III-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 54 (2021)

RIKEN Accel. Prog. Rep. 54 (2021)

Eﬀect of heavy-ion irradiation on survival rate of Torenia fournieri
A. Matsuta,∗1 M. Hatashita,∗2 K. Takagi,∗2 Y. Hayashi,∗3 T. Abe,∗3 K. Murai,∗1 and Y. Kazama∗1,∗3
A heavy-ion beam is a powerful mutagen that has
high linear energy transfer (LET). We previously demonstrated that the value of LET aﬀects the size and typeof the induced mutations in a model plant Arabidopsis
thaliana; a C-ion beam with an LET of 30.0 keV/µm
predominantly induce small mutations that aﬀect almost single genes, while an Ar-ion beam with an LET of290 keV/µm can induce chromosomal rearrangements including translocation and large inversions or deletions.1)
We focus on the induction of chromosomal rearrangements because they can aﬀect multiple genes resulting
in the induction of new phenotypic traits that have notbeen ever observed by single gene mutations.
To investigate such a broad spectrum of phenotypes,
we aim at observing the ﬂoral phenotypes of Torenia
fournieri after the irradiation of heavy-ion beams withhigh LET, because its simple ﬂoral structure with colorful petals allows us to observe the change in the phenotypes with high visibility.2) Moreover, T. fournieri isa useful plant for genetic analysis and molecular biology
because of its small genome size (2n = 18, 171 Mb), andbecause of the transformation techniques established.3,4)
Once an interesting mutant is isolated, mutations including rearrangements responsible for its phenotype can bedetermined by whole-genome resequencing. Dry seedirradiation and screening of seed propagated mutants
needs to be conducted using an inbred strain, to eﬃciently perform the investigation of the phenotypic mutation spectrum followed by whole-genome resequencing.
In this study, prior to performing mutant screening, dose
dependence on survival was examined and the eﬀect ofLETs on survival was compared.
The inbred line ‘Zairai murasaki’ was kindly provided
by Dr. Nishijima of the National Institute of Floricultural Science. Dry seeds of ‘Zairai murasaki’ were irradiated with Ar ions with LETs of 184 keV/µm and290 keV/µm, respectively, at a dose range of 25–200 Gy
100Survival rate (%)

8060
40Ar (184 keV/µm)
Ar (290 keV/µm)

200C (50 keV/µm)
050

100

150

200

250

300

350

400

450500

Dose (Gy)Fig. 1. LET-dependent eﬀect on survival rate in T. fournieri.
∗1
∗2
∗3

Department of Bioscience and Biotechnology, Fukui Prefectural University
Res. and Develop. Dept., The Wakasa Wan Energy ResearchCenterRIKEN Nishina Center

Fig. 2. Photographs of wild-type ﬂower (A); a mutant ﬂower
with a rough-edged pistil and petals (B), and a mutant
ﬂower without the yellow spot (C). Bars: 1 cm for wholephotos, 1 mm for the photos of pistils.

in the RIKEN RI-beam factory. C-ion beam irradiation with an LET of 50 keV/µm was carried out by
the Wakasa Wan Multi-purpose Accelerator with Synchrotron and Tandem at the Wakasa Wan Energy Research Center at a dose range of 100–500 Gy. IrradiatedM1 seeds were surface-sterilized and incubated on 0.7%
agar containing 1/2 MS medium supplemented with 3%
sucrose at 25◦ C under long-day conditions (16 h light,8 h dark). About 30–50 seeds were sown for each irradiation treatment. After 1.5 months, the survival rate (thenumber of plants having true leaves per total number ofgerminated plants) was determined.
In any LET, survival rates were decreased as the irradiation dose increased (Fig. 1). The eﬀect on survival
rate diﬀered among LET values; it increased as LET
values increased. The Ar ion beam with the LET of
290 keV/µm was the most eﬀective for the reductionof the survival rates, although the eﬀect of higher values of LET will need to be tested. Based on the ruleof thumb that a dose showing around 90% survival is
the most eﬀective in mutation induction, the most eﬀective doses on mutation induction were estimated fromthe current results; 300 Gy, 75 Gy, and 50 Gy for the
C ion (50 keV/µm), Ar ion (184 keV/µm), and Ar ion(290 keV/µm), respectively.
Mutant screening is carried out in the M2 generation
because a phenotype caused by recessive mutation is expressed in the M2 generation. However, we identiﬁedtwo mutants from 30 and 16 M1 plants after irradiation
with LETs of 290 keV/µm and 184 keV/µm, respectively.
The former has jagged pistil and petals (Fig. 2B), andthe latter has no yellow spot (Fig. 2C). These phenotypes were observed in all ﬂowers produced on the samebranch. The detection of mutations responsible for these
phenotypes is in progress.
References

1) Y. Kazama et al., Plant J. 92, 1020 (2017).
2) K. Sasaki et al., Plant Biotechnol. 25, 81 (2008).
3) S. Kikuchi et al., Chromosome Res. 14, 665 (2006).
4) R. Aida et al., Plant Sci. 153, 33 (2000).

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