Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 49 (2016)

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Robuststrains isolated by heavy-ion beam irradiation and endurance
screeningin the green algae, Haematococcus
pluvialis
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T. Takeshita,*1 K. Takita,*1 S. Ota,*1,*2 T. Yamazaki,*1,*2 Y. Kazama,*3 T. Abe,*3 and S. Kawano*1,*2EƵŵďĞƌŽĨĐŽůŽŶŝĞƐŽƐĞ ;'ǇͿFig. 1. Sensitivity testing with the heavy-ion beam.
Following heavy-ion beam irradiation of H. pluvials witheach dose of the C ion (ż), Ar ion (Ɣ) and Fe ion (Ƒ), 3,000cells from each culture were inoculated on an agar plate(n=4 or 5).
Department of Integrated Biosciences, Graduate School of Frontier
Sciences, University of Tokyo
䠆2JST, CREST.
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hsHaematococcus pluvialis is an egg-shaped or spherical
unicellular green microalga ranging from 20-50 ȝm indiameter, belonging to the green algal genus Volvocales(Chlorophyceae). After endospore formation, the alga
releases zoospores with two flagellars that grow andbecome the nonmotile palmela cell. The green cell becomesa cyst following exposure to an environmental stress factor
such as strong light or low nutrients, and accumulates
astaxanthin, which exerts a strong antioxidative effect, inthe cell1). If it is possible to obtain a strain with hightolerance to environmental stress (i.e., a robust strain), it
seems that the percentage of cell death is decreased, and itis therefore possible to improve productivity. In this study,
we attempted to isolate robust strains of H. pluvialis using
heavy-ion beam irradiation and endurance screening.
Heavy-ion beams of carbon (C), argon (Ar) or iron (Fe)were irradiated on H. pluvialis at various doses (C: 0̽200Gy; Ar: 0̽100 Gy; Fe: 0̽75 Gy), and the number of colony
forming units was counted (Fig. 1). The LETs were 22.5
keV/ȝm, 309 keV/ȝm, and 790 keV/ȝm, for C, Ar, and Fe,
respectively; thus, the number of colonies formed changedin the order of LETs in H. pluvialis, and the overkill
reported with Arabidopsis did not occur 2).
The cell culture irradiated with the heavy-ion beam ofeach ion species and dose was inoculated on an agar plate(Fig. 1). To isolate robust strains, the plates were subjected
to endurance screening, which included incubation for 3-12
months until the nutrient medium dried up. Most of the cells
were died to emit white fluorescence under UV irradiation.

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Fig. 2. Microscopic observation (A) and number of livingcells (B) in robust strains. WT (a, b), G4 (c, d), G7 (e, f), R1(g, h), R6 (i, j) in A. Number of living cells (total cell no. ×survival rate) in B. Dead cells are stained with Evans blueand emit the white fluorescence under UV irradiation.
After selecting robust strains that were still alive as the redor green cells from dead cells emitting white fluorescencethrough the use of microscopic observation, they werecultivated with continuous light, and each set of 10 strains
maintained a red or a green character. The red strain is acandidate for high astaxanthin accumulation and the green
strain is a candidate for high proliferation with chlorophylls.
The culture scale was increased to the BBM liquid medium
in 100-mL flasks under continuous light (125 μmol
photons·m-2·s-1), and sodium acetate solution (finalconcentration: 45 mM) was added to induce astaxanthin
accumulation. The survival rate of the wild type strain(WT1-3) at 7 days post-inoculation was 0-6.2% under these
conditions (Fig. 2Aa,b and B). The number of living cells,
as determined by cell count, and survival rate were high in
G4 (91.8%), G7 (74.9%), R1 (47.2%), and R6 (61.7%) (Fig.
2Ac̽h and B). Isolates G4, G7, R1, and R6 indicated highchlorophyll and carotenoid production rates, and G7 and R1
showed conspicuously high carotenoid production (data notshown).
The heavy-ion beams irradiated into the four robust
strains were the Ar ion beam at 25 Gy, and C ion beam at
100 Gy. Under these irradiation conditions, approximately
200 colonies formed on an agar plate (Fig. . It is thoughtthat the irradiation conditions are suitable for the variant
construction of H. pluvialis.
This study was supported by JST, CREST (to SK).

䠆1- 265 -References
1) Wayama et al., Plos One 8(1), e53618 (2013).
2) Kazama et al., Plant Biotechnol. 25(1), 113-117 (2008).