The Effect of Heavy-Ion Beams with High Linear Energy Transfer on Mutant Production in M₁ Generation of Torenia fournieri

Abstract

A high-LET heavy-ion beam has a severe effect on survival and effectively induces chromosomal rearrangements. In this study, the effect of high-LET heavy-ion irradiation on mutation induction in the M₁ generation was investigated in an inbred line of Torenia fournieri, which is a widely used horticultural plant. Dry seeds of the inbred line ‘Zairai murasaki’ were irradiated with a C-ion beam (LET: 50 keV µm⁻¹) or Ar-ion beams (LETs: 184 keV µm⁻¹ or 290 keV µm⁻¹) at different doses, and then sown on 1/2 MS plates. After determining the survival rates from each irradiation condition, appropriate doses of each beam were roughly determined to produce a survival rate of 90%: 300, 75, and 50 Gy for the C-ion beam with a LET of 50 keV µm⁻¹, Ar-ion beam with a LET of 184 keV µm⁻¹, and Ar-ion beam with a LET of 290 keV µm⁻¹, respectively. In the screening of branches with aberrant flowers, one and two aberrant plants were isolated from 16 and 30 M₁ plants after irradiation with LETs of 184 keV µm⁻¹ and 290 keV µm⁻¹, respectively. However, no aberrant plants were identified in M₁ plants after irradiation with a LET of 50 keV µm⁻¹. We concluded that high-LET heavy-ion beam irradiation is effective in inducing mutations even in the M₁ generation of inbred ornamental plants. This technique could be widely used for breeding ornamental plants that can be propagated vegetatively.

Torenia fournieri belongs to the family Scrophulariaceae and is widely used as a horticultural plant. This species originated in tropical and subtropical Asia and Africa (Yamazaki 1985), and has a high-temperature tolerance (Warner and Erwin 2005). Therefore, T. fournieri is a popular horticultural plant for decorating flower beds during the summer season. Since the ‘Crown’ series that has a variety of flower colors was released in 1988, new cultivars with different colors have been developed, leading to this species becoming commonly chosen for horticultural use. Irradiating in vitro cultured tissue with heavy-ion beams has also produced variations in flower color, providing several new varieties that are available for sale (Miyazaki et al. 2006, Sasaki et al. 2008, Kako et al. 2012).

T. fournieri has previously been used to study floral morphology and flower color. Various floral morphological mutants have been characterized (Sasaki et al. 2012, Nishijima et al. 2013, 2016), and genetic disruptions the flavonoid biosynthetic-related gene and floral homeotic genes through genome editing have been performed to determine their phenotypic effects (Nishihara et al. 2018, Sasaki and Ohtsubo 2020). Therefore, this species is now a model plant for studying ornamental characteristics. T. fournieri is also important in reproductive biology, as the pollen tube attractant peptide LURE is known to have been first discovered in this species (Okuda et al. 2009). This relates to the unique feature of the naked embryo sac that allows the establishment of an in vitro system for observing the guidance of pollen tubes (Higashiyama et al. 1998). Moreover, T. fournieri is a suitable plant for genomic analysis and molecular biology because of its small genome size (2n=18, 171 Mb), and the transformation techniques established (Aida et al. 2000, Kikuchi et al. 2006). If a relevant mutant is obtained, such as a plant with different flower shapes or colors, mutations responsible for its phenotype can be determined using whole-genome resequencing.

This study aimed to investigate the mutation effects of heavy-ion irradiation on an ornamental plant. Heavy-ion beams are powerful mutagens with high linear energy transfer (LET). The value of LET was found to influence several aspects of mutation induction in Arabidopsis thaliana. First, the survival rate was affected by the value of LET, and a beam with a LET of 290 keV µm⁻¹ resulted in the most severe effect on plant survival (Kazama et al. 2008b). Second, the mutation frequency in the M₂ generation was different depending on the value of LET, and a beam with a LET of 30 keV µm⁻¹ showed the highest mutation frequency (Kazama et al. 2011). Finally, the size and type of the induced mutations were also affected by the value of LET (Hirano et al. 2012, Kazama et al. 2013); when Ar ions with a LET of 290 keV µm⁻¹ were irradiated, chromosomal rearrangements, including translocations, large inversions, and deletions, were efficiently induced (Hirano et al. 2015, Kazama et al. 2017). Whole-genome re-sequencing followed by detection of mutants induced by Ar ions with a LET of 290 keV µm⁻¹ revealed that 26.9% of all Ar-induced homozygous mutations were rearrangements, which was seven times higher than that of C ions with a LET of 30 keV µm⁻¹ (3.4%) and 30 times higher than that of fast neutrons (0.9%), which often cause chromosomal aberrations (Belfield et al. 2012). These findings provoke further considerations as to the mechanism involved in high-LET irradiation affecting mutagenesis in ornamental plants. Chromosome rearrangements can have a strong effect on phenotypic changes (Kazama et al. 2018). T. fournieri is suitable for this analysis because of its simple floral structure with colorful petals, allowing us to readily observe phenotypic changes.

In this study, we investigated the effect of heavy-ion irradiation with different LET values on survival and phenotypic changes in the M₁ generation. We showed that high-LET irradiation can change the floral phenotype of T. fournieri in the M₁ generation.

Materials and methods

Plant materials

The inbred line ‘Zairai murasaki’ of T. fournieri was kindly provided by Dr. T. Nishijima of the National Institute of Floricultural Science.

Irradiation treatment and growth condition

Dry seeds of the inbred line ‘Zairai murasaki’ were irradiated with C ions at a dose range of 100 to 500 Gy using the Wakasa Wan Multi-Purpose Accelerator with Synchrotron and Tandem at the Wakasa Wan Energy Research Center. The LET value of the C-ion beam was 50 keV µm⁻¹ on the surface of the seeds. Ar-ion beam irradiation with LETs of 184 keV µm⁻¹ and 290 keV µm⁻¹, both of which were calculated at the surface of the seeds, were conducted in the RIKEN RI-beam factory. The dose ranges of the Ar-ion beams with 184 keV µm⁻¹ and 290 keV µm⁻¹ were 50 to 200 Gy and 25 to 150 Gy, respectively. To reduce the ion velocity and control the LET values, the ions were passed through a combination of absorbers (Ryuto et al. 2008). The irradiated seeds were surface-sterilized and incubated on 0.7% agar containing 1/2 MS medium (Murashige and Skoog 1962) supplemented with 3% (w/v) sucrose at 25°C under long-day conditions (16-h light/8-h dark). After 1.5 months, the survival rate (the number of plants with true leaves per total number of germinated plants) was determined.

Observation of floral phenotypes

After calculating the survival rate, M₁ plants were transplanted into vinyl pots containing soil and then placed in a growth chamber at 25°C under long-day conditions (16-h light/8-h dark). Floral phenotypes were observed for each plant, and the positions of aberrant flower openings were recorded for each plant. Normal and aberrant flowers were observed and photographed under a stereomicroscope (SZX16, Olympus, Tokyo, Japan) attached to a CCD camera (DP10, Olympus). For scanning electron microscopy (SEM), pistils were treated with an aqueous solution containing 0.5 M choline chloride and 0.5 M sodium methanesulfonate overnight. The treated pistils were observed using a scanning electron microscope with a cool stage at −20°C (S-3000N, Hitachi, Tokyo, Japan). The SEM was operated at 4 kV.

Results

Effect of heavy-ion irradiation with different LET values on the survival rate

Dry seeds of the inbred ‘Zairai murasaki’ line were irradiated with a C-ion beam (50 keV µm⁻¹) and Ar-ion beams (184 keV µm⁻¹ and 290 keV µm⁻¹) (see Materials and methods). The irradiated seeds were sterilized and sown on 1/2 MS plates and grown in a growth chamber (see Materials and methods). Germination rates were not affected by irradiation (data not shown), similar to previous studies on Arabidopsis thaliana (Kazama et al. 2008b). Survival rates are shown in Table 1, and the rates decreased as the irradiation dose increased, regardless of the LET values, although the effect on survival rates differed depending on the LET value. The Ar-ion beam with a LET of 290 keV µm⁻¹ was the most effective for reducing the survival rates among the LET values tested. The shoulder doses, in which 90% survival was observed, were 300, 75, and 50 Gy for the C (50 keV µm⁻¹), Ar (184 keV µm⁻¹), and Ar ion (290 keV µm⁻¹), respectively. Higher LET values correlated to lower shoulder doses. These doses are normally determined as appropriate for heavy-ion beam irradiation and generally show the highest mutation frequencies. For example, after irradiation at these doses with M₁ plants of Arabidopsis (Kazama et al. 2008b) those plants were then chosen for further investigations.

Table 1. Effect of heavy-ion irradiation on survival of T. fournieri.

LET (keV µm−1)	Dose (Gy)	No. of germinated seeds	No. of plants with true leaves	% Survival
Control	0	112	107	95.5
50	200	32	31	96.9
	300	26	25	96.2
	400	32	24	75.0
	500	34	19	55.9
184	50	77	71	92.2
	75	115	102	88.7
	100	87	82	94.3
	150	74	46	62.2
	200	17	0	0.0
290	25	119	107	89.9
	50	138	127	92.0
	75	100	86	86.0
	100	92	5	5.4
	150	45	0	0.0

Effect of Ar-ion irradiation on the mutation induction in the M₁ generation

We identified one and two plants with aberrant flowers from 16 and 30 M₁ plants after irradiation with LETs of 184 keV µm⁻¹ and 290 keV µm⁻¹, respectively (Table 2). In these plants, all flowers in at least one branch showed the same aberrant phenotypes, suggesting that these phenotypes were not derived from radiation injury. While the wild-type flower had two pale violet petals and three dark violet petals (Fig. 1A), each of the aberrant flowers showed different floral phenotypes; one had no yellow spot (Fig. 1B), another had white petals with violet edges (Fig. 1C), and the third had a jagged pistil and petals (Fig. 1D). The jagged pistil had a roughly edged stigma. Stereo microscopic observation revealed that the stigma of the mutant had a jagged, intricate structure, in which most of the cells protruded outward (Fig. 2). Although the heredity of the mutant phenotype remains to be investigated, only the mutant with the jagged stigma was fertile, while the others were sterile. The roughly edged stigma was maintained in vegetatively propagated mutant plants. These mutants with aberrant floral phenotypes were observed only in Ar-ion beam-irradiated M₁ plants. Such mutants were not observed in the 300 M₁ plants subjected to C-ion irradiation (Table 2).

Table 2. Summary of mutant screening in M₁ generation.

LET (keV µm−1)	Dose (Gy)	No. of plants screened	No. of plant with aberrant flowers
50	300	80	0
184	75	16	1
290	50	30	2

　

Fig. 1. Photographs of a wild-type flower (A), a mutant flower without the yellow spot (B), a mutant with increased petals with pale color (C), and a mutant flower with a rough-edged stigma and petals (D). Scale bars=2 mm.

Fig. 2. SEM observation of the wild-type stigma (A) and rough-edged stigma (B). The stigma of the mutant had a jagged, intricate structure. Scar bar=250 µm.

Discussion

Normally, mutant screening is performed in the M₂ generation in seed propagated plants because a phenotype caused by a recessive mutation is expressed in the M₂ generation. In the case of vegetative propagation plants such as T. fournieri, mutant screening in the M₁ generation can also be performed because the branch with mutant flowers can be propagated vegetatively, even if the mutated branch was only shown as chimaera in the whole mutant plant. Such a procedure shortens the breeding times. However, mutant screening in the M₁ generation is difficult when an inbred line is used because of the disappearance of phenotypes caused by recessive mutations. In the current study, we showed that high-LET irradiation can effectively induce mutants in the M₁ generation in the inbred T. fournieri line, ‘Zairai murasaki.’

The effect on survival (Table 1) was similar to that of A. thaliana, in which 290 keV µm⁻¹ was the most effective in reducing plant survival (Kazama et al. 2008a, b). In the case of A. thaliana, the LET that produces the most severe effect on plant survival can cause substantial alterations (Hirano et al. 2012, Kazama et al. 2017). It is therefore possible that such dynamic alterations are also efficiently induced by irradiation with a LET of 290 keV µm⁻¹ in T. fournieri.

Heavy-ion beam irradiation of ornamental plants is effective in generating a wide variety of floral traits. Heavy-ion irradiation can be applied to various vegetative tissues in many flowering plants, and its vegetative propagation has produced a variety of mutants (Miyazaki et al. 2006, Kanaya et al. 2008, Ishii et al. 2009, Abe et al. 2012, 2015, Hisamatsu et al. 2016, 2020, Hayashi et al. 2017, Ochiai et al. 2019). All occurred in the M₁ generation and were subsequently propagated vegetatively. The current results suggest that floral mutants can be obtained in the M₁ generation even with irradiation of seeds of an inbred line. When chemical mutagens are applied to seeds, mutant isolation in M₁ plants was reported (Jain et al. 1968, Alcantara et al. 1996). Heavy-ion irradiation of fertilized egg cells produced recessive homozygous albino mutants of Nicotiana tabacum L. (Abe et al. 2000) and a flower color mutant of Petunia altiplana in M₁ plants (Suzuki et al. 1999). When the seeds were irradiated with heavy-ion beams, recessive homozygous dwarf and xthanta-like mutants were observed in sweet peppers (Honda et al. 2006), recessive homozygous anantha and xthanta mutants in ‘Micro-Tom’ tomato (Imanishi et al. 2007, 2010), and mutants with aberrant floral morphologies were produced in ‘Xanthi’ tobacco (Kazama et al. 2008a) in the M₁ generation. Although these phenomena appeared to be unique to Solanaceae plants, mutants with different floral phenotypes were observed in Delphinium (Utoyama et al. 2009) and Torenia in the current study. The previous mutants isolated in the M₁ generation were induced by irradiation with C, N, or Ne ions, which had lower LETs than those in this study. The higher LET with Ar ions induces chromosomal rearrangements, which may have a higher mutagenic effect on the M₁ generation, as shown here. Chromosome rearrangements in wheat root tip cells in the M₁ generation occurred more frequently with heavy-ion irradiation than X-ray irradiation (Kikuchi et al. 2009). In the case of the dioecious plant Silene latifolia, the mutants with partial deletions on the Y chromosome showed elevated gene expression levels on X chromosome-linked genes (Krasovec et al. 2019). It is possible that chromosomal rearrangements cause global changes in gene expression levels, leading to alterations in floral phenotypes in the M₁ generation. Since the number of mutants was small in this study, further sowing of irradiated seeds and observation of the mutations is required. Additional quantitative analysis and irradiation experiments in various species are needed to elucidate the effectiveness of mutagenesis in the M₁ generation of higher LET with heavy-ion irradiation.

Acknowledgments

We thank Dr. Takaaki Nishijima for his generous gift of ‘Zairai murasaki’ seeds. We also thank the Wakasa Wan Energy Research Center, RIKEN Nishina Center, and the Center for Nuclear Study, University of Tokyo, for the operation of the RIKEN RI-beam factory for heavy-ion irradiation in this study. This research was supported by JSPS KAKENHI Grant Numbers 20H03297 and 20K21449 to Y.K. This research was also partly supported by the Joint Research Program of Fukui Prefectural University and the WERC.

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