Ion beam irradiation mutagenesis in rye ( Secale cereale L . ) , linseed ( Linum usitatissimum L . ) and faba bean ( Vicia faba L .

Ion beam irradiation is a potential tool for inducing novel mutations in plants. We chose three crop species (rye, linseed, and faba bean) to determine the potential of nitrogen ion beam irradiation for inducing mutations. We tested ion beam irradiation with nitrogen ions at six different fluencies (5×105, 1×106, 5×106, 1×107, 5×107, and 1×108 N-ion cm-2) on dry grains. The three studied crop species had different sensitivities to the irradiation. Increased doses of ion irradiation had more effect on survival than on germination. Rye seedlings had the lowest survival rate at high doses of irradiation and significantly higher off-type plant phenotypes than the other two species. In M1 seedlings, stunted growth, failure to complete the plant life cycle and chlorophyll mutants were observed in all three species. Terminal-inflorescence mutations and sectional chimeras in faba bean were observed in the M2 generation. We conclude that ion beam irradiation is an effective tool for mutation breeding of diverse crop species when the appropriate dose is defined.


Introduction
Plant breeding depends on the existence of variation in useful traits, and where the amount of natural variation is not adequate in available germplasm, novel mutations may be induced by irradiation or chemical mutagenesis.Favourable phenotypes are then selected for subsequent breeding objectives, during which the desirable characteristics are generally stabilized in subsequent generations.Physical and chemical mutagens have demonstrated their usefulness in plant mutation induction (Oladosu et al. 2016).The favoured forms of irradiation for mutation have been fast neutron bombardment (Le et al. 2001), X-, and gamma-ray irradiation (Beyaz and Yildiz 2017).Key problems with mutagenesis include the shortage of physical and chemical mutagen sources, especially in Finland, and the low viability of seeds or other plant parts after neutron and gamma-ray irradiation.A further problem is that suitable radiation sources are becoming less readily available to plant breeders and replacements need to be found.Chemical mutagens such as ethyl methane sulphonate (EMS) are considered objectionable from an occupational health viewpoint, environmental issues and post treatment handling (Neel 1970, Predieri and Di Virgilio 2007, Oladosu et al. 2016).
During the last two decades, the use of ion beam irradiation has emerged as an effective and unique technique for inducing mutations in plants (Tanaka et al. 2010).Ion beams provide high linear energy transfer (LET) compared to low LET radiation such as gamma-ray and X-ray, which simply means applying a large amount of energy in a small area, when aligned along the direction of the beam.The higher LET radiation has greater biological effects on plants than lower LET radiation.The remarkable advantages of ion beams for mutation plant breeding over the other mutagens include high mutation rates, high survival rates, wide phenotypic variation, and targeted-trait specificity (Tanaka et al. 2010, Abe et al. 2012).The application of ion beams in plant mutation breeding was introduced in the 1990s in Japan.Ion bean irradiation has since been used on several plant species mainly in Japan (reviewed in Abe et al. 2012Abe et al. , 2015) ) and China (reviewed in Dong et al. 2016).The effectiveness of ion beam mutation has been studied in several food crops (e.g.Maekawa et al. 2003, Yamaguchi et al. 2009a, Ishikawa et al. 2012), ornamentals (e.g.Yamaguchi et al. 2003, Yamaguchi et al. 2009b, Yamaguchi 2018), and Arabidopsis thaliana as a model plant species (e.g.Hirano et al. 2015, Du et al. 2018).The method has not been tested on arable crops grown in the cool-temperate zone.
To be effective, the ion beam has to penetrate the seed coat and reach meristematic tissue in the embryo.Seed anatomy is important in this regard.In the small-grain cereals such as rye (Secale cereale L.), the embryo is presented at the proximal end of the grain, and is easily irradiated through the outer layers of the caryopsis.In dicotyledonous crops, the embryo is partly protected by the cotyledons as well as the seed coat.In linseed (Linum usitatissimum L.), an example oilseed, the seeds are small and the seed coat is relatively thin, whereas in faba bean (Vicia faba L.), an example grain legume, the dimensions are much larger.
Hence we chose these three species as suitable test cases in a study to determine the potential of nitrogen ion beam irradiation for inducing mutation in crop species that had not yet been investigated with this mutation technique.
Dry seeds were carefully placed into plastic petri dishes (35 mm ∅ × 10 mm, Fig. S1) using forceps and held in place with Blu Tack mounting putty (Bostik Smart Adhesives, https://www.bostik.com/)with the embryo on the top surface, presented to the ion beam, ensuring high and uniform irradiation.Irradiation was conducted at the JYFL cyclotron beam facility (Department of Physics, University of Jyväskylä, Finland) and the chosen ions were 14 N 6+ (Gorelick et al. 2007).For the range of irradiation, fluencies were 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , and 1×10 8 ions cm -2 accelerated to 310 MeV.The controls for the three studied species (non-irradiated seeds) were prepared the same way as other treatments.The window of the instrument was 40 mm in diameter, limiting the number of seeds that could be irradiated.This was not a problem for the small seeds of both rye and linseed, but it was for faba bean since its seeds are larger than those of rye and linseed, so several dishes had to be prepared.The sample size was 100 seeds of rye and linseed and 50 of faba bean.Each dose was replicated three times for all three species used in this study, giving seven treatments × three replicates × three species.
After irradiation, seedlings were transplanted into a standard peat-soil mixture (White 420 W; Kekkilä Oy, Vantaa, Finland) in plastic 1020 Trays (54 × 28 × 6 cm), and grown in a climate-controlled glasshouse of the Department of Agricultural Sciences, University of Helsinki, Finland.The photoperiod was set to 16h day / 8h night, and the temperature was maintained at 21 °C day /18 °C night.The minimum photosynthetic photon flux density was 300 µmol m -2 s -1 .The relative humidity was maintained at 60%.Survival rate (%) was calculated as the number of seedlings from the irradiated seeds divided by number of seedlings from the non-irradiated control.M 1 plants were scored for visible phenotypic variations (mutants) from control plants and after 6 weeks each individual mutant was transferred into a 1 l plastic pot filled with the same peat-soil medium for secure seed harvest.Only faba bean plants (treatments 5×10 7 ions cm -2 and 1×10 8 ions cm -2 ) were taken to the M 2 generation.Two-hundred faba bean M 2 plants were grown in individual 4 l pots in insect-proof cages, and the frequency of chlorophyll deficient and terminal-inflorescence mutants were recorded.

Results and discussion
The germination rate was not affected by irradiation at the doses tested for faba bean and linseed, but rye grains exposed to the highest dose of ion irradiation (1×10 8 ions cm -2 ) had 17% poorer germination (Table 1).The germination rate of linseed in all treatments including control was lower than initially, which might be due to physical damage of the seeds by the forceps.Similarly, in soybean (Glycine max [L.] Merr., Im et al. 2017) and tobacco (Nicotiana tabacum L., Kazama et al. 2008), germination rate remained unchanged when the irradiation dose on dry seeds was increased.In rice (Oryza sativa L., Maekawa et al. 2003) and einkorn wheat (Triticum monococcum L., Murai et al. 2013), with similar grain shapes to rye, the rate of germination dropped when the irradiation doses were increased.The highest doses of irradiation (5×10 7 cm -2 and 1×10 8 ions cm -2 ) induced more mutant phenotypes in linseed and faba bean M 1 seedlings, although the same trend followed at lower dose rates in rye with considerably more offtype phenotypes (Table 1).In all three species the survival rates significantly decreased at 5×10 7 ions cm -2 dose of irradiation (Fig. 1), as noted in several other crop species (see Abe et al. 2012).Rye seedlings had the lowest and linseed seedlings had the highest survival rates (Fig. 1).The higher doses of ion irradiation also resulted in slower growth of all three crops particularly on faba beans (Figs.2A, B and C).The slow growth rate of faba beans at doses of 5×10 7 cm -2 and 1×10 8 ions cm -2 most likely is due to radiation injury on seeds in the M 1 generation.For example, in mung bean (Vigna radiata Wilcz.), seedling elongation / growth rate was negatively correlated with irradiation dose (Grasso et al. 2016).
A wide range of abnormal phenotypes was observed in M 1 plants (Fig. 3A-N).In general, aberrant phenotypes observed in M 1 generation can be due to a dominant mutation, a homozygous recessive mutation, or non-genetic radiation injury.In most mutagenesis, visible phenotypes in the M 1 generation are attributed to dominant mutations (see Abe et al. 2012).Some of the present mutations, however, were more typical of homozygous recessive changes.For example, the radiation-induced terminal-inflorescence mutation in faba bean (Fig. 3J) is generally controlled by a single recessive gene (Sjödin 1971, Benlloch et al. 2015).Ion beam irradiation has been shown to induce homozygous recessive mutations that can be detected already in the M 1 generation (  N atom per cm 2 ; (c) 1×10 6 N atom per cm 2 ; (d) 5×10 6 N atom per cm 2 ; (e) 1×10 7 N atom per cm 2 ; (f) 5×10 7 N atom per cm 2 ; (g) 1×10 8 N atom per cm 2 .
The molecular nature of mutations induced by ion beam mutagenesis has been extensively studied in mammals, where it generally has produced double-stranded DNA breaks.In Arabidopsis, it has been shown that ion beams led to clustered DNA damage in the chromosomes, and that they have great potential to induce complicated intra-chromosomal reshuffles (Shikazono et al. 2005, Hirano et al. 2015).The point-like mutations and intergenic rearrangements such as deletions and insertions are reported as being more frequent in mutants induced by ion beams than in those induced by ionizing radiations such as gamma rays (Abe et al. 2012, Yamaguchi 2018).
In this study faba bean only was taken to the M 2 generation.Sectional chimera (Fig. 3H), maculate leaf shape (Fig. 3I) and terminal-inflorescence (Fig. 3J) phenotypes were observed in these seedlings.The chlorophylldeficient mutants (albino and sectional chimera) had the highest frequency (4%), following by terminal-inflorescence phenotype (2%).Considering the small sample size in this study, the above numbers confirm a relatively high mutation rate and a broad spectrum of phenotypes.For example, the percentage of albino mutants in a large population of einkorn wheat was lower than ours (Murai et al. 2013).Our results indicated the potential of ion beam irradiation as an ideal mutation method for faba bean breeding.The high mutation rate in small mutation populations would reduce the cost and labour intensiveness for phenotyping in mutation breeding programs.
Since Stadler's (1928) report that mutation was induced in barley (Hordeum vulgare L.) by X-rays, radiation mutagenesis has been considered as an important tool for generating new plant phenotypes for plant breeding and genetic studies.Ion beam irradiation has now been shown to be a valuable tool for mutation breeding of cool-temperate crops.The ion beam induced numerous mutations in rye at relatively low doses, with a slight effect on germination.In faba bean, the mutagenesis did not affect germination, but induced some mutations.The seed coat of faba bean is thicker than those of the other two species (Rowland 1977, Abbo et al. 2014, Smýkal et al. 2014), which could shield the embryo from some of the effects of the radiation.Due to the high mutation frequency observed in this study at M 1 , we suggest that this technique is useful to increase the genetic diversity in target plant germplasm in an early mutation generation to produce mutant crops with agronomically important traits for particular purposes.highly acknowledged.The work was financed through the EU FP7 project 'Legume Futures' (Grant 245216CP-FP).

Fig. 1 .Fig. 2 .
Fig. 1.Effect of ion beam irradiation on the survival rate of rye, linseed and faba bean.The error bars show ± 1 standard error of the mean.

Fig. 3 .
Fig. 3. Variation in phenotypes of M 1 and M 2 seedlings produced by ion beam irradiation in studied species.(A) albino plant of rye; (B) curly leaf in rye; (C) coleoptile mutant in rye; (D) first leaf stunt growth in rye; (E) control (left) vs mutant (right), naked spike in rye; (F) mutant spike of rye; (G) rusty leaves in faba bean; (H) sectional chimera leaf in faba bean at M 2 ; (I) Maculata leaflet in faba bean at M 2 ; (J) terminal inflorescence mutation in faba bean which was observed at M 1 and M 2 ; (K) multi-branching linseed seedling; (L) foliage leaves showing early damage in linseed; (M) early damage in linseed; (N) pollen diversity in linseed; bar = 1 cm

Fig. S1 .
Fig. S1.Dry seed preparation for ion beam irradiation.(a) overview on all three species, (b) view from above and (c) view from side in rye

Table 1 .
Germination rate and frequencies of mutant plants at M1 on the three studied crop species Number of seeds sown were: 300 rye, 300 linseed and 150 faba bean for each treatment (over three replicates)