Plant Mutation Breeding with Heavy Ion Irradiation at IMP

The Heavy Ion Research Facility in Lanzhou (HIRFL) is one of the ion-beam acceleration facilities intensively used at IMP, founded as national laboratory and opened for user in world from 1992. Since then, a lot of experiments irradiated by heavy ion beam have been carried out in the HIRFL, including plant mutation breeding. In this review, the biological effects induced by heavy ions and their corresponding mechanisms were reported from the point of view of cytological, morphological and molecular levels. To date, a large number of mutants were isolated using heavy ion irradiation IMP, such as early maturity, flower color and shape, high yield and disease resistant. In conclusion, heavy ion beam irradiation is an efficient mutagen and has significant phenotypic variations in plant. Our research will be further focused on transformation of scientific and technological achievements and mutagenic mechanism of heavy ion beam on high plant at the molecular level in the recent future.


Introduction
Since Muller (1927) and Stadler (1928) discovered that X-rays can induce mutations in drosophila and barley, respectively, the use of ionizing radiation such as X-rays and gamma rays to induce variation has become an established technology (Ahloowalia & Maluszynski, 2001).Ion beams have been applied as a nuclear technique since the late 1950s, especially in the field of surface modification on various materials (Cui & Luo, 1999).In 1986, Chinese scientists studied the biological effect of ion implantation and first applied it to rice breeding.Since then, a new research field Ion Beam Bioengineering has been established (Wu et al., 1999).The high linear energy transfer (LET) heavy-ion beam has recently been used on many plants, resulting fruitful achievements (Yu et al., 1991), such as the carnation (Okamura et al., 2003), chrysanthemum (Yamaguchi et al., 2010), wheat (Wei et al., 1998), buckwheat (Morishita et al., 2003) and Arabidopsis (Kazama et al., 2008), because of its higher mutation rate and wider mutation spectrum with lower damage to irradiated materials compared to the traditional low LET radiation methods, (Abe et al., 2000;Tanaka, 1999).Thus, more and more researchers pay attention to the application of heavy ion beam irradiation in plant mutation breeding.
The Institute of Modern Physics (IMP), affiliated with the Chinese Academy of Sciences, was founded in 1957 in Lanzhou, China.It is now an institute making important contributions to basic research as well as the applications of heavy-ion physics and nuclear techniques (Li, 2007;Wang, 2006).Aiming at the scientific front and strategy demand on modern agriculture, the researchers of IMP have been carrying out mutagenic improvement on higher plant since the middle of 1990s.In this review, we introduce IMP accelerators facility and focus on the plant breeding research with heavy ions at the IMP accelerators.

HIRFL-The Platform of Mutation Breeding Induced by Ion Beam at IMP
The Heavy Ion Research Facility in Lanzhou (HIRFL) is one of the ion-beam acceleration facilities intensively used in the past two decades at IMP (Sun et al., 1996).A lot of experiments irradiated by heavy ion beam have been carried out in the HIRFL.
The HIFRL was found as national laboratory and opened for user in world from 1992 (Wei, 1989).Then, it was upgraded many times, especially, the new upgrade project of Cooling Storage Ring (CSR) at the beginning of the udied, dicals and inhibit seedling growth of wheat seeds, leading to a great variety of chromosomal aberrations in root tip cells of irradiated seeds and high aberration frequencies compared to the corresponding control.In order to analyze the relationship between plant growth and cytological effects, wheat dry seeds were exposed to various doses of 12 C 6+ beams and the biological endpoints reflecting plants growth and root apical meristem activities were investigated (Liu et al., 2013).The results showed that the plant survival rate descended at higher doses and various types of chromosome aberrations were observed in the mitotic cells.The frequencies of mitotic cells with lagging chromosomes and these with anaphase bridges reached the peak around 60 Gy, while the frequencies of fragments increased as the irradiation doses increased up to 200 Gy.Thus, the total frequencies of mitotic cells with chromosome aberrations induced by irradiation increased significantly with the increasing doses.To further investigate the mutagenic mechanism, Liu et al. (2012) comparatively studied the differences in pollen viability and pollen mother cells meiosis between KFJT-1 and KFJT-CK, which was isolated from KFJT-CK after carbon ion beam irradiation in sweet sorghum.The results showed that the total number of pollens and pollen viability of KFJT-1 were more than that of KFJT-CK.Triad, unequal separation of chromosome and asychronization of chromosomes segregation were found in KFJT-1 meiosis, but the most of chromosomal aberrations induced by carbon ions were repaired, the aberration rate of KFJT-1 being only 4.5%.In the case of phenotypic effects induced by heavy ion beam irradiation, Saintpaulia ionahta (Zhou et al., 2006), sweet sorghum (Dong & Li, 2012;Dong et al., 2015) and Arabidopsis thaliana (Du et al., 2014) were studied at IMP, respectively.For Saintpaulia ionahta, in vitro leaf explants samples were irradiated with carbon ion beams with linear energy transfer (LET) values in the range of 31-151 keV/um at different doses.It was found that the fresh weight increased and surviving fraction decreased dramatically with increasing LET at the same doses.In addition, malformed shoots, including curliness, carniation, nicks and chlorophyll deficiency, occurred in carbon ion beam.Eventually, a chlorophyll-deficent mutant and other malformations were obtained.For sweet sorghum, the carbon ions had a marked stimulatory effect on the survival rate at low doses, with the value at 30 Gy being 86%, higher than that of non-irradiated seeds.The carbon ion irradiation resulted in physiological damage of the KFJT-CK seeds and the surviving fraction in the field decreased as the dose increased above 30 Gy.The results demonstrated that disappearance of the growth point, curing of the leaf tip, plant withering and etiolated seedlings were observed in some mutants.Other mutants showed stalk thickening, spikes and larger grain.However, an early-maturity mutant, KFJT-1, was isolated at 80 Gy, which the growth period was shortened by about 20 days compared to the wild type KFJT-CK.In addition, the seed coat color of KFJT-1 was dark yellow brown than that of KFJT-CK.Physiological analysis showed that the proline content of KFJT-1 was increased by 11.05% while the malondialdehyde content was significantly lower than that of KFJT-CK.In terms of Arabidopsis thaliana, a total of 1363 lines of plants from 28062 M2 populations displayed alterations in the leaf, stem, flower, or life cycle, with abnormal leaves and a premature life cycle as the main phenotypic variations.The total mutation rate was 4.77%.In the 1363 mutant lines, 64% were displaying one conspicuous phenotype, 13.3% displayed two conspicuous phenotypes, and 13.1% showed at least three mutant traits.
The reason heavy ions are more suitable for induced mutagenesis is well explained by Liu et al. (2008) and Tanaka et al. (2010).However, mutation mechanism for heavy ion irradiation remains unknown at molecular levels.In recent years, preliminary studies were carried out to reveal the molecular nature of phenotype variation in higher plants after heavy ion irradiation.In the previous research, Hd1, a homologue of CO, was found to be a major determinant of photoperiod response (Laurie et al., 2004) while Hd3a was shown to be a homologue of FT (Yano et al., 2001).Therefore, we analyzed the Hd1 and Hd3a genes between early-maturity mutant and wild type in sweet sorghum (Dong et al., 2015), which indicated that there was no difference for the Hd1 gene, but 2 bp insertions were clearly observed in early-maturity mutant compared to wild type in the case of the Hd3a gene.This finding might provide the first insight into the mechanism of the photoperiodic control of flowering time in an early-maturity mutant induced by the heavy ion beam irradiation.In the post-genomic era, researchers not only identify the mutated genes but also explore the function and the molecular regulation of those genes.At IMP, five mutants with transmittable phenotypes were roughly mapped based on the positional cloning technology in Arabidopsis thaliana (Du et al., 2014), which we discovered that mutants #164, #60 and #352 correspond to previously described genes, while mutants #197 and #357 correspond to genes that have not been reported before.
In addition, there are two mutational sites in the #197 genome.Above researches indicated that the heavy-ion irradiation induced more complicated mutations.Further verification and detailed molecular analysis will be carried out using gene expression methods and RNA sequencing technology in the near future.

Table 1 .
Effects of heavy ion beam on chromosome aberrations, growth of different irradiated materials

Table 2 .
The mutant types induced by heavy ion beam irradiation at IMP ience n beam irradiat ionahta.The f