2021 Volume 86 Issue 4 Pages 291-295
In Undaria pinnatifida, an effective method for mutant screening in sporophytes has not been established. The present study developed a novel mutant screening method for Undaria sporophyte by combining gametophyte mutagenesis with heavy-ion beam and land-based tank culture system. When we irradiated gametophytes and sporophytes with carbon- and argon-ion beams, survival rates of the female gametophytes and the sporophytes decreased with increasing dose. However, those of the male gametophyte did not decrease after both of the irradiations. Mutant screening during the sporophyte development was performed by using a land-based tank culture system. High-growth plants were selected in the first mutant (M1) population derived from the irradiated materials. We successfully obtained mutant candidates with higher growth than the wild type in the M2 generation obtained from brother-sister inbreeding of selected M1 plants. Four high-growth mutant candidate lines were selected from M2 populations of 48 lines. The mutant candidates were derived from 3 lines of the gametophyte irradiation and 1 line of the sporophyte irradiation, suggesting that the materials for the irradiation are applicable for mutant induction. The mutant screening method and the selected mutant candidates would advance the breeding and molecular biology in U. pinnatifida.
Seaweed aquaculture has several potentials for food production, sequestration of CO2, and buffering against anthropogenic pollution (e.g., Froehlich et al. 2019). Undaria pinnatifida (Harvey) Suringar (Alariaceae, Laminariales) has been cultivated as a leading industrial species in Japan, Korea, and China after the cultivation method was established in the 1950s (Yamanaka and Akiyama 1993). However, the yield is unstable, and improvement of cultivation techniques is required to meet market demand. Moreover, in the fishery community in Japan, a cultivar with higher productivity and quality during a shorter growing period is strongly needed because the number of fishery workers is decreasing, and they are aging.
In previous studies of breeding for brown macroalgae, heterosis in U. pinnatifida (Hara and Akiyama 1985) and Macrocystis pyrifera (Westermeier et al. 2010) was confirmed. In China, 24–27% higher productivity than ordinary lines was obtained by crossing Saccharina japonica and S. longissima and selecting for 5 generations (Zhang et al. 2011). In Tokushima and Hyogo prefectures in Japan, an earlier variety was obtained by crossing 1 male and 1 female gametophyte originating in 1 spore (Dan et al. 2015, Niwa and Harada 2016). In China, an elite cultivar was developed through gametophyte clone crossing and consecutive selection, and the yield indicated more 40% higher than the commonly cultivated population (Shan et al. 2016). These cultivars can currently meet the fisheries requirements; however, higher growth cultivars are needed in response to future climate change and the decrease in the number of producers.
Recently, mutagenesis using ion beams as mutagen has received attention. This method can induce mutations with high frequency at a relatively low dose at which almost all plants survive and with a wide range of variations (Tanaka et al. 2010, Abe et al. 2012, Murai and Abe 2021). Many elite lines have been obtained by ion beam mutagenesis. For instance, using the heavy-ion beam of the RIKEN RI-Beam Factory, commercial varieties of 35 lines of terrestrial plants and 2 lines of sake yeast have been derived (Abe et al. 2015, Abe 2021). High-growth or large-organ mutants were obtained by ion-beam irradiation, including Japanese toad lily with large flowers (Nakano et al. 2010), Artemisia annua L. with heavy leave (Inthima et al. 2014), high-yielding rice with long grains (Morita et al. 2019), Arabidopsis thaliana with large flower and seeds (Nhat et al. 2021), and large rotifers (Tsuneizumi et al. 2021).
Niwa et al. (2009) obtained color mutants of Neopyropia yezoensis f. narawaensis by irradiating C-ions in macroalgae. Moreover, we have established a rapid evaluation method to determine the effects of ion mutagenesis on U. pinnatifida zoospore by observing gametophyte mutants (Hirano et al. 2020). However, an effective method for mutant screening in sporophyte of U. pinnatifida has not been established. Since the harvest time of sporophylls required for the zoospore irradiation is limited in U. pinnatifida, materials that can be irradiated at any time are required. The present study developed a novel mutant screening method by combining gametophyte mutagenesis with heavy-ion beam and land-based culture system. We demonstrated that mutant candidates with increased growth could be selected using a new screening method.
The mother plant for the irradiation was collected in Hirota Bay, Iwate prefecture. According to the method described by Hirano et al. (2020), the zoospores were induced and cultured to gametophytes. After 6 months, gametophytes that had grown to 2 cm in diameter were irradiated with C ions (135 AMeV, LET, 30.0 keV·µm−1) at a dose of 0–25 Gy or Ar ions (95 AMeV, LET, 284 keV·µm−1) at a dose of 0–10 Gy. The irradiated gametophytes were cut into fragments with a homogenizer, and fragments of sizes ranging 42–100 µm were collected by sieving through micro meshes. One male gametophyte and 1 female gametophyte were picked up and placed into 1 well of a 48-well micro-plate with 1 mL PESI medium (Tatewaki 1966). Gametophytes were incubated at 20°C with a 12-h photoperiod at a photon flux density of 10 µmol m−2 s−1. Gametophyte size was measured after 8 weeks of culture. Gametophytes with cell filament less than 100 µm long and whitened were defined as dead, and survival rates of irradiated male and female gametophytes were calculated.
Heavy-ion beam irradiation to sporophytesYoung sporophytes of U. pinnatifida, 1 mm in length, were transferred into 15-mL plastic tubes for C ion irradiation and into plastic bags for Ar ion irradiation in sterilized seawater. The irradiation experiments were carried out in the same way as described above. Each tube or plastic bag contained 50 sporophytes. After irradiation, the sporophytes were cultured in 300-mL aeration flasks with 1/4 PESI medium and aeration at 15°C with a photoperiod of 12-h/12-h (light/dark), and 90 µmol photons m−2 s−1. The survival rate was measured after 3 weeks of culture.
The growth of sporophytes and selection of high-growth plants in M1 generationAll sporophytes germinated from gametophytes subjected to heavy-ion beam irradiation, and all sporophytes with heavy-ion beam irradiation with all ions and doses were cultured in a 7-L aquarium for approximately 2 weeks with running seawater. The seawater temperature and light intensity were set at 10°C and 100 µmol photons m−2 s−1, respectively. Then, sporophytes were cultured in a cyclone and floating culture system (CFCS; Sato et al. 2017b) until sporophyll formation. The total length and leaf weight of sporophytes cultured in the CFCS for 100 days after irradiation were measured. The plants were selected based on a 100 cm or more length and 40 g or more weight without deformity, and spores were induced from their sporophylls to gametophytes. These were used for screening in the M2 generation.
The screening for mutant candidates with higher growth in the M2 generationSpores induced from sporophylls of the selected M1 plants were poured into plastic dishes filled with 30 mL of PESI medium. These dishes were incubated at 20°C with 12-h photoperiods and 20 µmol photons m−2 s−1. After 1 month, when germinated gametophytes reached 1 cm in diameter as a colony, ten colonies of females and one colony of males were selected and transferred to a well of a plastic petri dish with six wells. The 6-well dishes were incubated at a light intensity of 20 µmol photons m−2 s−1 with 12-h photoperiods for gemination. Sporophytes were cultured in the 300-mL aeration flask in 1/4 PESI for 3 weeks at a light intensity of 20 µmol photons m−2 s−1 with a 12-h photoperiod. The seawater temperature was decreased by 1°C a week to mimic culture conditions in the ocean. Sporophytes were cultured in a 7-L aquarium at 10°C and 50 µmol photons m−2 s−1 for 20 days. All sporophytes were placed in a plastic tray filled with sterilized seawater without overlapping and photographed with a digital camera. The total length of all individuals was measured, and the plants with values 3 times higher than average values were selected as mutant candidates with increased growth. The candidates and unirradiated control plants were cultured in CFCS, and total length and leaf weights were measured 100 days after germination.
Although the survival rates of female gametophytes irradiated with heavy-ion beams with C and Ar ions decreased with increasing dose, those of male gametophytes did not decrease after both irradiations (Fig. 1a). The survival rates of sporophytes irradiated with heavy-ion beams were decreased at ＞12.5 Gy of C and ＞0.2 Gy of Ar (Fig. 1b). The plants that withered and died after a leaf curled during culture in the marine flask were observed with irradiation dose elevation.
Sporophytes derived from the gametophytes and sporophytes irradiated with Ar-ion beam at 10 Gy were dead by 50 days after irradiation. Smaller plants were observed at higher doses of irradiation with both C and Ar ions. Based on the length and weight (see Materials and methods), we selected 18 plants out of 210 plants derived from gametophyte irradiation and 30 plants out of 297 plants derived from sporophyte irradiation (Table 1). Zoospores were induced from the sporophylls of each selected plant to yield gametophytes. These were used for screening in the M2 generation.
| Ion | Dose (Gy) | Number of M1 plants applied to the screening | Number of M2 lines applied to the screening | Number of M2 lines included mutant candidates | |||
|---|---|---|---|---|---|---|---|
| Gametophytes | Sporophytes | Gametophytes | Sporophytes | Gametophytes | Sporophytes | ||
| 12C6+ | 0.5 | 41 | 41 | 0 | 2 | 0 | 0 |
| 2 | 27 | 41 | 4 | 13 | 1 | 0 | |
| 5 | 19 | 23 | 4 | 6 | 0 | 1 | |
| 12.5 | 15 | 21 | 1 | 1 | 0 | 0 | |
| 40Ar17+ | 0.2 | 30 | 50 | 3 | 1 | 1 | 0 |
| 1 | 31 | 44 | 3 | 4 | 0 | 0 | |
| 2.5 | 30 | 40 | 3 | 2 | 1 | 0 | |
| 5 | 17 | 37 | 0 | 1 | 0 | 0 | |
As screening for high-growth mutant, mutant candidates with total lengths markedly higher than those of others were selected from 1302G-R68, 1302G-B60, 1302G-W40 lines derived from the gametophyte irradiation with C ions at 5 Gy, Ar ions at 0.2 Gy, and Ar ions at 2.5 Gy, respectively (Table 1). When these 3 lines were cultured in the CFCS, 4 plants (B169–172) in 1302G-R68, 2 plants (Y163, 164) in 1302G-B60, and 7 plants (B235–B242) in 1302G-W40 were observed to be markedly larger than other plants in each line (Fig. 2a–c). In addition to the gametophyte irradiation, the sporophyte irradiation also induced the high growth mutant. When 1302S-R40 line derived from the sporophyte irradiation with C ions at 2 Gy was cultured in the CFCS, 7 plants (B219, B221, B222, B247, B248, B251, B254) of both lines were markedly larger than other plants (Fig. 2d). The sporophyte weights of four mutant candidates were higher than that of the wild type 100 days after germination (Fig. 3).
In heavy-ion mutagenesis, a unit of linear energy transfer (LET; keV µm−1) affects the efficiency of mutation induction and the molecular nature of mutations (Kazama et al. 2008, 2011, 2017, Hirano et al. 2012, 2015). The LET-dependent effect was also observed in U. pinnatifida, and the Ar-ion irradiation also exhibited high biological effects on the cell division and growth in zoospore irradiation (Hirano et al. 2020). A similar tendency was observed on the sporophyte irradiation, and a decrease of the survival rates was observed at low doses in Ar-ion irradiation (Fig. 1b). However, interestingly, the male gametophytes did not show a dose-dependent decrease of the survival rate both of C- and Ar-ion irradiation (Fig. 1a). These results suggested that there would be tissue or organ-specific sensitivity to heavy-ion beams regardless of haploid or diploid nuclear phases.
Screening of mutant candidates in the M2 generationIn the present study, we successfully obtained the mutant candidates by the screening method using the CFCS. Since land-based cultivation by the CFCS enabled plants with sporophylls without loss due to the strong wave motion experienced in ocean culture (Sato et al. 2017b), all plants irradiated with heavy-ion beams could be applied for the mutant screening, and subsequent generations were effectively induced. Three mutant candidates with high growth were obtained from gametophyte irradiation (Table 1). Therefore, gametophyte irradiation is thought to be one of the effective methods for mutant screening in sporophytes. The sporophyte irradiation was also able to induce the mutant candidate. Including the zoospore irradiation (Hirano et al. 2020), further study is required to reveal the efficiency of mutant induction by each irradiation. Since a high-energy Ar-ion beam (160 AMeV) was available to seaweeds by establishing of WACAME line in RIBF (Fukunishi et al. 2015), large-scale experiments with Ar-ion beam could be applied to U. pinnatifida. The LET-dependent effects in U. pinnatifida will be elucidated by using the WACAME line.
The mutant candidates should also be subjected to progeny testing and phenotypic analysis in the M3 generation. As a preliminary experiment, progenies (M3 generation) of Y163 and Y164, the selected mutant candidates from 1302G-B60 (Figs. 2, 3), showed similar phenotypes in M2 generation (data not shown). The M3 analysis will discuss the inheritance of the phenotypes in the mutant candidates. In the aquaculture trial, it is revealed that the use of early-maturing and late-maturing strains of U. pinnatifida derived from regional strains enable dual cropping at the same cultivation rope and contribute to increasing yield (Sato et al. 2017a). This is because the concentration of the workforce for the harvest can be avoided, especially in Japan, where the number of fishers is declining. Various high-growth strains are required to utilize this method in practical aquaculture and to spread out the fishing period. Therefore, it is expected that the mutant candidates with high growth could be used in the industry for higher productivity.
Our study revealed that C- and Ar-ion irradiation to gametophytes and sporophytes of U. pinnatifida could be a powerful tool to get candidates resources for elite cultivars. The combination with the CFCS tank system, screening methods for mutant candidates, phenotypic analysis, and genome information would advance the breeding and molecular biology of U. pinnatifida.
We thank the RIKEN Nishina Center and the Center for Nuclear Study, University of Tokyo for the operation of RIBF for performing the ion-beam irradiations in this study. This study was partly supported by the Formation of Tohoku Marine Science Center Project (Technical Development That Leads to the Creation of New Industries) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and JST-OPERA Program Grant Number JPMJOP1832, Japan.
