Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 47 (2014)

Rapid evaluation of mutational effects resulting from heavy-ion irradiation ofUndaria pinnatifida

T. Hirano,＊1 Y. Sato,＊1,＊2 K. Ichinose,＊3 Y. Hayashi,＊3 N. Fukunishi,＊3 and T. Abe＊1,＊3Undaria pinnatifida, called wakame in Japan, is a majorcommercial seaweed. Recently, there has been a demand for
new cultivars with enhanced properties, such as high yield,
high environmental adaptability, or high concentration ofavailable contents for human health, to expand the market
for wakame. Therefore, we performed the mutation
breeding of U. pinnatifida through heavy-ion-beamirradiation.1) Optimization of the dose and linear energy
fransfer (LET) in heavy-ion mutagenesis is essential for
efficient mutant induction.2) However, a method forevaluating mutation frequency has never established in U.
pinnatifida, and the optimization is still difficult. Moreover,in macroalgae, the analysis of mutation frequency in M2
generation consumes much time and requires large space. In
the present study, we irradiated zoospores with heavy-ion
beams. Because the female and male gametophytes
developed from the zoospores are in the haploid stage of its
life cycle, mutant screening can be performed in M1
generation. Through the mutant screening and investigationof the mutant phenotypes, we tried to develop a method forthe effectiveness of heavy-ion mutagenesis.
Samples (3 cm × 3 cm) of sporophylls of U. pinnatifida
were irradiated with C ions (LET: 30.0 keV/μm) at a doseof 0–25 Gy or Ar ions (LET: 280 keV/μm) at a dose of 0–10
Gy. The irradiated pieces were placed in beakers filled withsterilized seawater, and zoospores were induced. The
zoospore suspension were diluted with Provasoli's enrichedseawater with Iodine3) and poured into plastic dishes. The
dishes were incubated at 20 ºC with 12 h photoperiods anda light intensity of 5 μmol m-2 s-1. The gametophyte size
was measured after 3 weeks of culture. The gametophyte
over 100 μm in size of the longest cell filament was defined
as a developed gametophyte, and the formation rate ofdeveloped gametophyte was calculated. Mutant screening
was performed after 5 weeks of culture.
When we investigated the growth of the gametophytes
developed from the irradiated zoospores, the number ofdeveloped gametophytes decreased with increasing
irradiated dose (Fig. 1). A comparison between C-ion andA-ion irradiation revealed that the Ar-ion irradiation has
high biological effect to the cell division or cell growth.
After 5 weeks of culture, some mutants in cell shape, cellsize, and intracellular structure were observed. One of themutants showed a reduction in cell elongation (Fig. 2C, D).
In the untreated control, the female gametophyte cells werelarger than the male gametophyte cells (Fig. 2A, B). There
were at least two types of cell elongation mutants:
RIKEN Innovation Center
Riken Food Co., Ltd.
＊3RIKEN Nishina Center＊1＊2- 299 -small-cell type (Fig. 2C) and large-cell type (Fig. 2D). One
possible interpretation is that the small-cell type and thelarge-cell type cells were induced in male and female
gametophytes, respectively. The mutation frequency for thecell elongation mutant tended to increase in adose-dependent manner (data not shown). Therefore, themutants can be used as an indicator for investigating theeffectiveness of heavy-ion mutagenesis. We will evaluate
LET-dependent effects for mutation induction in U.
pinnatifida using this method.

Fig. 1 Effect of heavy-ion irradiation on gametophyte
development. The formation rates after 3 weeks of culture
are expressed as the mean of two individual experiments.

Fig. 2 Morphological mutants induced by C-ion irradiation.
The male (A) and female gametophytes (B) fromunirradiated zoospores were cultured for 5 weeks. The cellelongation mutants after 5 weeks of culture derived fromthe C-ion irradiation at 5 Gy (C) and 12.5 Gy (D). Bars
indicate 20 μm.
References

1) Y. Sato et al.: RIKEN Accel. Prog. Rep. 46, 267 (2012).
2) Y. Kazama et al.: Plant Biotechnol. 25, 113 (2008).
3) M. Tatewaki: Phycologia 6, 62 (1966).