Brief Overview of Sweet Sorghum Irradiated by Carbon Ion Beam

Sweet sorghum [Sorghum bicolor (L.) Moench] is a C4 plant characterized by a high photosynthetic efficiency and a high biomassand sugaryielding crop. However, the current varieties of sweet sorghum cannot meet the rapid growth demand for bio-ethanol production because of its low sugar content in China. To breed novel varieties to provide excellent raw materials for bio-ethanol production, the dry seeds were irradiated by carbon ion beam irradiation with different doses in sweet sorghum, resulting in acquiring an early-maturity mutant at 80 Gy dose, which the growth period was stably shortened for around 20 days compared to wild-type plant. In this paper, we briefly summarized the biological effects induced by carbon ion beam, the characters of early-maturity mutant, and revealed corresponding mechanisms from the point of view of morphological, physiological and molecular levels. In conclusion, there were significant effects on sweet sorghum irradiated by carbon ion beam.


Introduction
As is known to all, heavy ions irradiation is expected to increase mutation frequency and mutation spectrum, characterized by high linear energy transfer (LET) and relative biological effectiveness (RBE) (Zhou et al., 2006).Heavy ion beam has been used on many plants, such as the Artemisia annua (Inthima et al., 2014), chrysanthemum (Yamaguchi et al., 2010), wheat (Liu et al., 2013), Lotus japonicus (Luo et al., 2016), Green wandering jew (Liu et al., 2016) and Arabidopsis (Du et al., 2014;Yoshihiro et al., 2017), etc.In plants, the biological effects of heavy-ion radiation encompassed a wide range of alterations, including developmental abnormalities (Kranz, 1994), chromosomal aberrations (Ritter & Durante, 2010;Kikuchi et al., 2009;Wei et al., 2006) and increased mutation rates (Du et al., 2014).Heavy-ion radiation-induced variations have also been found in the plant genome (Xu et al., 2006).Shikazono et al. (2005) reported that more structural alterations were induced by carbon ions in DNA than low-LET radiation.At the same time, the study also revealed that deletions were more often found in the DNA sequences of the rejoined site because of carbon ions irradiation.Sweet sorghum [Sorghum bicolor (L.) Moench] is a C 4 plant characterized by a high photosynthetic efficiency and a high biomass-and sugar-yielding crop (Billa et al., 1997), a conclusion is drawn that sweet sorghum is a potential useful energy crop.The primary advantage of sweet sorghum is its high ethanol productivity, 3700 to 5600 liter/ha per year (Gibbons, 1986).However, the current varieties of sweet sorghum cannot meet the rapid growth demand in China for bio-ethanol production owing to its low sugar content.To breed novel varieties to provide excellent raw materials for bio-ethanol production from sweet sorghum, more work has been done for mutation breeding with carbon ion beam irradiation in sweet sorghum since 2006 (Dong et al., 2007;Dong et al., 2015).In this paper, we briefly summarized the biological effects induced by carbon ions, characters of early-maturity mutant, and revealed corresponding mechanism from the point of view of morphological, physiological and molecular levels.

Biological Effects Induced by Carbon Ion Beam in Sweet Sorghum
Ionizing radiation is known to have several effects on plant growth and development, ranging from stimulatory effects at low doses, harmful consequences at intermediate levels to pronounced detrimental outcomes at high doses (Cucinotta & Durante, 2006;Durante & Cucinotta, 2008;Veronica et al., 2001).For sweet sorghum, the dry seeds were firstly irradiated by carbon ion beam with different doses, including 10, 15, 20, 30, 40, 50, 80 Gy, respectively (Dong et al., 2007).The results indicated that the survival fraction of sweet sorghum presented "saddle like model" that the survival rate decreased, then increased and decreased again with irradiation doses increased.According to M1 generation effects in this irradiation experiment, the optimal mutagenic dose, 40-60 Gy, was recommended.However, the recommended dose is too low to inhibit the development of sweet sorghum.Based on above results, the dry seeds of sweet sorghum were secondly irradiated by carbon ions with doses of 120, 160, 200, and 240 Gy (Dong et al., 2008), which there was significant inhibition for germination and seedling growth after irradiation.The percentage germination and surviving fraction decreased when the dose increased, but also the seedling growth was obviously retarded at 200 and 240 Gy.From the two experiments (Dong et al., 2007;Dong et al., 2008), the carbon ions had a marked stimulatory effect on the survival rate at low doses, with the value being 86% when the dose was at 30 Gy, higher than that of wild-type seeds.The carbon ion irradiation led to physiological damage and the surviving fraction in the field decreased as the dose increased above 30 Gy. Especially, the physiological damage obviously increased above 200 Gy.Ultimately, the median lethal dose was estimated to be 120 Gy for sweet sorghum.
To further validated the mutagenic effects of the carbon ions, correlation analysis was carried out between the irradiation dose and the main agronomic traits according to indexes which were measured at the mature stage, including node number, plant height, stalk diameter, sugar content and single stem weight (Dong et al., 2009;Dong et al., 2015).The analysis demonstrated that the irradiation dose was negatively correlated with both the node number and the sugar content.More specifically, there was a significant negative correlation of 0.93 between irradiation dose and sugar content (p < 0.01).However, there were positive correlations between the irradiation dose and other agronomic traits, such as plant height, stalk diameter and single plant weight (Table 1).Note. * Significant correlation at p < 0.05 level, ** Significant correlation at p < 0.01 level.

Phenotypic Characters of Early-Maturity Mutant in Sweet Sorghum
After carbon ion irradiation, some abnormal mutants were firstly isolated at the seedling stage, such as growing point vanishing, tip curling, plant withering and etiolation (Figure 1, Dong et al., 2015).In addition, higher mutation frequencies of 9.1%, 8.1% and 8.5%, were found for stalk thickening, sugar content and plant withering, respectively.Interestingly, an early-maturity mutant, KFJT-1, was obtained after carbon ion irradiation at 80 Gy, which the growth period was stably shortened for around 20 days than wild-type plant, KFJT-CK (Figure 2, Dong et al., 2012).
jas.ccsenet.2006).It is well known that rice is a short-day plant that induces transition from the vegetative phase to the reproductive phase when it senses a decrease in day length.Signals from light and circadian clocks are received by OsGI, the rice orthologue of Arabidopsis GIGANTEA, and it regulates expression of Heading date 1 (Hd1) (Hayama et al., 2002;Hayama et al., 2003;Song et al., 2007).Transcription of Heading date 3a (Hd3a) is strongly suppressed in the Hd1 under short day conditions, suggesting that Hd1 can up-regulate Hd3a expression under short day conditions (Yano et al., 2001;Kojima et al., 2002).Hd3a is a rice orthologue of Arabidopsis FLOWERING LOCUS T (FT), which encode florigens that can move from the leaf to the shoot apical meristem and induce flowering in plants (Komiya et al., 2008).These genes were shown to encode a mobile flowering signal (Mathieu et al., 2007;Kobayashi, 2007).Sweet sorghum is a short-day plant and the photoperiod-sensitive sorghum variety will not flower under long days.Based on these researches, Hd1 and Hd3a genes were cloned from KFTJ-1 and KFJT-CK.Analysis of the PCR amplification and sequence alignment of the Hd1 and Hd3a genes revealed that there was no difference between KFJT-CK and KFTJ-1 in the case of Hd1 gene.In contrast, an insert fragment GA was found in KFTJ-1 and the corresponding fragment was not found in KFJT-CK for the Hd3a gene (Dong et al., 2015).Generally, the majority of the induced mutations were deletions in the mutants due to carbon ion irradiation (Hirano et al., 2012).A similar study (Tanaka et al., 2010) also found that 11 mutations were 1-100 bp deletions, one was a 1 bp insertion and two had base substitutions in the 14 point-like mutations induced by carbon ions.Nonetheless, our study indicated that an early-maturity mutant, KFJT-1, not only flowered but also produced mature grain.We inferred that the photoperiod sensitivity might have been altered to photoperiod non-sensitivity by the 2 bp insertions of Hd3a in KFTJ-1.The finding of the Hd3a gene variation provides preliminary insight into the mechanism of the photoperiodic control of flowering time in an early-maturity mutant induced by the carbon ion beam irradiation in sweet sorghum.Importantly, this speculation needs further validation in the future.
To obtain a global view of the tissue specific characteristics at the transcriptional level between KFJT-1 and KFJT-CK at seedling stage, total six DGE libraries from roots, stems and leaves were sequenced with Solexa/Illumina DGE analysis, respectively (Dong et al., 2017).In root, a total of 557 genes were up-regulated and 160 genes were down-regulated.In stem, a total of 1,232 genes were up-regulated and 928 genes were down-regulated.In leaf, total 1,577 genes were up-regulated and 754 genes were down-regulated.The total numbers of the tags-mapped genes were 717, 2160 and 2331 in root, stem and leaf, respectively.Functional analysis of gene ontology indicated that genes involved flowering time were also enriched such as "response to far red light", "pollen development" including "auxin transport", which consist with the short growth cycle of KFJT-1 compared to KFJT-CK.Specially, the metabolic pathway, "polysaccharide metabolic process", "carboxylic acid biosynthetic process" and "developmental maturation" was founded and suggested that KFJT-1 fixed higher carbon than KFJT-CK in young leaf.The enrichment analysis of biological metabolic pathways revealed that the circadian rhythm was over-represented at seedling stage.In young root, the genes involved in the circadian rhythm were mostly up-regulated including PHYA, PHYB, TOC1, APR3, GI, LHY, CCA1 and WNK1.Whereas, in young stem, most of the genes were down-regulated including PHYA, PHYB, ARP7, CHS, TOC1 and CCA.In young leaf, six genes were involved, which three genes (PHYA, TOC1 and APR3) were up-regulated and the other three genes (WNK1 and CK2α, CK2β) were down-regulated.The gene GI which is a typical disruption of the PHYB signal transduction pathway was also slightly up-regulated in young root.

Conclusions
The conventional breeding method takes several years to develop a new cultivar/variety from wild species.Comparatively speaking, heavy ion beam irradiation is an effective technique for mutation breeding to produce new cultivars (Cerrone et al., 2012).In this review, the optimal mutagenic dose was recommended for mutation breeding in sweet sorghum according to dose response curve.Moreover, early-maturity mutant, KFJT-1, has been identified by the Gansu Provincial Variety Approval Committee after regional trial and production test, resulting in achieving economic benefits for local governments and enterprises (Dong et al., 2016).As far as I know, this variety of sweet sorghum is firstly created using heavy ion beam mutation breeding technology in the world.Importantly, the corresponding mechanisms of biological effects, flowering time key genes and tissue-specific expression at seeding stage were systematically investigated, founding that there was a distinct biological effect on sweet sorghum irradiated by carbon ions.In brief, a marked stimulatory effect was not only discovered after carbon ion irradiation at low doses, but also significant expression differences were achieved in roots, stem, leaves for genes involved flowering time between KFTJ-1 and KFJT-CK at seeding stage.
Figure 1.E, F a

Table 1 .
The correlation coefficient between irradiation dose and the main agronomic traits of sweet sorghum