Ⅲ-4. Radiation Chemistry & Biology
RIKEN
Accel. Prog. Rep. 56 (2023)

RIKEN Accel. Prog. Rep. 56 (2023)

Sample preparation for ion-beam irradiation for biologicalexperiments
Y. Hayashi∗1 and T. Abe∗1
RIKEN has developed a unique technology for mutation induction by using heavy-ion beams from particle accelerators at the RI Beam Factory (RIBF). At
relatively low doses, ion beams induced mutations ata high rate without severely inhibiting growth. Theirradiation treatment given to the various plant materials is quick, lasting between a few seconds and a
few minutes, but is suﬃcient to induce mutation. By
using this method, RIKEN has already put 39 newvarieties on the market in Japan, the United States,
and the European Union since 2001.1,2) Furthermore,high-yielding rice, good growth wakame (Undaria pinnatiﬁda), lettuce with low browning, microalgae withhigh oil content, and larger rotifer were identiﬁed as
beneﬁcial mutants.
We have developed an automatic irradiation system
consisting of a range shifter and an automatic sample
changer to cope with numerous requests with various
biological samples such as dry seeds, wet seeds, callus,
cultured tissue materials, pollens, seedings, cuttings
etc.3) Biological samples are stored in sample containers. Typical sample containers include plant boxes forregenerated plants and seedlings, 35-, 60-, and 90 mmdiameter plastic petri dishes for cultured tissues andseeds, and square plastic boxes for cuttings. The sample containers containing biological samples are attached to unique frames that support each sample containers.4) Irregularly shaped samples placed in zipper
bags or hybri-bags are ﬁxed directly to the frame in linewith the irradiation position. For example, Arabidopsis seeds were placed in a hybri-bag and arranged in asingle layer by deaeration, then we found the most effective LET for mutation induction.5) A uniform dose
distribution is important for reproducible treatment
on biological materials using ions with stable LET.
The thickness of the sample is limited by the irradiation conditions. Therefore, it is necessary to place
the sample in the most suitable container and attach
Table 1.

Samples∗1Containers
Petri dish, Plant box

Cuttings

Zipper bag, Square plastic box

LiquidFig. 1. Various samples set in the frame of automatic sample changer.
a: seed in the zipper bag, b: liquid sample in the hybribag, c: centrifuge tube ﬁxed in the cassette, d: micro
tube and PCR tube ﬁxed in the cassette.

Sample containers available in the frames of automatic sample changer.

Cultured tissues
Seeds

the container to the frame.
We built a new beam line, ‘WACAME,’ to increaseavailable nuclides with higher LETs and longer ranges.
The Ar-ion beams are accelerated by RRC and IRC
to 160 MeV/nucleon and sent to the E5 beam lines.
Ar-ion irradiation is now available in tubes, increasing the number of microbes and microalgae users. We
have created two types of cassettes for liquid culturesof microbes and microalgae (Fig. 1). One is for 15 mL
centrifuge tube, and the other is for 1.5 mL micro and200 µL PCR tubes. The ﬁrst one is also available forcuttings that do not ﬁt into square plastic boxes. The
second one is rubber lined to prevent slipping down oftubes. Samples are ﬁxed by springs to adapt various
size of samples in both cassettes. Holes were drilled in
the frame to check the sample’s center. Each cassettealso has a hole, and by aligning it with the hole in theframe, accurate placement in the irradiation position
was made possible. In addition, yellow stickers wereplaced on the plates so that the irradiation ﬁeld could
be seen. We will continue to develop new frames andcassettes that promote eﬃciency in irradiation experiments as a tailor-made mutagen treatment technology
to meet user requirements.

Petri dish, Zipper bag, Square plastic box, Hybri-bag
Centrifuge tube (15 ml), Micro tube (1.5-2 ml), 8 strips PCR tube*1, Hybri-bag*2*1: Use 6 strips in the middle and cut off the both ends.
*2: The thickness of sample for Fe-ion irradiation must be less than 2 mm.

RIKEN Nishina Center- 187 -

Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 56 (2023)

References
1) T. Abe et al., in Ion beam radiation mutagenesis inPlant Mutation Breeding and Biotechnology, (Oxfordshire UK, 2012), p. 99.
2) T. Abe et al., Nucl. Phys. News 25, 30 (2015).
3) H. Ryuto et al., J. Biomed. Nanotechnol. 2, 88 (2006).
4) H. Ryuto et al., Plant Biotechnol. 25, 119 (2008).
5) Y. Kazama et al., Plant Biotechnol. 25, 113 (2008).

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