 kg in 3 years) [73]. However, the production of this species is based on the capture of wild individuals in nature; thus, only a control of the entire life cycle could ensure the sustainability of the industry through a reduction in its reliance on

The strong increase of the aquaculture production since the early 1980s has relied chiefly on the domestication of an increasing number of fish species [12, 13, 81–85]. Nevertheless, only a limited number has reached a high level of domestication (**Table 1**), such as the rainbow trout,

**Figure 7.** Global aquaculture production from 1950 to 2016 of the seabass *Dicentrarchus labrax* (data from the FAO database).

The seabass has become in less than three decades the second most produced fish species in the Mediterranean Sea, just after the sea bream *Sparus aurata* (whose production has followed a similar trend).

#### **3.3. The Atlantic bluefin tuna (***Thunnus thynnus***)**

The farming of the Atlantic bluefin tuna is recent [9]. The first trials of farming date back to the 1970s in Canada, Japan, and Australia [71]. Nevertheless, the aquaculture production truly started in the middle of the 1980s in the Mediterranean Sea, with the evolution of new technics allowing to provide fish to fattening farms [71, 72]. Despite significant progress, notably thanks to the work of a consortium of European researchers, the reliable control of the life cycle of the Atlantic bluefin tuna in captivity was never reached [73, 74]. Consequently, this

**Figure 8.** Global aquaculture production from 1950 to 2016 of the Atlantic bluefin tuna *Thunnus thynnus* (data from the FAO database).

industry is entirely based on the stocking of wild-caught specimens, which are reared in cages during a period varying from few months to 2 or 3 years [72–76].

Despite the lack of control of the full life cycle in captivity, the aquaculture production has evolved very quickly from the year 1990 to reach 4080 tons in 2010 (**Figure 8**). More than 10 countries around the Mediterranean Sea currently produce this species, that is, around 60–70 farms [72 - 74]. The leading producers are Croatia, Malta, Turkey, Spain, and Tunisia [77, 78]. The first kind of production, which dates back to the 1980s, is based on the capture of breeders, whose weight varies between 40 and 400 kg, during the migration season, most often close to the spawning areas [73]. Those large individuals are then transported at low speed (1–1.5 knots) over distances ranging from few to 100 km, which might sometimes take several weeks, before being transferred to very large sea cages [73, 79]. Within those very large rearing cages, which may reach 50–60 m of diameter (sometimes even larger than 100 m) and 20–35 m deep, fish are fattened during several months, time required to rebuild muscle fat content that confers to the high-quality flesh researched on the Japanese market of sushi and sashimi [71, 73, 76]. During the middle of 1990s, a second type of production was initiated and consists of capturing immature individuals (8–20 kg in body weight), which are then reared during about 2–3 years in smaller sea cages than for breeders (50–60 m of diameter for about 20 m deep) to get fish of 30–50 kg [73, 76]. This type of production is mainly developed in the Adriatic Sea, particularly in Croatia [71, 76]. In all rearing systems, tunas are most often feed small forage fishes, among which sardinella (*Sardinella aurita*), pilchard (*Sardina pilchardus*), herring (*Clupea harengus*), mackerel (*Scomber scombrus*), chub mackerel (*Scomber japonicas*), horse mackerel (*Trachurus* sp.), bogue (*Boops boops*), and some cephalopods [71, 73, 75, 78, 79]. The feed conversion rates, which are estimated on a wet feed/wet tuna biomass, are most often high and vary between 10 and 20:1 [73, 78]. In other words, it requires 20 kg of forage fish to make 1 kg of tuna [73]. These feed conversion rates may be even beyond 40:1 for large specimens [73]. Two close relative species to the Atlantic bluefin tuna are also currently produced in other geographic areas using similar methods, namely the Northern Pacific bluefin tuna (*Thunnus orientalis*) in Mexico [78, 79] and the Southern bluefin tuna (*Thunnus maccoyii*) in Australia [80]. In 2002, Japanese scientists were able to obtain eggs and larvae of *T. orientalis* in an artificial setting; yet, there is not a broad knowledge of how to culture tuna in captive conditions, and much research is needed to consistently control the entire life cycle in captivity [78].

In conclusion, the aquaculture production of Atlantic bluefin tuna has strongly increased within the past two decades, mainly driven by the Japanese market [78], its high commercial value associated with its high growth (30 kg in 3 years) [73]. However, the production of this species is based on the capture of wild individuals in nature; thus, only a control of the entire life cycle could ensure the sustainability of the industry through a reduction in its reliance on wild stocks [72, 73, 80].

### **4. Conclusions**

**Figure 8.** Global aquaculture production from 1950 to 2016 of the Atlantic bluefin tuna *Thunnus thynnus* (data from the

The seabass has become in less than three decades the second most produced fish species in the Mediterranean Sea, just after the sea bream *Sparus aurata* (whose production has fol-

**Figure 7.** Global aquaculture production from 1950 to 2016 of the seabass *Dicentrarchus labrax* (data from the FAO

The farming of the Atlantic bluefin tuna is recent [9]. The first trials of farming date back to the 1970s in Canada, Japan, and Australia [71]. Nevertheless, the aquaculture production truly started in the middle of the 1980s in the Mediterranean Sea, with the evolution of new technics allowing to provide fish to fattening farms [71, 72]. Despite significant progress, notably thanks to the work of a consortium of European researchers, the reliable control of the life cycle of the Atlantic bluefin tuna in captivity was never reached [73, 74]. Consequently, this

FAO database).

lowed a similar trend).

database).

80 Animal Domestication

**3.3. The Atlantic bluefin tuna (***Thunnus thynnus***)**

The strong increase of the aquaculture production since the early 1980s has relied chiefly on the domestication of an increasing number of fish species [12, 13, 81–85]. Nevertheless, only a limited number has reached a high level of domestication (**Table 1**), such as the rainbow trout, the Nile tilapia, or the Atlantic salmon [83–85]. The 35 species classified at Level 5 [12] belong to 10 families, among which Cyprinidae (*n* = 10 species), Salmonidae (*n* = 8), and Acipenseridae (*n* = 5) [83]. For these species, the entire life cycle is controlled in captivity, and breeding programs have allowed improving, among others, growth, with average genetic gains comprised between 10 and 15% per generation [37, 58, 85–87]. Today, it is estimated that about 10% of the global production is based on improved individuals [37, 87–89]. Nevertheless, very often, even for the species that have reached Level 4 or 5 (**Table 1**), a significant part of global production is based on the introduction of wild individuals. Conversely to these few domesticated species, or more accurately domesticated populations, the majority of farmed fish species still rely on the regular inputs of wild individuals (**Table 1**); thus, there is no strong dichotomy within the same species between wild individuals (coming from fisheries) and farmed individuals (produced in aquaculture) [90–93]. Besides, for numerous species, aquaculture is not a true alternative to capture fisheries but rather a mean to produce wild individuals to a certain commercial size by strongly decreasing the high-mortality rate characteristics of wild populations [90, 94]. Most farmed fish are thus still relatively similar to their wild congeners [95, 96].

intensity level of farming, from extensive to highly intensive, and industrialization are also very diverse [18]. From an activity mainly artisanal, aquaculture has evolved to include very large companies that export in numerous countries [17, 103]. According to FAO, the annual production by fish farmer also strongly varies from less than 1 ton in Indonesia to 4 tons in

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India, 7 tons in China, 35 tons in Chile, and 187 tons in Norway [19].

Address all correspondence to<EMAIL_ADDRESS>
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[4] Diana JS, Egna HS, Chopin T, Peterson MS, Cao L, Pomeroy R, Verdegem M, Slack WT, Bondad-Reantaso MG, Cabello F. Responsible aquaculture in 2050: Valuing local condi-

[5] Balon EK. Origin and domestication of the wild carp, *Cyprinus carpio*: From roman gour-

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**Conflict of interest**

**Author details**

Teletchea Fabrice

**References**

2002;**59**:15-23

The author declares no conflict of interest.

Even though the number of farmed aquatic species (including fish, molluscs, and crustaceans) has strongly increased from 1950 to 2010, from about 72 to more than 500 [19, 20], only few species ensure the bulk of the production today [30, 83, 97]. For fish only, 15 species ensure more than 85% of the global production in 2005 [30], despite the number of farmed species rose from 43 to 219 between 1950 and 2005 [97]. In 2009, this trend was confirmed with more than 90% of the global production relying on 20 species only [83]. Only in Europe, most of the aquaculture production is based on the rearing of 10 species only [34, 98]. For some species, which have a very high production today, their farming is quite recent, dating back only to two or three decades only, such as the striped catfish or the Atlantic salmon [30, 97]. Among the 33 species with more than 100,000 tons in 2005, about one-quarter was not produced 40 years ago [97], which illustrates that new species can contribute strongly to the global production [99–101]. Conversely, most farming trials of new species realized within the past decades, either failed or resulted in low production volumes, about tens of tons. This demonstrates how difficult it is to farm a new species, whose development depends on the interaction of various factors, among which biological (availability of wild individuals, ability to control the life cycle in captivity), economical (acceptability by consumers, competition with other animal products), and environmental ones (availability of suitable sites and water, competition with other resources) [12, 18, 84, 91]. More recently, it has also become evident that climate change, which may result, among others, in global warming, saline water intrusion, and ocean acidification, may affect aquaculture [102]. Therefore, aquaculture should use genetically improved and robust animals not suffering from inbreeding depression, resulting from well-managed selective breeding programs with proper inbreeding control and breeding goals [102]. The leading species for aquaculture production have been extensively introduced across the world, particularly in the past century, resulting in that the bulk of aquaculture production relied on the farming of these very few alien species in numerous countries [11, 15]. Yet, the contribution of native species to global aquaculture will perhaps improve resulting in a more diversified and even production than today [99–101]. At least the intensity level of farming, from extensive to highly intensive, and industrialization are also very diverse [18]. From an activity mainly artisanal, aquaculture has evolved to include very large companies that export in numerous countries [17, 103]. According to FAO, the annual production by fish farmer also strongly varies from less than 1 ton in Indonesia to 4 tons in India, 7 tons in China, 35 tons in Chile, and 187 tons in Norway [19].