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Mutation breeding in the marine crop Porphyra yezoensis (Bangiales, Rhodophyta): Cultivation experiment of the artificial red mutant isolated by heavy-ion beam mutagenesis
Aquaculture 314 (2011) 182–187
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Mutation breeding in the marine crop Porphyra yezoensis (Bangiales, Rhodophyta): Cultivation experiment of the artificial red mutant isolated by heavy-ion beam mutagenesis Kyosuke Niwa a,⁎, Takeshi Yamamoto b, Hirofumi Furuita b, Tomoko Abe c a b c
Fisheries Technology Institute, Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries, Akashi, Hyogo, 674–0093, Japan Inland Station, National Research Institute of Aquaculture, Fisheries Research Agency, Tamaki, Mie, 519–0423, Japan RIKEN Nishina Center, Wako, Saitama, 351–0198, Japan
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Article history: Received 27 May 2010 Received in revised form 1 February 2011 Accepted 2 February 2011 Available online 4 March 2011 Keywords: Artificial mutant Free amino acid (FAA) Heavy-ion beam Mutation breeding Photosynthetic pigment Porphyra yezoensis
a b s t r a c t We carried out a cultivation experiment using an artificial red mutant of Porphyra yezoensis isolated by heavy-ion beam mutagenesis to evaluate whether the red mutant has potential as a new cultivar. The blade length of the red mutant was shorter than that of a wild-type HG-511 at the first harvest. However, the results of the present study suggest that the red mutant tends to increase the contents of phycoerythrin, total free amino acid (FAA), alanine and taurine in comparison with the wild type. These tendencies toward the FAA contents were strongly supported by the laboratory culture experiment. From these superior characteristics of pigment and FAA contents, the red mutant IBY-R1 may become a new cultivar in nori cultivation, although it is possible that the mutant harvest will be lower than that of other cultivars. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The marine crop Porphyra, commonly known as nori, is mainly cultivated in Japan, Korea and China. This genus usually has a heteromorphic life cycle with a haploid gametophytic blade and a sporophytic filament referred to as the conchocelis phase. In Japan, Porphyra yezoensis Ueda has been extensively cultivated in nori farms (Miura and Aruga, 1987; Miura, 1988; Niwa and Aruga, 2006), and most of the cultivated strains have been bred by selective breeding. Although the breeding method contributed to isolating vigorously growing cultivars with more elongate blades (Miura, 1984; Niwa et al., 2004, 2008a), intensive selective breeding has led to genetic uniformity in nori farms (Niwa and Aruga, 2003, 2006). Recently, nori cultivators have requested new breeds of cultivars with a better flavor of dried nori, higher tolerance to disease or high seawater temperature, and a deeper color even under low-nutrient conditions in nori farms; therefore, to develop new cultivars, it is important to attempt mutation breeding in Porphyra as one of the breeding methods (Niwa et al., 2009). In Porphyra, artificial pigmentation mutants have been induced by chemical mutagens (e.g. Mitman and van der Meer, 1994; Yan et al., ⁎ Corresponding author. Tel.: + 81 78 941 8601; fax: + 81 78 941 8604. E-mail address: [email protected] (K. Niwa). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.02.007
2000) and gamma rays (Wang et al., 2000; Yan et al., 2005). Since gametophytic blades of P. yezoensis are haploid, recessive mutations appear directly in the phenotypes of the blades. In addition, P. yezoensis propagates by an asexual subcycle through archeospores released from gametophytic blades. It is therefore easy to isolate pure lines (complete homozygous conchocelis strains) with mutational genotypes via the self-fertilization of blades developed from archeospores released from mutated cell clusters in haploid blades. Our previous study demonstrated that heavy-ion beam mutagenesis is an effective tool for the isolation of artificial pigmentation mutants of P. yezoensis (Niwa et al., 2009), and these mutants were genetically characterized by cross-fertilization experiments (Niwa, 2010). Of the isolated mutants, gametophytic blades of the artificial red mutant IBY-R1 showed significantly higher phycoerythrin (PE) content than wildtype HG-511 in the laboratory culture (Niwa et al., 2009). Since the red mutant possessed a high PE content, it is possible that the free amino acid (FAA) contents, known to be the most important substances among the components affecting taste in Porphyra blades (Noda et al., 1975; Harada et al., 1990; Yoshie et al., 1993), may also differ between the red mutant and the wild type. Therefore, to evaluate whether the red mutant has potential as a new cultivar, its growth characteristics, photosynthetic pigment contents and free amino acid (FAA) contents were compared with those of the wild type in nori farms of two cultivator groups. In addition to the cultivation experiment, FAA
K. Niwa et al. / Aquaculture 314 (2011) 182–187
contents of the red mutant and wild-type blades were examined in the laboratory culture. 2. Materials and methods The artificial red mutant IBY-R1 of P. yezoensis, which was isolated from wild-type HG-511 by heavy-ion beam mutagenesis, was used in the present study (Niwa et al., 2009): the conchocelis strain of the red mutant was established by self-fertilization of a red-colored blade developed from an archeospore that was released from a red-mutated cell cluster in the HG-511 blades irradiated by heavy-ion beams. Wild-type HG-511 was also used for comparison with the red mutant. The cultivation experiment was carried out by two nori cultivator groups at nori farms in Akashi, Hyogo Prefecture, Japan. The cultivation method was as described by Niwa et al. (2008a). Briefly, after seeding of conchospores of the red mutant and the wild type using the land seeding facility, the germlings (gametophytic blades) on the nori nets were cultivated by the floating culture facility in the nori farms for approximately 20 days. For germling culture, the nori nets were raised on the floating culture structures with nursery “U”-shaped frames. The nets were emersed on these floating structures for about 2–4 hours every morning (except for rainy days) to minimize diatom attachment and disease. After the germling culture, the nori nets were dried and stored at −25 °C. The nori nets were then placed again on the floating culture structures without nursery frames in the nori farms, and the
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gametophytic blades on the nori nets were grown without emersion for harvest. Blade samples of the red mutant and the wild type were collected at the first and third harvests. The length and width of the samples were measured using a vernier caliper. The contents of three major photosynthetic pigments (chlorophyll a, phycoerythrin and phycocyanin) in the gametophytic blades were determined by the same procedure, described in Niwa et al. (2009). Briefly, the blade portion was thoroughly ground in 0.1 M phosphate buffer (pH 6.8) with a glass homogenizer. The extract was centrifuged at 10,000 ×g for 10 min, and the supernatant was used to determine PE and PC contents. The contents were determined spectrophotometrically using the formulas described by Beer and Eshel (1985). Using tissue pellets after centrifugation for phycobilins, chlorophyll a (Chl. a) was extracted by 90% acetone for 2 h at 4 °C in darkness. After centrifugation at 5,000 ×g for 10 min, the supernatant was used to determine the Chl. a content spectrophotometrically using the formula of Jeffrey and Humphrey (1975). Free amino acid compositions of the blade samples were determined using an automatic amino acid analyzer L-8500 (Hitachi, Tokyo, Japan) after the samples had been extracted in 0.6 N perchloric acid, based on the methods of Ogata and Murai (1994) and Yamamoto et al. (2000). The moisture of the samples was determined by 10 h drying at 110 °C. In addition to the cultivation experiment, gametophytic blades of the red mutant IBY-R1 and wild-type HG-511 were cultured for comparison of the FAA contents in the laboratory under the same conditions. Mature conchocelis colonies of the red mutant and the wild type were cultured in 300 mL flasks with NPM medium (Niwa and Aruga, 2003) and agitated by aeration at 15 °C under 80 μmol photons · m− 2 · s− 1 in 10:14 h (light:dark). Vinylon monofilaments (about 6 cm long) were placed in the flasks for conchospore attachment. After their attachment to monofilaments, conchocelis colonies were removed from the flasks. The culture medium was renewed twice a week. Blades that developed from conchospores were detached from the monofilaments when they had grown to 2–3 cm long. The detached blades were transferred to 1 L flasks and further cultured to obtain blade samples for FAA analysis. Data were tested statistically using Student's t-test. When the S.D. was significantly different among populations, the Welch t-test was applied. Two-way multiple analysis of variance (MANOVA) was performed to investigate the potential differences by the strain, cultivator group and harvest time. Difference between means was compared by Fisher's protected least significant difference test. The statistical analyses were compared out using the StatView (SAS Institute, Cary, NC, USA), and a probability level of less than 0.05 was considered as significant. 3. Results 3.1. Growth characteristics Fig. 1 shows the gametophytic blades of the red mutant IBY-R1 and wild-type HG-511 at the first harvest of two nori cultivator groups. Both of the red mutant blades in the two groups grew more reddish in color than the wild-type blades. The data on their growth are shown in Table 1 Blade length, blade width and blade length-to-width ratios of the artificial red mutant IBY-R1 and wild-type HG-511 of Porphyra yezoensis at the first harvest by the two nori cultivator groups.
Nori cultivator group 1 Nori cultivator group 2 Fig. 1. Gametophytic blades of the artificial red mutant IBY-R1 and wild-type HG-511 of Porphyra yezoensis at the first harvest by the two nori cultivator groups. Three blades on left side, red mutant IBY-R1; three blades on right side, wild-type HG-511. Scale bar, 5 cm.
Strain
Blade length
Blade width
Length-to-width
IBY-R1 HG-511 IBY-R1 HG-511
12.2 ± 1.0⁎ 13.9 ± 1.9 11.6 ± 1.7⁎ 16.2 ± 2.8
1.1 ± 0.2⁎
11.9 ± 2.8⁎ 15.8 ± 3.3 11.0 ± 2.9⁎ 16.5 ± 3.9
0.9 ± 0.2 1.1 ± 0.3 1.0 ± 0.1
Asterisk indicates that the red mutant is significantly different from the wild type (p b 0.05).
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50
50
a
Nori cultivator group 1
b
Nori cultivator group 1
*
Pigment contents (µg/cm2)
Pigment contents (µg/cm2)
First harvest
40 *
30
20
10
0 50
Chl. a
PE
30
20
10
50
c
*
0
PC
Nori cultivator group 2
Chl. a
PE
d
Pigment contents (µg/cm2)
Pigment contents (µg/cm2)
*
30
20
10
PC
Nori cultivator group 2
First harvest
40
Third harvest
40
Third harvest
40
30
20
10 *
0
Chl. a
PE
PC
0
Chl. a
PE
PC
Fig. 2. Contents of chlorophyll a (Chl. a), phycoerythrin (PE) and phycocyanin (PC) in the artificial red mutant IBY-R1 (■) and wild-type HG-511 (□) of Porphyra yezoensis at the first and third harvests by the two nori cultivator groups. Average± SD (n= 3). Asterisk indicates that the red mutant is significantly different from the wild type (p b 0.05).
Table 1. At the first harvest of the two nori cultivator groups, the blade length was significantly shorter in the red mutant than in the wild type. Blade width was slightly wider in the red mutant than in the wild type, and the blade length-to-width ratio was significantly lower in the red mutant than in the wild type at the first harvest of the two cultivator groups. The ratios indicate that the blade of the red mutant was wider relative to the length than the blade of the wild type in the nori farms. 3.2. Pigment contents Fig. 2 shows the contents of three major photosynthetic pigments, chlorophyll a (Chl. a), phycoerythrin (PE) and phycocyanin (PC), in the gametophytic blades of the red mutant and the wild type at the first and third harvests of the two cultivator groups. In both cultivations, the PE content was remarkably higher in the red mutant than in the wild type at the two different harvest times, even though the PE content at the first harvest by cultivator group 2 was not significantly different between the Table 2 Summary of MANOVA on photosynthetic pigment contents of the artificial red mutant IBY-R1 and wild-type HG-511 of Porphyra yezoensis depending on the strain and harvest time in each nori cultivator group.
Nori cultivator group 1
Nori cultivator group 2
Source of variance
Chl. a
PE
PC
Strain Harvest time Strain × Harvest time Strain Harvest time Strain × Harvest time
0.5491 0.0698 0.7234 0.0684 0.8510 0.6130
0.0005 0.3249 0.2074 0.0007 0.3804 0.5080
0.0131 0.0190 0.2196 0.0110 0.0248 0.3171
Bold indicates significant difference with no interaction between the factors (p b 0.05).
two strains. In particular, the difference in PE content between the two strains was larger at the third harvest than at the first harvest in both cultivations. The result of MANOVA also indicated that PE content of the red mutant throughout the two harvest times was significantly higher than that of the wild type in each cultivator group (Table 2). On the other hand, no clear tendencies in the Chl. a and PC contents were observed between the two strains as shown in Fig. 2, although PC content throughout the two harvest times was significantly lower in the red mutant than in the wild type in each cultivator group (Table 2). 3.3. Free amino acid contents Table 3 shows total free amino acid (FAA) contents in the gametophytic blades of the red mutant and the wild type at the first and third harvests of the two cultivator groups. The total FAA content was higher in the red mutant than in the wild type at each harvest time by the two cultivator groups, although the difference at the first harvest
Table 3 Total free amino acid contents in the artificial red mutant IBY-R1 and wild-type HG-511 of Porphyra yezoensis at the first and third harvests by the two nori cultivator groups.
Nori cultivator group 1 Nori cultivator group 2
Strain
First harvest
Third harvest
IBY-R1 HG-511 IBY-R1 HG-511
59.70 ± 1.01⁎
56.30 ± 3.73⁎ 43.10 ± 1.33 49.18 ± 1.50⁎ 38.94 ± 1.62
50.91 ± 1.72 59.77 ± 2.38 54.72 ± 2.67
Asterisk indicates that the red mutant is significantly different from the wild type (p b 0.05).
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Table 4 Summary of MANOVA on free amino acid contents of the artificial red mutant IBY-R1 and wild-type HG-511 of Porphyra yezoensis depending on the strain and nori cultivator group in each harvest time.
First harvest
Third harvest
Source of variance
Total FAA
Asp
Glu
Ala
Tau
Strain Nori cultivator group Strain × Nori cultivator group Strain Nori cultivator group Strain × Nori cultivator group
0.0004⁎ 0.1265 0.1651 b 0.0001⁎ 0.0026⁎ 0.2912
0.1426 0.3122 0.9876 b 0.0001⁎ b 0.0001⁎ 0.2877
b 0.0001⁎ b 0.0001⁎ 0.0004⁎ 0.1085 b 0.0001⁎ 0.0029⁎
0.0204⁎ b0.0001⁎ 0.0002⁎ b0.0001⁎ 0.0236⁎ 0.0543
b0.0001⁎ 0.1944 0.6022 b0.0001⁎ 0.0431⁎ 0.7829
Significant difference is shown as asterisk (p b 0.05). Bold indicates significant difference with no interaction between the factors.
by nori cultivator group 2 was not significant. The result of statistical analysis in Table 4 also indicated that total FAA content throughout the two cultivator groups was significantly higher in the red mutant than in the wild type at each harvest time. The contents of the four major FAA, aspartic acid (Asp), glutamic acid (Glu), alanine (Ala) and taurine (Tau), are shown in Fig. 3. At the first harvest by nori cultivator group 1, the Glu content was significantly lower in the red mutant than in the wild type, and the Asp content was similar between the two strains (Fig. 3a); however, the Ala and Tau contents of the red mutant were significantly higher than those of the wild type at the first harvest (Fig. 3a). Furthermore, all four major FAA contents at the third harvest by nori cultivator group 1 were significantly higher in the red mutant than in the wild type, and clear differences between the two strains were found in the Ala and Tau contents (Fig. 3b). On the other hand, at the first harvest by cultivator group 2, the Glu content was significantly lower in the red mutant than in the wild type, and the Asp and Ala contents were not significantly different between the two strains (Fig. 3c); however, the Tau content of the red mutant was significantly higher at the first harvest (Fig. 3c). At the third harvest by cultivator group 2, the three major FAA (Asp, Ala and Tau) contents were significantly higher in the red mutant than in the wild type (Fig. 3d). In MANOVA, Tau content of
25
a
the red mutant throughout the two cultivator groups was significantly higher than that of the wild type in each harvest time (Table 4). However, the statistical differences were not clear between the other major FAA contents due to interactions between factors. In addition to the FAA contents in the cultivation experiment, the FAA contents of the red mutant were compared with those of the wild type in laboratory culture. The total FAA content in the laboratory experiment was 46.59 ± 1.67 mg/gDM in the red mutant and 29.62 ± 0.88 mg/gDM in the wild type (average ± SD, n = 3), indicating that the total FAA content was significantly higher in the red mutant than in the wild type (p b 0.05). The four major FAA contents in the laboratory experiment are shown in Fig. 4. All four major FAA contents were significantly higher in the red mutant than in the wild type, and clear differences between the two strains were observed in the Asp, Ala and Tau contents. 4. Discussion In nori farming, cultivators usually harvest several times from the same nori net. It is therefore necessary to compare the blade length and shape among different strains before cutting at the first harvest
25 Nori cultivator group 1
*
First harvest
20
b
Nori cultivator group 1 *
Third harvest
20
*
mg/g DM
mg/g DM
*
15 10 *
5
10
*
5
0
Asp
Glu
Ala
*
0
Tau
25
Asp
Glu
Ala
Tau
25
c
d
*
Nori cultivator group 2 First harvest
Nori cultivator group 2
15 10
*
Third harvest
20
mg/g DM
20
mg/g DM
15
*
15 10
*
5
5 *
0
Asp
Glu
Ala
Tau
0
Asp
Glu
Ala
Tau
Fig. 3. Free amino acid contents in the artificial red mutant IBY-R1 (■) and wild-type HG-511 (□) of Porphyra yezoensis at the first and third harvests by the two nori cultivator groups. Average ± SD (n = 3). Asterisk indicates that the red mutant is significantly different from the wild type (p b 0.05).
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14 *
12 *
mg/g DM
10
*
8 6 4
*
2 0
Asp
Glu
Ala
Tau
Fig. 4. Free amino acid contents in the artificial red mutant IBY-R1 (■) and wild-type HG-511 (□) of Porphyra yezoensis after 7 weeks of laboratory culture under the same conditions. Average ± SD (n = 3). Asterisk indicates that the red mutant is significantly different from the wild type (p b 0.05).
(Niwa et al., 2008a). Although the results of our previous study showed that the blade length increase was similar between the red mutant IBY-R1 and wild-type HG-511 during the experiment period in the laboratory culture (Niwa et al., 2009), the blade length of the red mutant IBY-R1 was significantly shorter than that of wild-type HG-511 at the first harvest by the two nori cultivator groups (Table 1). These results by the two cultivator groups suggest that the harvest of the red mutant is lower than that of the wild type. To assess clearly the harvest characteristics, further study will be needed to investigate the yield as the number of harvests progresses in large-scale cultivation. It is well known that the blade color changes with the environmental conditions and blade age (Aruga, 1974); however, the red mutant blades were always more reddish than those of the wild type at the first and third harvests by each cultivator group, and the color difference between the two strains was clearer at the third than at the first harvest. These results indicate that the color phenotype of the red mutant was genetically stable even in nori farms under changeable environments. Furthermore, it is suggested that the PE content of the red mutant was markedly higher than that of the wild type in the two nori cultivations (Fig. 2), and PE content of the mutant throughout the two harvest times was also significantly higher than that of the wild type in each cultivator group (Table 2): these characteristics are in good agreement with that of our previous laboratory experiment (Niwa et al., 2009). It is therefore inferred that the color of the red mutant resulted from the higher content of the red pigment PE in comparison with the wild type. In addition to the PE content of the red mutant, the color difference between the red mutant and the wild type increased at the third harvest because the difference in PE content between the two strains was greater at the third than at the first harvest in both cultivations (Fig. 2). Recently, as one of breeding objectives, it is important to breed the cultivar with a better flavor of dried nori (Niwa et al., 2005; Niwa et al., 2008b). Among the components affecting taste in Porphyra gametophytic blades, free amino acids (FAA) are known to be the most important substances, among which aspartic acid (Asp), glutamic acid (Glu), alanine (Ala) and taurine (Tau) are predominant in the FAA (Noda et al., 1975; Harada et al., 1990; Yoshie et al., 1993). In the four major FAA, Glu and Ala were considered to be the most important FAA regarding the taste of dried nori (Noda et al., 1975). On the other hand, it is unclear whether Tau is important for the taste in Porphyra blades. In the present study, total FAA content of the red mutant tended to be higher than that of the wild type at the examined harvest times in the two nori cultivator groups (Table 3). The result of MANOVA also supported the findings (Table 4). Among the FAA contents, Ala and Tau were especially higher in the red mutant than in the wild type, except
for the Ala content at the first harvest by nori cultivator group 2 (Fig. 3). Table 4 also indicates that the red mutant has higher Tau content, although Ala content had no clear pattern as due to an interaction between the factors in the first harvest. Furthermore, total FAA content in the laboratory experiment was significantly higher in the red mutant than in the wild type and, in particular, the Ala and Tau contents of the red mutant were higher (Fig. 4). These results suggest that the red mutant tends to have a higher total FAA content and, above all, Ala and Tau contents than the wild type, although it is possible that these characteristics are changed by the environmental conditions in nori farms. Thus, the red mutant seems to have potential as a cultivar with a better flavor of dried nori. In addition, the difference in PE content between the two strains was greater at the third than first harvest, and the respective differences in Ala and Tau contents between the two strains also increased at the third harvest; therefore, the high contents of the two FAA may correlate with the high PE content. On the other hand, no clear relationship in the Glu content was observed between the two strains from the cultivation experiment (Fig. 3). In our previous study (Niwa et al., 2008a), the Glu content alone decreased with the number of harvests among the four major FAA contents; however, this tendency was not found in the present study, presumably because of the different environmental conditions in nori farms. Nori cultivation is one of the most profitable aquacultures in Japan, Korea and China. The annual production value of nori in Japan amounts to almost US$1bn. In determining the quality of commercially dried nori sheets, their color is very important: a deep black color is considered to be high grade in Japan. Furthermore, it is known that the deeper the black of the dried nori sheets, the higher the contents of the three major photosynthetic pigments (Chl. a, PE and PC) and the FAA (Aruga, 1974; Saitoh et al., 1975; Miura and Shin, 1989). On the other hand, until now, reddish nori sheets have been considered to have a commercially low value in nori industry, because the blades change to a reddish color when the nutrients in seawater begin to decrease in nori farms. Before isolating the red mutant IBY-R1 by heavy-ion beam mutagenesis, some spontaneous and artificial red mutants (previous red mutants) were isolated, but were not clearly characterized as having high PE content (Aruga and Miura, 1984; Yan et al., 2000); therefore, the previous red mutants are not used by nori cultivators in Japan. However, the present study suggests that the red mutant IBY-R1 tends to have higher contents of PE, total FAA, Ala and Tau than the wild type in nori cultivation. Furthermore, it is easy to discriminate the red mutant from the other cultivars with the naked eye. From these superior and distinguishing characteristics, the red mutant IBY-R1 may become a new cultivar in nori cultivation, even though it is possible that the mutant harvest will be lower than that of other cultivars. Acknowledgements We thank the Hyogo Nori Institute for seeding nori nets with conchospores of the red mutant IBY-R1 and wild-type HG-511, and the nori cultivator groups of Akashiura Fishermen's Cooperative for cultivating the gametophytic blades in their nori farms.
References Aruga, Y., 1974. Color of the cultivated Porphyra. Our Nori Res. 23, 1–14 (in Japanese). Aruga, Y., Miura, A., 1984. In vivo absorption spectra and pigment contents of the two types of color mutants of Porphyra. Jpn. J. Phycol. 32, 243–250. Beer, S., Eshel, A., 1985. Determining phycoerythrin and phycocyanin concentrations in aqueous crude extract of red algae. Aust. J. Mar. Freshw. Res. 36, 785–792. Harada, K., Osumi, Y., Fukuda, N., Amano, H., Noda, H., 1990. Changes of amino acid compositions of ‘Nori’, Porphyra spp. during storage. Nippon Suisan Gakkaishi (Bull. Jpn Soc. Sci. Fish). 56, 607–612. Jeffrey, S.W., Humphrey, G.F., 1975. New spectrophotometric equations for determining chlorophylls a, b, c1, c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanz. 167, 191–194. Mitman, G.G., van der Meer, J.P., 1994. Meiosis, blade development, and sex determination in Porphyra purpurea (Rhodophyta). J. Phycol. 30, 147–159.
K. Niwa et al. / Aquaculture 314 (2011) 182–187 Miura, A., 1984. A new variety and a new form of Porphyra (Bangiales, Rhodophyta) from Japan: Porphyra tenera Kjellman var. tamatsuensis Miura, var. nov. and P. yezoensis Ueda form. narawaensis Miura, form. nov. J. Tokyo Univ. Fish. 71, 1–14. Miura, A., Aruga, Y., 1987. Distribution of Porphyra in Japan as affected by cultivation. J. Tokyo Univ. Fish. 74, 41–50. Miura, A., 1988. Taxonomic studies of Porphyra species cultivated in Japan, referring to their transition to the cultivated variety. J. Tokyo Univ. Fish. 75, 311–325. Miura, A., Shin, J.-A., 1989. Crossbreeding in cultivars of Porphyra yezoensis (Bangiales, Rhodophyta) – Preliminary report. Korean J. Phycol. 4, 207–211. Niwa, K., Aruga, Y., 2003. Rapid DNA extraction from conchocelis and ITS-1 rDNA sequences of seven strains of cultivated Porphyra yezoensis (Bangiales, Rhodophyta). J. Appl. Phycol. 15, 29–35. Niwa, K., Kikuchi, N., Iwabuchi, M., Aruga, Y., 2004. Morphological and AFLP variation of Porphyra yezoensis Ueda form. narawaensis Miura (Bangiales, Rhodophyta). Phycol. Res. 52, 180–190. Niwa, K., Kikuchi, N., Aruga, Y., 2005. Morphological and molecular analysis of the endangered species Porphyra tenera (Bangiales, Rhodophyta). J. Phycol. 41, 294–304. Niwa, K., Aruga, Y., 2006. Identification of currently cultivated Porphyra species by PCR-RFLP analysis. Fish. Sci. 72, 143–148. Niwa, K., Furuita, H., Yamamoto, T., 2008a. Changes of growth characteristics and free amino acid content of cultivated Porphyra yezoensis Ueda (Bangiales, Rhodophyta) blades with the progression of the number of harvests in a nori farm. J. Appl. Phycol. 20, 687–693. Niwa, K., Furuita, H., Yamamoto, T., Kobiyama, A., 2008b. Identification and characterization of a green-type mutant of Porphyra tenera Kjellman var. tamatsuensis Miura (Bangiales, Rhodophyta). Aquaculture 274, 126–131. Niwa, K., Hayashi, Y., Abe, T., Aruga, Y., 2009. Induction and isolation of pigmentation mutants of Porphyra yezoensis (Bangiales, Rhodophyta) by heavy-ion beam irradiation. Phycol. Res. 57, 194–202.
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Niwa, K., 2010. Genetic analysis of artificial green and red mutants of Porphyra yezoensis Ueda. Aquaculture 308, 6–12. Noda, H., Horiguti, Y., Araki, S., 1975. Studies on the flavor substances of ‘Nori’, the dried laver Porphyra spp.-II. Free amino acids and 5’-nucleotides. Nippon Suisan Gakkaishi (Bull. Jpn Soc. Sci. Fish). 41, 1299–1303. Ogata, H., Murai, T., 1994. White muscle of masu salmon, Oncorhynchus masou masou, smolts possesses a strong buffering capacity due to a high level of anserine. Fish Physiol. Biochem. 13, 285–293. Saitoh, M., Araki, S., Sakurai, T., Oohusa, T., 1975. Variations in contents of photosynthetic pigments, total nitrogen, total free amino acids and total free sugars in dried lavers obtained at different culture grounds and harvesting times. Nippon Suisan Gakkaishi (Bull. Jpn Soc. Sci. Fish). 41, 365–370 (in Japanese with English abstract). Wang, S.J., Zheng, Y.Z., Ma, L.B., Xu, P., Zhu, J.Y., 2000. Gamma-rays induction of mutation in conchocelis of Porphyra yezoensis. Chin. J. Oceanol. Limnol. 18, 47–53. Yamamoto, T., Unuma, T., Akiyama, T., 2000. The influence of dietary protein and fat levels on tissue free amino acid levels of fingerling rainbow trout (Oncorhynchus mykiss). Aquaculture 182, 353–372. Yan, X.H., Fujita, Y., Aruga, Y., 2000. Induction and characterization of pigmentation mutants in Porphyra yezoensis (Bangiales, Rhodophyta). J. Appl. Phycol. 12, 69–81. Yan, X.H., Liang, Z.Q., Song, W.L., Huang, J., Ma, P., Aruga, Y., 2005. Induction and isolation of artificial pigmentation mutants in Porphyra haitanensis Chang et Zheng (Bangiales, Rhodophyta). J. Fish. Chin. 29, 166–172 (in Chinese with English abstract). Yoshie, Y., Suzuki, T., Shirai, T., Hirano, T., 1993. Free amino acids and fatty acid composition in dried nori of various culture locations and prices. Nippon Suisan Gakkaishi (Bull. Jpn Soc. Sci. Fish). 59, 1769–1775 (in Japanese with English abstract).
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Mutation breeding in the marine crop Porphyra yezoensis (Bangiales, Rhodophyta): Cultivation experiment of the artificial red mutant isolated by heavy-ion beam mutagenesis Kyosuke Niwa a,⁎, Takeshi Yamamoto b, Hirofumi Furuita b, Tomoko Abe c a b c
Fisheries Technology Institute, Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries, Akashi, Hyogo, 674–0093, Japan Inland Station, National Research Institute of Aquaculture, Fisheries Research Agency, Tamaki, Mie, 519–0423, Japan RIKEN Nishina Center, Wako, Saitama, 351–0198, Japan
a r t i c l e
i n f o
Article history: Received 27 May 2010 Received in revised form 1 February 2011 Accepted 2 February 2011 Available online 4 March 2011 Keywords: Artificial mutant Free amino acid (FAA) Heavy-ion beam Mutation breeding Photosynthetic pigment Porphyra yezoensis
a b s t r a c t We carried out a cultivation experiment using an artificial red mutant of Porphyra yezoensis isolated by heavy-ion beam mutagenesis to evaluate whether the red mutant has potential as a new cultivar. The blade length of the red mutant was shorter than that of a wild-type HG-511 at the first harvest. However, the results of the present study suggest that the red mutant tends to increase the contents of phycoerythrin, total free amino acid (FAA), alanine and taurine in comparison with the wild type. These tendencies toward the FAA contents were strongly supported by the laboratory culture experiment. From these superior characteristics of pigment and FAA contents, the red mutant IBY-R1 may become a new cultivar in nori cultivation, although it is possible that the mutant harvest will be lower than that of other cultivars. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The marine crop Porphyra, commonly known as nori, is mainly cultivated in Japan, Korea and China. This genus usually has a heteromorphic life cycle with a haploid gametophytic blade and a sporophytic filament referred to as the conchocelis phase. In Japan, Porphyra yezoensis Ueda has been extensively cultivated in nori farms (Miura and Aruga, 1987; Miura, 1988; Niwa and Aruga, 2006), and most of the cultivated strains have been bred by selective breeding. Although the breeding method contributed to isolating vigorously growing cultivars with more elongate blades (Miura, 1984; Niwa et al., 2004, 2008a), intensive selective breeding has led to genetic uniformity in nori farms (Niwa and Aruga, 2003, 2006). Recently, nori cultivators have requested new breeds of cultivars with a better flavor of dried nori, higher tolerance to disease or high seawater temperature, and a deeper color even under low-nutrient conditions in nori farms; therefore, to develop new cultivars, it is important to attempt mutation breeding in Porphyra as one of the breeding methods (Niwa et al., 2009). In Porphyra, artificial pigmentation mutants have been induced by chemical mutagens (e.g. Mitman and van der Meer, 1994; Yan et al., ⁎ Corresponding author. Tel.: + 81 78 941 8601; fax: + 81 78 941 8604. E-mail address: [email protected] (K. Niwa). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.02.007
2000) and gamma rays (Wang et al., 2000; Yan et al., 2005). Since gametophytic blades of P. yezoensis are haploid, recessive mutations appear directly in the phenotypes of the blades. In addition, P. yezoensis propagates by an asexual subcycle through archeospores released from gametophytic blades. It is therefore easy to isolate pure lines (complete homozygous conchocelis strains) with mutational genotypes via the self-fertilization of blades developed from archeospores released from mutated cell clusters in haploid blades. Our previous study demonstrated that heavy-ion beam mutagenesis is an effective tool for the isolation of artificial pigmentation mutants of P. yezoensis (Niwa et al., 2009), and these mutants were genetically characterized by cross-fertilization experiments (Niwa, 2010). Of the isolated mutants, gametophytic blades of the artificial red mutant IBY-R1 showed significantly higher phycoerythrin (PE) content than wildtype HG-511 in the laboratory culture (Niwa et al., 2009). Since the red mutant possessed a high PE content, it is possible that the free amino acid (FAA) contents, known to be the most important substances among the components affecting taste in Porphyra blades (Noda et al., 1975; Harada et al., 1990; Yoshie et al., 1993), may also differ between the red mutant and the wild type. Therefore, to evaluate whether the red mutant has potential as a new cultivar, its growth characteristics, photosynthetic pigment contents and free amino acid (FAA) contents were compared with those of the wild type in nori farms of two cultivator groups. In addition to the cultivation experiment, FAA
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contents of the red mutant and wild-type blades were examined in the laboratory culture. 2. Materials and methods The artificial red mutant IBY-R1 of P. yezoensis, which was isolated from wild-type HG-511 by heavy-ion beam mutagenesis, was used in the present study (Niwa et al., 2009): the conchocelis strain of the red mutant was established by self-fertilization of a red-colored blade developed from an archeospore that was released from a red-mutated cell cluster in the HG-511 blades irradiated by heavy-ion beams. Wild-type HG-511 was also used for comparison with the red mutant. The cultivation experiment was carried out by two nori cultivator groups at nori farms in Akashi, Hyogo Prefecture, Japan. The cultivation method was as described by Niwa et al. (2008a). Briefly, after seeding of conchospores of the red mutant and the wild type using the land seeding facility, the germlings (gametophytic blades) on the nori nets were cultivated by the floating culture facility in the nori farms for approximately 20 days. For germling culture, the nori nets were raised on the floating culture structures with nursery “U”-shaped frames. The nets were emersed on these floating structures for about 2–4 hours every morning (except for rainy days) to minimize diatom attachment and disease. After the germling culture, the nori nets were dried and stored at −25 °C. The nori nets were then placed again on the floating culture structures without nursery frames in the nori farms, and the
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gametophytic blades on the nori nets were grown without emersion for harvest. Blade samples of the red mutant and the wild type were collected at the first and third harvests. The length and width of the samples were measured using a vernier caliper. The contents of three major photosynthetic pigments (chlorophyll a, phycoerythrin and phycocyanin) in the gametophytic blades were determined by the same procedure, described in Niwa et al. (2009). Briefly, the blade portion was thoroughly ground in 0.1 M phosphate buffer (pH 6.8) with a glass homogenizer. The extract was centrifuged at 10,000 ×g for 10 min, and the supernatant was used to determine PE and PC contents. The contents were determined spectrophotometrically using the formulas described by Beer and Eshel (1985). Using tissue pellets after centrifugation for phycobilins, chlorophyll a (Chl. a) was extracted by 90% acetone for 2 h at 4 °C in darkness. After centrifugation at 5,000 ×g for 10 min, the supernatant was used to determine the Chl. a content spectrophotometrically using the formula of Jeffrey and Humphrey (1975). Free amino acid compositions of the blade samples were determined using an automatic amino acid analyzer L-8500 (Hitachi, Tokyo, Japan) after the samples had been extracted in 0.6 N perchloric acid, based on the methods of Ogata and Murai (1994) and Yamamoto et al. (2000). The moisture of the samples was determined by 10 h drying at 110 °C. In addition to the cultivation experiment, gametophytic blades of the red mutant IBY-R1 and wild-type HG-511 were cultured for comparison of the FAA contents in the laboratory under the same conditions. Mature conchocelis colonies of the red mutant and the wild type were cultured in 300 mL flasks with NPM medium (Niwa and Aruga, 2003) and agitated by aeration at 15 °C under 80 μmol photons · m− 2 · s− 1 in 10:14 h (light:dark). Vinylon monofilaments (about 6 cm long) were placed in the flasks for conchospore attachment. After their attachment to monofilaments, conchocelis colonies were removed from the flasks. The culture medium was renewed twice a week. Blades that developed from conchospores were detached from the monofilaments when they had grown to 2–3 cm long. The detached blades were transferred to 1 L flasks and further cultured to obtain blade samples for FAA analysis. Data were tested statistically using Student's t-test. When the S.D. was significantly different among populations, the Welch t-test was applied. Two-way multiple analysis of variance (MANOVA) was performed to investigate the potential differences by the strain, cultivator group and harvest time. Difference between means was compared by Fisher's protected least significant difference test. The statistical analyses were compared out using the StatView (SAS Institute, Cary, NC, USA), and a probability level of less than 0.05 was considered as significant. 3. Results 3.1. Growth characteristics Fig. 1 shows the gametophytic blades of the red mutant IBY-R1 and wild-type HG-511 at the first harvest of two nori cultivator groups. Both of the red mutant blades in the two groups grew more reddish in color than the wild-type blades. The data on their growth are shown in Table 1 Blade length, blade width and blade length-to-width ratios of the artificial red mutant IBY-R1 and wild-type HG-511 of Porphyra yezoensis at the first harvest by the two nori cultivator groups.
Nori cultivator group 1 Nori cultivator group 2 Fig. 1. Gametophytic blades of the artificial red mutant IBY-R1 and wild-type HG-511 of Porphyra yezoensis at the first harvest by the two nori cultivator groups. Three blades on left side, red mutant IBY-R1; three blades on right side, wild-type HG-511. Scale bar, 5 cm.
Strain
Blade length
Blade width
Length-to-width
IBY-R1 HG-511 IBY-R1 HG-511
12.2 ± 1.0⁎ 13.9 ± 1.9 11.6 ± 1.7⁎ 16.2 ± 2.8
1.1 ± 0.2⁎
11.9 ± 2.8⁎ 15.8 ± 3.3 11.0 ± 2.9⁎ 16.5 ± 3.9
0.9 ± 0.2 1.1 ± 0.3 1.0 ± 0.1
Asterisk indicates that the red mutant is significantly different from the wild type (p b 0.05).
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50
50
a
Nori cultivator group 1
b
Nori cultivator group 1
*
Pigment contents (µg/cm2)
Pigment contents (µg/cm2)
First harvest
40 *
30
20
10
0 50
Chl. a
PE
30
20
10
50
c
*
0
PC
Nori cultivator group 2
Chl. a
PE
d
Pigment contents (µg/cm2)
Pigment contents (µg/cm2)
*
30
20
10
PC
Nori cultivator group 2
First harvest
40
Third harvest
40
Third harvest
40
30
20
10 *
0
Chl. a
PE
PC
0
Chl. a
PE
PC
Fig. 2. Contents of chlorophyll a (Chl. a), phycoerythrin (PE) and phycocyanin (PC) in the artificial red mutant IBY-R1 (■) and wild-type HG-511 (□) of Porphyra yezoensis at the first and third harvests by the two nori cultivator groups. Average± SD (n= 3). Asterisk indicates that the red mutant is significantly different from the wild type (p b 0.05).
Table 1. At the first harvest of the two nori cultivator groups, the blade length was significantly shorter in the red mutant than in the wild type. Blade width was slightly wider in the red mutant than in the wild type, and the blade length-to-width ratio was significantly lower in the red mutant than in the wild type at the first harvest of the two cultivator groups. The ratios indicate that the blade of the red mutant was wider relative to the length than the blade of the wild type in the nori farms. 3.2. Pigment contents Fig. 2 shows the contents of three major photosynthetic pigments, chlorophyll a (Chl. a), phycoerythrin (PE) and phycocyanin (PC), in the gametophytic blades of the red mutant and the wild type at the first and third harvests of the two cultivator groups. In both cultivations, the PE content was remarkably higher in the red mutant than in the wild type at the two different harvest times, even though the PE content at the first harvest by cultivator group 2 was not significantly different between the Table 2 Summary of MANOVA on photosynthetic pigment contents of the artificial red mutant IBY-R1 and wild-type HG-511 of Porphyra yezoensis depending on the strain and harvest time in each nori cultivator group.
Nori cultivator group 1
Nori cultivator group 2
Source of variance
Chl. a
PE
PC
Strain Harvest time Strain × Harvest time Strain Harvest time Strain × Harvest time
0.5491 0.0698 0.7234 0.0684 0.8510 0.6130
0.0005 0.3249 0.2074 0.0007 0.3804 0.5080
0.0131 0.0190 0.2196 0.0110 0.0248 0.3171
Bold indicates significant difference with no interaction between the factors (p b 0.05).
two strains. In particular, the difference in PE content between the two strains was larger at the third harvest than at the first harvest in both cultivations. The result of MANOVA also indicated that PE content of the red mutant throughout the two harvest times was significantly higher than that of the wild type in each cultivator group (Table 2). On the other hand, no clear tendencies in the Chl. a and PC contents were observed between the two strains as shown in Fig. 2, although PC content throughout the two harvest times was significantly lower in the red mutant than in the wild type in each cultivator group (Table 2). 3.3. Free amino acid contents Table 3 shows total free amino acid (FAA) contents in the gametophytic blades of the red mutant and the wild type at the first and third harvests of the two cultivator groups. The total FAA content was higher in the red mutant than in the wild type at each harvest time by the two cultivator groups, although the difference at the first harvest
Table 3 Total free amino acid contents in the artificial red mutant IBY-R1 and wild-type HG-511 of Porphyra yezoensis at the first and third harvests by the two nori cultivator groups.
Nori cultivator group 1 Nori cultivator group 2
Strain
First harvest
Third harvest
IBY-R1 HG-511 IBY-R1 HG-511
59.70 ± 1.01⁎
56.30 ± 3.73⁎ 43.10 ± 1.33 49.18 ± 1.50⁎ 38.94 ± 1.62
50.91 ± 1.72 59.77 ± 2.38 54.72 ± 2.67
Asterisk indicates that the red mutant is significantly different from the wild type (p b 0.05).
K. Niwa et al. / Aquaculture 314 (2011) 182–187
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Table 4 Summary of MANOVA on free amino acid contents of the artificial red mutant IBY-R1 and wild-type HG-511 of Porphyra yezoensis depending on the strain and nori cultivator group in each harvest time.
First harvest
Third harvest
Source of variance
Total FAA
Asp
Glu
Ala
Tau
Strain Nori cultivator group Strain × Nori cultivator group Strain Nori cultivator group Strain × Nori cultivator group
0.0004⁎ 0.1265 0.1651 b 0.0001⁎ 0.0026⁎ 0.2912
0.1426 0.3122 0.9876 b 0.0001⁎ b 0.0001⁎ 0.2877
b 0.0001⁎ b 0.0001⁎ 0.0004⁎ 0.1085 b 0.0001⁎ 0.0029⁎
0.0204⁎ b0.0001⁎ 0.0002⁎ b0.0001⁎ 0.0236⁎ 0.0543
b0.0001⁎ 0.1944 0.6022 b0.0001⁎ 0.0431⁎ 0.7829
Significant difference is shown as asterisk (p b 0.05). Bold indicates significant difference with no interaction between the factors.
by nori cultivator group 2 was not significant. The result of statistical analysis in Table 4 also indicated that total FAA content throughout the two cultivator groups was significantly higher in the red mutant than in the wild type at each harvest time. The contents of the four major FAA, aspartic acid (Asp), glutamic acid (Glu), alanine (Ala) and taurine (Tau), are shown in Fig. 3. At the first harvest by nori cultivator group 1, the Glu content was significantly lower in the red mutant than in the wild type, and the Asp content was similar between the two strains (Fig. 3a); however, the Ala and Tau contents of the red mutant were significantly higher than those of the wild type at the first harvest (Fig. 3a). Furthermore, all four major FAA contents at the third harvest by nori cultivator group 1 were significantly higher in the red mutant than in the wild type, and clear differences between the two strains were found in the Ala and Tau contents (Fig. 3b). On the other hand, at the first harvest by cultivator group 2, the Glu content was significantly lower in the red mutant than in the wild type, and the Asp and Ala contents were not significantly different between the two strains (Fig. 3c); however, the Tau content of the red mutant was significantly higher at the first harvest (Fig. 3c). At the third harvest by cultivator group 2, the three major FAA (Asp, Ala and Tau) contents were significantly higher in the red mutant than in the wild type (Fig. 3d). In MANOVA, Tau content of
25
a
the red mutant throughout the two cultivator groups was significantly higher than that of the wild type in each harvest time (Table 4). However, the statistical differences were not clear between the other major FAA contents due to interactions between factors. In addition to the FAA contents in the cultivation experiment, the FAA contents of the red mutant were compared with those of the wild type in laboratory culture. The total FAA content in the laboratory experiment was 46.59 ± 1.67 mg/gDM in the red mutant and 29.62 ± 0.88 mg/gDM in the wild type (average ± SD, n = 3), indicating that the total FAA content was significantly higher in the red mutant than in the wild type (p b 0.05). The four major FAA contents in the laboratory experiment are shown in Fig. 4. All four major FAA contents were significantly higher in the red mutant than in the wild type, and clear differences between the two strains were observed in the Asp, Ala and Tau contents. 4. Discussion In nori farming, cultivators usually harvest several times from the same nori net. It is therefore necessary to compare the blade length and shape among different strains before cutting at the first harvest
25 Nori cultivator group 1
*
First harvest
20
b
Nori cultivator group 1 *
Third harvest
20
*
mg/g DM
mg/g DM
*
15 10 *
5
10
*
5
0
Asp
Glu
Ala
*
0
Tau
25
Asp
Glu
Ala
Tau
25
c
d
*
Nori cultivator group 2 First harvest
Nori cultivator group 2
15 10
*
Third harvest
20
mg/g DM
20
mg/g DM
15
*
15 10
*
5
5 *
0
Asp
Glu
Ala
Tau
0
Asp
Glu
Ala
Tau
Fig. 3. Free amino acid contents in the artificial red mutant IBY-R1 (■) and wild-type HG-511 (□) of Porphyra yezoensis at the first and third harvests by the two nori cultivator groups. Average ± SD (n = 3). Asterisk indicates that the red mutant is significantly different from the wild type (p b 0.05).
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14 *
12 *
mg/g DM
10
*
8 6 4
*
2 0
Asp
Glu
Ala
Tau
Fig. 4. Free amino acid contents in the artificial red mutant IBY-R1 (■) and wild-type HG-511 (□) of Porphyra yezoensis after 7 weeks of laboratory culture under the same conditions. Average ± SD (n = 3). Asterisk indicates that the red mutant is significantly different from the wild type (p b 0.05).
(Niwa et al., 2008a). Although the results of our previous study showed that the blade length increase was similar between the red mutant IBY-R1 and wild-type HG-511 during the experiment period in the laboratory culture (Niwa et al., 2009), the blade length of the red mutant IBY-R1 was significantly shorter than that of wild-type HG-511 at the first harvest by the two nori cultivator groups (Table 1). These results by the two cultivator groups suggest that the harvest of the red mutant is lower than that of the wild type. To assess clearly the harvest characteristics, further study will be needed to investigate the yield as the number of harvests progresses in large-scale cultivation. It is well known that the blade color changes with the environmental conditions and blade age (Aruga, 1974); however, the red mutant blades were always more reddish than those of the wild type at the first and third harvests by each cultivator group, and the color difference between the two strains was clearer at the third than at the first harvest. These results indicate that the color phenotype of the red mutant was genetically stable even in nori farms under changeable environments. Furthermore, it is suggested that the PE content of the red mutant was markedly higher than that of the wild type in the two nori cultivations (Fig. 2), and PE content of the mutant throughout the two harvest times was also significantly higher than that of the wild type in each cultivator group (Table 2): these characteristics are in good agreement with that of our previous laboratory experiment (Niwa et al., 2009). It is therefore inferred that the color of the red mutant resulted from the higher content of the red pigment PE in comparison with the wild type. In addition to the PE content of the red mutant, the color difference between the red mutant and the wild type increased at the third harvest because the difference in PE content between the two strains was greater at the third than at the first harvest in both cultivations (Fig. 2). Recently, as one of breeding objectives, it is important to breed the cultivar with a better flavor of dried nori (Niwa et al., 2005; Niwa et al., 2008b). Among the components affecting taste in Porphyra gametophytic blades, free amino acids (FAA) are known to be the most important substances, among which aspartic acid (Asp), glutamic acid (Glu), alanine (Ala) and taurine (Tau) are predominant in the FAA (Noda et al., 1975; Harada et al., 1990; Yoshie et al., 1993). In the four major FAA, Glu and Ala were considered to be the most important FAA regarding the taste of dried nori (Noda et al., 1975). On the other hand, it is unclear whether Tau is important for the taste in Porphyra blades. In the present study, total FAA content of the red mutant tended to be higher than that of the wild type at the examined harvest times in the two nori cultivator groups (Table 3). The result of MANOVA also supported the findings (Table 4). Among the FAA contents, Ala and Tau were especially higher in the red mutant than in the wild type, except
for the Ala content at the first harvest by nori cultivator group 2 (Fig. 3). Table 4 also indicates that the red mutant has higher Tau content, although Ala content had no clear pattern as due to an interaction between the factors in the first harvest. Furthermore, total FAA content in the laboratory experiment was significantly higher in the red mutant than in the wild type and, in particular, the Ala and Tau contents of the red mutant were higher (Fig. 4). These results suggest that the red mutant tends to have a higher total FAA content and, above all, Ala and Tau contents than the wild type, although it is possible that these characteristics are changed by the environmental conditions in nori farms. Thus, the red mutant seems to have potential as a cultivar with a better flavor of dried nori. In addition, the difference in PE content between the two strains was greater at the third than first harvest, and the respective differences in Ala and Tau contents between the two strains also increased at the third harvest; therefore, the high contents of the two FAA may correlate with the high PE content. On the other hand, no clear relationship in the Glu content was observed between the two strains from the cultivation experiment (Fig. 3). In our previous study (Niwa et al., 2008a), the Glu content alone decreased with the number of harvests among the four major FAA contents; however, this tendency was not found in the present study, presumably because of the different environmental conditions in nori farms. Nori cultivation is one of the most profitable aquacultures in Japan, Korea and China. The annual production value of nori in Japan amounts to almost US$1bn. In determining the quality of commercially dried nori sheets, their color is very important: a deep black color is considered to be high grade in Japan. Furthermore, it is known that the deeper the black of the dried nori sheets, the higher the contents of the three major photosynthetic pigments (Chl. a, PE and PC) and the FAA (Aruga, 1974; Saitoh et al., 1975; Miura and Shin, 1989). On the other hand, until now, reddish nori sheets have been considered to have a commercially low value in nori industry, because the blades change to a reddish color when the nutrients in seawater begin to decrease in nori farms. Before isolating the red mutant IBY-R1 by heavy-ion beam mutagenesis, some spontaneous and artificial red mutants (previous red mutants) were isolated, but were not clearly characterized as having high PE content (Aruga and Miura, 1984; Yan et al., 2000); therefore, the previous red mutants are not used by nori cultivators in Japan. However, the present study suggests that the red mutant IBY-R1 tends to have higher contents of PE, total FAA, Ala and Tau than the wild type in nori cultivation. Furthermore, it is easy to discriminate the red mutant from the other cultivars with the naked eye. From these superior and distinguishing characteristics, the red mutant IBY-R1 may become a new cultivar in nori cultivation, even though it is possible that the mutant harvest will be lower than that of other cultivars. Acknowledgements We thank the Hyogo Nori Institute for seeding nori nets with conchospores of the red mutant IBY-R1 and wild-type HG-511, and the nori cultivator groups of Akashiura Fishermen's Cooperative for cultivating the gametophytic blades in their nori farms.
References Aruga, Y., 1974. Color of the cultivated Porphyra. Our Nori Res. 23, 1–14 (in Japanese). Aruga, Y., Miura, A., 1984. In vivo absorption spectra and pigment contents of the two types of color mutants of Porphyra. Jpn. J. Phycol. 32, 243–250. Beer, S., Eshel, A., 1985. Determining phycoerythrin and phycocyanin concentrations in aqueous crude extract of red algae. Aust. J. Mar. Freshw. Res. 36, 785–792. Harada, K., Osumi, Y., Fukuda, N., Amano, H., Noda, H., 1990. Changes of amino acid compositions of ‘Nori’, Porphyra spp. during storage. Nippon Suisan Gakkaishi (Bull. Jpn Soc. Sci. Fish). 56, 607–612. Jeffrey, S.W., Humphrey, G.F., 1975. New spectrophotometric equations for determining chlorophylls a, b, c1, c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanz. 167, 191–194. Mitman, G.G., van der Meer, J.P., 1994. Meiosis, blade development, and sex determination in Porphyra purpurea (Rhodophyta). J. Phycol. 30, 147–159.
K. Niwa et al. / Aquaculture 314 (2011) 182–187 Miura, A., 1984. A new variety and a new form of Porphyra (Bangiales, Rhodophyta) from Japan: Porphyra tenera Kjellman var. tamatsuensis Miura, var. nov. and P. yezoensis Ueda form. narawaensis Miura, form. nov. J. Tokyo Univ. Fish. 71, 1–14. Miura, A., Aruga, Y., 1987. Distribution of Porphyra in Japan as affected by cultivation. J. Tokyo Univ. Fish. 74, 41–50. Miura, A., 1988. Taxonomic studies of Porphyra species cultivated in Japan, referring to their transition to the cultivated variety. J. Tokyo Univ. Fish. 75, 311–325. Miura, A., Shin, J.-A., 1989. Crossbreeding in cultivars of Porphyra yezoensis (Bangiales, Rhodophyta) – Preliminary report. Korean J. Phycol. 4, 207–211. Niwa, K., Aruga, Y., 2003. Rapid DNA extraction from conchocelis and ITS-1 rDNA sequences of seven strains of cultivated Porphyra yezoensis (Bangiales, Rhodophyta). J. Appl. Phycol. 15, 29–35. Niwa, K., Kikuchi, N., Iwabuchi, M., Aruga, Y., 2004. Morphological and AFLP variation of Porphyra yezoensis Ueda form. narawaensis Miura (Bangiales, Rhodophyta). Phycol. Res. 52, 180–190. Niwa, K., Kikuchi, N., Aruga, Y., 2005. Morphological and molecular analysis of the endangered species Porphyra tenera (Bangiales, Rhodophyta). J. Phycol. 41, 294–304. Niwa, K., Aruga, Y., 2006. Identification of currently cultivated Porphyra species by PCR-RFLP analysis. Fish. Sci. 72, 143–148. Niwa, K., Furuita, H., Yamamoto, T., 2008a. Changes of growth characteristics and free amino acid content of cultivated Porphyra yezoensis Ueda (Bangiales, Rhodophyta) blades with the progression of the number of harvests in a nori farm. J. Appl. Phycol. 20, 687–693. Niwa, K., Furuita, H., Yamamoto, T., Kobiyama, A., 2008b. Identification and characterization of a green-type mutant of Porphyra tenera Kjellman var. tamatsuensis Miura (Bangiales, Rhodophyta). Aquaculture 274, 126–131. Niwa, K., Hayashi, Y., Abe, T., Aruga, Y., 2009. Induction and isolation of pigmentation mutants of Porphyra yezoensis (Bangiales, Rhodophyta) by heavy-ion beam irradiation. Phycol. Res. 57, 194–202.
187
Niwa, K., 2010. Genetic analysis of artificial green and red mutants of Porphyra yezoensis Ueda. Aquaculture 308, 6–12. Noda, H., Horiguti, Y., Araki, S., 1975. Studies on the flavor substances of ‘Nori’, the dried laver Porphyra spp.-II. Free amino acids and 5’-nucleotides. Nippon Suisan Gakkaishi (Bull. Jpn Soc. Sci. Fish). 41, 1299–1303. Ogata, H., Murai, T., 1994. White muscle of masu salmon, Oncorhynchus masou masou, smolts possesses a strong buffering capacity due to a high level of anserine. Fish Physiol. Biochem. 13, 285–293. Saitoh, M., Araki, S., Sakurai, T., Oohusa, T., 1975. Variations in contents of photosynthetic pigments, total nitrogen, total free amino acids and total free sugars in dried lavers obtained at different culture grounds and harvesting times. Nippon Suisan Gakkaishi (Bull. Jpn Soc. Sci. Fish). 41, 365–370 (in Japanese with English abstract). Wang, S.J., Zheng, Y.Z., Ma, L.B., Xu, P., Zhu, J.Y., 2000. Gamma-rays induction of mutation in conchocelis of Porphyra yezoensis. Chin. J. Oceanol. Limnol. 18, 47–53. Yamamoto, T., Unuma, T., Akiyama, T., 2000. The influence of dietary protein and fat levels on tissue free amino acid levels of fingerling rainbow trout (Oncorhynchus mykiss). Aquaculture 182, 353–372. Yan, X.H., Fujita, Y., Aruga, Y., 2000. Induction and characterization of pigmentation mutants in Porphyra yezoensis (Bangiales, Rhodophyta). J. Appl. Phycol. 12, 69–81. Yan, X.H., Liang, Z.Q., Song, W.L., Huang, J., Ma, P., Aruga, Y., 2005. Induction and isolation of artificial pigmentation mutants in Porphyra haitanensis Chang et Zheng (Bangiales, Rhodophyta). J. Fish. Chin. 29, 166–172 (in Chinese with English abstract). Yoshie, Y., Suzuki, T., Shirai, T., Hirano, T., 1993. Free amino acids and fatty acid composition in dried nori of various culture locations and prices. Nippon Suisan Gakkaishi (Bull. Jpn Soc. Sci. Fish). 59, 1769–1775 (in Japanese with English abstract).