100 Gy 60Co γ-Ray Induced Novel Mutations in Tetraploid Wheat

10 accessions of tetraploid wheat were radiated with 100 Gy 60Co γ-ray. The germination energy, germination rate, special characters (secondary tillering, stalk with wax powder, and dwarf), meiotic process, and high-molecular-weight glutenin subunits (HMW-GSs) were observed. Different species has different radiation sensibility. With 1 seed germinated (5%), T. dicoccum (PI434999) is the most sensitive to this dose of radiation. With a seed germination rate of 35% and 40%, this dose also affected T. polonicum (As304) and T. carthlicum (As293). Two mutant dwarf plants, T. turgidum (As2255) 253-10 and T. polonicum (As302) 224-14, were detected. Abnormal chromosome pairings were observed in pollen mother cells of both T. dicoccoides (As835) 237-9 and T. dicoccoides (As838) 239-8 with HMW-GS 1Ax silent in seeds from them. Compared with the unirradiated seed of T. polonicum (As304) CK, a novel HMW-GS was detected in seed of T. polonicum (As304) 230-7 and its electrophoretic mobility was between 1By8 and 1Dy12 which were the HMW-GSs of Chinese Spring. These mutant materials would be resources for wheat breeding.


Introduction
Human activities and natural calamities decreased the biological diversity and narrowed the genetic variability that limits crop breeding. Novel mutations in plants, which are crucial for improving resistance/tolerance to environmental stress, enhancing quality and yield traits, and facilitating the seed set of hybrid, have been created, such as in Arabidopsis [1], rice [2], maize [3], wheat [4], and some horticultural plants [5].
Since the 1970s, -rays, sodium azide, and ethyl methane sulfonate (EMS) have been used for wheat breeding [4]. Inducing mutation with 60 Co -ray is an effective way and had bred some hexaploid wheat cultivars. Guinness/1322 (Bulgaria), for an example, was mutationally bred from Katya (a hexaploid wheat cultivar from Bulgaria) by 50 Gy 60 Co -ray [6]. Compared with Katya, Guinness/1322 shows better lodging and shedding resistance, better ecological adaptability of drought tolerance, and higher productivity [7]. Inducing mutation with 60 Co -ray was also used for tetraploid wheat breeding, but only two cultivars, Yavor (Bulgaria) and Implus (Turkey), were bred from durum wheat (AABB, 2 = 4 = 28) and different frequencies of induced mutations were observed under 100 Gy 60 Co -ray [7,8].
Tetraploid wheat (AABB, 2 = 4 = 28) distributes widely and adapts extensively to the environment and contains considerable wealth of genetic and morphological variation [9], such as high abilities of powdery mildew resistance in Triticum dicoccoides Körne [10], abundant genetic diversity of storage proteins in T. dicoccoides [10] and Triticum turgidum L. [11], valuable genes contributing to the grains per spike in Triticum carthlicum Nevski [12], dwarf genes in Triticum polonicum L. [13], and high content of gluten and tolerance to the saline in Triticum durum Desf. [14,15]. Tetraploid wheat with AB genomes is important natural resources for breeding [16]. Therefore, creating novel mutation through radiation in tetraploid or hexaploid wheat may be an effective way for wheat breeding.
In the present study, 10 accessions of tetraploid wheat were radiated with 100 Gy 60 Co -ray. Following the radiation, mutations of the agronomic traits, cytogenetics and 2 The Scientific World Journal high-molecular-weight glutenin subunits (HMW-GSs) were observed, which could be used for further selection and utilization of the radiated progenies.

Materials and Methods
2.1. Materials. All seeds of the accessions were deposited at Triticeae Research Institute, Sichuan Agricultural University, Sichuan, China. Information of the accessions was listed in Table 1.

2.2.
Radiation. 20 seeds of each accession were radiated with 100 Gy 60 Co -ray at the Institute of Biological and Nuclear Technology, Sichuan Academy of Agricultural Sciences, China. Dose rate was 1.1 Gy/min, and unirradiated seeds were used as a control (CK).

Seed Germination.
Respective 20 radiated and CK seeds of each accession were exposed with 4 ∘ C for 24 hours and germinated with 25 ∘ C. The germination energy (percentage of the seeds germinated in 10 days) and germination rate were calculated as follows: Germination rate = Number of germinated seeds Total number of seeds (20) × 100%. (1)

Agronomic Characters Identification.
Radiated seedlings and CK ones were planted in the field. Agronomic characters including plant height, tiller number, seed set, and other special characters (secondary tillering, stalk with wax powder, and dwarf) were observed.

Meiotic Analysis.
Young spikes were fixed in Carnoy's solution II (ethanol : chloroform : acetic acid = 6 : 3 : 1 V/V) and stored at 4 ∘ C. The pollen mother cells were stained with improved phenol fuchsin. Observations of the chromosome pairing of meiosis were made and documented with an Olympus BX-51 microscope coupled with a Photometric SenSys CCD camera. 60 cells of each accession were counted to confirm the pairing in the meiotic process.

High-Molecular-Weight Glutenin Subunits (HMW-GSs).
HMW-GSs of radiated and CK seeds were analyzed according to the method of Wan et al. [17]; eight seeds were tested in every single plant. The HMW-GSs of Chinese Spring (Null, 7 + 8, 2 + 12) were used as marker.

Discussion
Different species has different suitable dose of radiation intensity, such as 300 to 700 Gy 60 Co -ray in Sorghum bicolor   (L.) Moench [18], less than 200 Gy in Roegneria [19]. Suitable dose of radiation is various among different species in same genus [19]. In the present study, 100 Gy 60 Co -ray differently induced mutations in the tetraploid wheat accessions. The results of germination energy and germination rate suggest that T. dicoccum (PI434999) is the most sensitive to the treatment and this dose of radiation is lethal dose to it. As to most tetraploid wheat, 100 Gy 60 Co -ray radiation is an insufficient dose to induce mutation. Chromosomal translocation, chromosome breakage, and deletions in chromosome which came from radiation mutation may lead to defects in chromosome pairing [20]. In this study, the abnormal chromosome pairings in meiotic process, such as univalents, trivalents, quadrivalents, and lagging chromosomes, were observed in pollen mother cells of some radiated plants, suggesting that 100 Gy 60 Co -ray might create some mutations at chromosome level. Univalents were observed in T. dicoccoides (As838) 239-7 with 11 secondary tillerings and stalk with wax powder. Meanwhile, quadrivalent in T. dicoccoides (As835) 237-9, trivalents in T. dicoccoides (As838) 239-8, and lagging chromosomes in T. polonicum (As304) 230-7 were observed with HMW-GS mutations.
Thus, the abnormal chromosome pairing of meiotic process reflected radiation mutations. Meiotic process observation could be used as a tool for mutation identification at wheat earing stage.
HMW-GSs are important storage proteins in wheat and its related species and 10% of endosperm proteins are HMW-GSs [21][22][23]. Theoretically, tetraploid wheat should contain 4 different HMW-GSs, 1Ax, 1Ay, 1Bx and 1By [23], but only one or two, no more than three subunits, were expressed due to gene silencing. Different HMW-GSs combinations have different effect on flour quality [23]. In the present study, compared with CK, 1Ax was silent in T. dicoccoides (As835) 237-9-5 and T. dicoccoides (As838) 239-8-2. HMW-GS gene silencing might be caused by specific nucleotide substitutions in the promoter region [21] and single repeat changes or repeat indels or large deletions in codon region [22,23]. A novel HMW-GS was detected in T. polonicum (As304) 230-7-1 and its electrophoretic mobility was between 1By8 and 1Dy12 which were the HMW-GSs of Chinese Spring. Single nucleotide mutation or repeat deletions could restore the expression of genes; homoeologous recombination might be a novel pathway for allelic variation or molecular evolution of The Scientific World Journal    HMW-GSs [22,24]. The mechanism of mutations in HMW-GS is under research. Dwarf genes were found to be affecting architecture of rice plant [25]. GID1 gibberellin receptors affect the plant height of Arabidopsis [26]. 10 dwarfing genes/alleles have been discovered from tetraploid wheat [13,[27][28][29]. Associating with an extreme dwarf trait, only a few dwarfing genes have been used for wheat breeding worldwide [30]. Digging new plant height reducinggene is more and more important for wheat dwarf breeding. In the present study, significant plant dwarf was observed in both radiated plants T. polonicum Inducing mutations for genetic improvement in breeding resources has been successfully and widely used for plant breeding. Sodium azide, EMS, and -rays are major tools for mutation. Sodium azide was widely used for mutation and breeding in rice [31], barley [32], tomato [33], and maize [3] but was not an effective mutagen in Arabidopsis [34]. EMS mainly induced single nucleotide mutations in Arabidopsis thaliana [1], hexaploid wheat, and Triticale [35]. Inducing mutations through chromosome aberration and single nucleotide mutant enriched the gene banks of the species [33]. During the past fifty years, about 130 wheat cultivars bred from mutation have been widely produced in China [36]. In the present study, some novel mutations in several tetraploid wheat cultivars were induced by 100 Gy 60 Co -ray, such as HMW-GS and dwarf trait, which could be used as resources for theoretical study and future wheat breeding.

Conclusion
In the present study, 100 Gy 60 Co -ray differently induced mutations in accessions of tetraploid wheat. Following the radiation the germinated ratio of the materials varied from 5% to 95% and this dose of radiation is lethal dose to The Scientific World Journal 7 T. dicoccum (PI434999). The effects of radiation on the meiotic process of pollen mother cells and HMW-GSs were observed. Univalents, trivalents, quadrivalents, and lagging chromosomes in meiosis were detected in few cells of the observed accessions. As to HMW-GS, 1Ax was silent in T. dicoccoides (As835) 237-9-5 and T. dicoccoides (As838) 239-8-2 and a novel HMW-GS was detected in T. polonicum (As304) 230-7-1 whose electrophoretic mobility was between 1By8 and 1Dy12 which were the HMW-GSs of Chinese Spring. Compared to the CK, T. dicoccoides (As838) 239-7 had 11 secondary tillerings and stalk with wax powder. Plant dwarfs were also observed; the height of the radiated T. turgidum (As2255) 253-10 is 68.5 cm and T. polonicum (As302) 224-14 is 98.1 cm. These mutations would be resources for the future wheat breeding. 8 The Scientific World Journal