EFFECT OF HIGH ENERGY PROTON AND CARBON ION BEAMS ON FENUGREEK (Trigonella foenum-graecum L.) SEED IRRADIATION

P.D.ABEYTILAKARATHNA¹ and K.TAKAGI²

¹  Regional Agricultural Research and Development Centre, Bandarawela, Sri Lanka

²  The Wakasa Wan Energy Research Center, 64-52-1, Nagatani, Tsuruga, Fukui, 914-0192, Japan

ABSTRACT

 An experiment was conducted to find out the effect of high energy proton and carbon ion beams on fenugreek seeds irradiation at the Wakasa Wan Energy Research Center in Japan. Fenugreek Seeds were exposed to six proton beam doses; 0, 50, 150, 200, 300 and 400 Gy and five carbon beam doses; 0, 25, 50, 150 and 200 Gy. Proton beam irradiated seeds were established on planting trays using randomized complete block design with 3 replicates in the green house under the control environment. Seven groups of seeds; non-irradiated seeds, 50 Gy, 150 Gy, and 200 Gy of proton-beams irradiated, and 50 Gy, 150 Gy, and 200 Gy of carbon-beams irradiated fenugreek seeds were grown on aseptic laminar medium. Radio sensitivity parameters were recorded and data were subjected to statistical analysis using R software.

The 400 Gy dose was the optimum dose of proton beams for the irradiation fenugreek seeds for induce mutations. Carbon beam doses of 50, 150, and 200 Gy and proton beam doses of 150, 200 Gy were caused to reduce the hypocotyls and total lengths of hypocotyls and roots. Proton beam dose of 50 Gy was increased the hypocotyls and total lengths of hypocotyls and roots.

KEYWORDS: Carbon beams, Fenugreek, Mutation breeding, Proton beams, Seeds Irradiation

INTRODUCTION

Fenugreek is more important crop due to it uses for medicinal purposes as well as spice for food in Sri Lankan from very long time. Fenugreek is an annual legume native to the Mediterranean region. From ancient days, it has been grown in India, Argentina, Egypt and Mediterranean countries like Southern France, Morocco and Lebanon (Gupta, 2014).The leaves and seeds of fenugreek are consumed specially for medicinal purposes such as lowering blood sugar and cholesterol level, anti-cancer, anti-microbial etc. (Sadeghzadeh-Ahari et al., 2009). In addition, it is very useful legume crop that have ability to fix the nitrogen and fit to short-term rotation (Sadeghzadeh-Ahari et al., 2009). According to the studies of Neelakantan et al. (2014), fenugreek seeds support to control the glycemic in persons with diabetes.

The aim of the crop breeding is to obtain a reasonable number of desired mutations for a trait of interest while inflicting the least unintended disruption to the genotypic integrity of the crop. This ensures to obtain desirable induced mutant without presence of unintended induced deleterious alleles which would require additional interventions such as backcrossing with elite starting genotype (Mba, 2013; Magori et al., 2010). In order to produce high yielding, pest and disease resistance or tolerance promising fenugreek varieties with desirable traits, it is important to use modern mutation technologies. A mutation is a sudden heritable change in the DNA in a living cell, not caused by genetic segregation or genetic recombination (Van Harten, 1998). Mutagenesis is more important in genetic studies as well as selective breeding. Successful mutant isolation largely relies on the use of efficient mutagens. The ion beam mutagenesis is the one of the good technology and the ion beam is a physical mutagen that has just recently come into use for plants. In this type of mutagenesis, positively charged ions are accelerated at a high speed (around 20–80%of the speed of light) and used to irradiate target cells (Magori et al., 2010).

Plant mutations are carried out with high-energy (220 MeV) ion beam in Japan (Kondo et al., 2009) while low-energy (30 keV) ion beams use in China (Feng et al., 2009). In Japan, ion beam irradiation facilities are available in the Wakasa Wan Energy Research Center, at Tsuruga using Multi-purpose Accelerator with Synchrotron and Tandem (W-MAST). Moreover, ion beam facilities for plant mutation are available at TIARA of the Japan Atomic Energy Agency (JAEA), the RIKEN Accelerator Research Facility (RARF), and the Heavy Ion Medical Accelerator in Chiba (HIMAC) of National Institute of Radiological Sciences (NIRS) (Tanaka, 2009). Linear electron transport (LET) which is the energy deposited to target materials when an ionizing particle passing through them is very important in ion beam irradiation due to the effectiveness of ion beams as a mutagen might not be determined by the species of ions, but mostly by the LET of ions and ion beams with LET of around 10–500 keV/u appear to be suitable. (Magori et al., 2010). The unit of LET is in kilo electron volts per micrometer (keV/mm), which represents the average amount of energy lost per unit distance. Ion beams have a relatively high LET (around 10–1000 keV/mm or higher), while X-rays, gamma rays and electrons have low LETs (around 0.2 keV/mm). Therefore, ion beams have ability to make more severe damage to living cells and high relative biological effectiveness than other forms radiation (Blakey, 1992; Lett, 1992; Magori et al., 2010). Ion beams induce predominantly single- or double-strand DNA breaks with damaged end groups that are unable to be repaired easily due to ion beams deposit high energy on a local target (Goodhead, 1995). In addition to ion beams, the space crop breeding using cosmic radiation is a one of the novel plant mutation breeding techniques (Guo et al., 2010; Hu et al., 2010; Ou et al., 2010).

MATERILS AND METHODS

The fenugreek seeds were irradiated using 220 MeV proton beam and 660 MeV carbon beam at the Wakasa Wan Energy Research Center (WERC) at Tsuruga, Japan on 2014. Seeds were exposed to six proton irradiation doses; 0, 50, 150, 200, 300 and 400 Gy and five carbon irradiation doses; 0, 25, 50, 150 and 200 Gy.

Irradiation Sensitivity Test

Irradiated fenugreek seeds were established in 30cm x 30cm size planting trays using the flat method (Asncion, 2004) which consists of 25 of square shape planting holes of 4.5 cm width and 5.5 cm of depth, at the green house of WERC under the control environment. The irradiated seeds were established in three replicates of RCBD using the first 3 replicates of irradiated seeds. The planting trays were filled with the sterilized media containing vermiculite and coir dust and seeds were established. Irradiation sensitivity parameters such as Seeds germination, plant height at 14 after emergence, leaf abnormality of shots/ primary leaves were recorded. The data were subjected to analysis using open source scripts of R version 3.1.2 software.

Seven groups of seeds were used, each of which was non-irradiated seeds, 50 Gy, 150 Gy, and 200 Gy of proton-beams irradiated, and 50 Gy, 150 Gy, and 200 Gy of carbon-beams irradiated fenugreek seeds to compare the effect of carbon beams and proton beams. Seeds in each group were sterilized by immersing 5 minutes in 70% ethanol and subsequently immersed for 10 minutes in 10 % antiformin (effective chlorine concentration: ca. 0.5%) with 0.1% Tween 20,  and washed with sterile distilled water. Then seeds in each group were sown on aseptic laminar medium, consisted of 1/2 MS with 2% sucrose and 0.3% gellungum, in a square dishes. Dishes were vertically kept to facilitate rooting downward on the surface of a medium. Digital photographs of dishes were taken. Root length and other root characters of the fenugreek seeds were measured after digital photos using ImageJ software.

  

RESULTS AND DISCUSSION

Effect of Proton beams on Fenugreek Seedling Height

According to Owoseni et al. (2007), radio sensitivity or determination of the optimum dose of radiation is a term describing a relative measure of the quantity of recognizable effects of a radiation exposure on the irradiated material. Tshilenge-Lukanda et al. (2012), described that the optimum mutation doses can be determined using seedling survival rate and seedling height. Determination of first generation mutant (M₁) injury level using seedling height and survival should be a routine procedure in mutation breeding, because it has been established that these characters are correlated with M₁ mutation frequency (Asncion, 2004). In this study, the difference between height of fenugreek seedling at 13 days after planting was highly significant (p<0.001) with proton irradiation dose. The highest seedling heights were observed in both non-irradiated and 50 Gy proton irradiated seedlings (7.7 cm and 7.4 cm respectively). The seedling heights were reduced in both 150 and 200 Gy levels of irradiation (5.5 cm and 4.7 cm respectively) than the non-irradiated seedling. The lowest seeding heights were observed in both 300 and 400 Gy proton irradiation levels (2.8 cm and 2.7 cm respectively) (Table 01 & Figure 01).  According to Mba, (2013), the universally adopted norm is to select a dosage that results in reductions of 30 to 50 or 40 to 60 percent in growth or survival rates respectively of the first generation mutant (M₁) seedlings compared to the seedlings of untreated seeds. Seedling of which seeds were irradiated using 300 and 400 Gy proton doses were fallen in the range of 30 to 50 percent of height reduction than the seedling of non- irradiated seeds (Figure 01). 

 Effect of Proton Beams on Seedling Survival Rate

Seedlings were not dead up to 200 Gy irradiation dose of proton beam. The plant survival rates as percentage to the untreated seedling were started to decline after 200 Gy irradiation (Figure 02). According to recent studies of Magori et al.( 2010), irradiation dose at the shoulder end of the survival curves (200 and 1000Gy for carbon ions and electrons, respectively) or less than these doses is more efficient for ion beams . Brown (2013) also stated that the negative effects of radiation overdoses such as deletions of DNA nucleotide sequences that may cause reading-frame shifts, inactive protein products, or faulty transcripts. This would subsequently lead into null mutations, in which a particular gene may be inactivated. According to Mba et al. (2010), the dose of mutagen that is regarded as the optimal is one that achieves the optimum mutation frequency. The lethal dosage (LD50) was also used to determine the optimum irradiation dose. (Meyer,1996; Owoseni et al., 2007; Magori et al., 2010). The seedlings of which seeds were irradiated using 400 Gy proton dose was in the range of 40 to 60 percent of survival rates (Figure 02).

Effect of Carbon Beams and Proton Beams on Roots and Hypocotyls Lengths

Root lengths of fenugreek seedling at 6 days after sowing of seeds which irradiated with 0 to 200 Gy of proton beams and carbon beams were not seen significant different statistically at p < 0.05. The highest hypocotyls length (25.2 mm) was observed in the seedling of seed irradiated using  50 Gy proton beams while the shortest hypocotyls  length was seen in the seedling of seed irradiated using  50 Gy carbon beams. However, hypocotyls lengths were not affected significantly at p<0.05, up to 150 Gy proton beams. Likewise, 50 to 200 Gy carbon beams were also not showed a significant different at p<0.05 (Table 02).

The dosage of 50 Gy proton beam was caused to increase the hypocotyls lengths by 10.5 percent than the seedling of non-irradiated seeds while same dose of carbon beam was caused to reduce the hypocotyls length by 30.7 percent at 6 days after seed sowing. At the 150 Gy dose of carbon beam was caused to reduce the hypocotyls lengths by 2.7 fold than the same dose of proton beams. But, 0.7 fold reduction of hypocotyls was observed at 200 Gy carbon beam dose than same dose of proton beams (Figure 03).

The proton beam doses of 50, 150, 200 Gy and carbon beam dose of 150 Gy were effected to increase the root lengths  of the seedling at 6 days after irradiated seed sowing by 6.1, 8.7, 5.2 and 12.2 percent respectively than untreated seedlings. But, 50 and 200 Gy doses of carbon beams were caused to reduce root lengths by 7.8 & 34.8 percent respectively than non-irradiated seedlings (Figure 04).

The total length of roots and hypocotyls were seen significantly different at p<0.05 at 6 days after sowing the seeds but it was not significantly different at 8 days after sowing. Carbon beam 50 to 200 Gy doses and Proton beam 200 Gy dose were reasoned to reduce the total length of root and hypocotyls. The highest total length of root and hypocotyls was observed in 50 Gy dose of proton beams (37.4 mm) while the lowest length was observed in 50 Gy of carbon beams dose (table 03).  

Total lengths of root and hypocotyls at 50 Gy of proton beam was facilitated to increase the length by 8.7 percent than the non- irradiated seedlings while same dose of carbon beam facilitated to reduce the length by 23.3 percent. Carbon beam doses of 150 and 200 Gy were also caused to reduce the total length by 3.7 and 1.5 fold respectively than the same dose of proton beams (Figure 05).

CONCLUSIONS

The optimum proton beam dose for the irradiation of the fenugreek seeds for induce mutations was 400 Gy according to the radio sensitivity parameters such as seedling height and seedling survival rate. Hypocotyls lengths of irradiated fenugreek seedslings were reduced by 30.7, 23.7, 18.4, 8.8, 26.8  percent after expose to 50, 150 , 200 Gy carbon beams and 150, 200 Gy proton beams respectively. Hypocotyls length of the seedlings was increased by 10.5 percent after irradiated with 50 Gy proton beam. Roots lengths of the seedlings were reduced by 6.1, 8.7, 5.2, 7.8, 34.8 after irradiated with 50, 150, 200 Gy proton beams and 50, 200 Gy carbon beams respectively as well. In addition, roots length was increased by 12.2 percent after treated with 150 Gy of carbon beams. Total lengths of roots and hypocotyls were reduced by 3.2, 16.3, 23.26, 11.92, 24.13 percent after exposed to 150, 200 Gy of proton beams  and 50, 150, 200 Gy of carbon beams respectively while total lengths of roots and hypocotyls were increased by 8.7 percent when treated with 50 Gy proton beam.

ACKNOWLEDGEMENTS

We wish to express our thank to Fukui International Human Resource Development Center (FIHRDC) and the Wakasa Wan Energy Research Center (WERC) in Japan for funding this study under “the atomic energy researchers and research students acceptance program, FY 2014”

REFERENCES

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Table 01. Proton beam irradiation dose (Gy) and the fenugreek seedling (cm) height at 13 days after planting.

 

*** Significantly different at p=0.001

   

Means of the same letters in superscripts are not significantly different at Duncan’s p=0.01

  

Table 02. Root lengths and hypocotyls lengths of fenugreek seedling with different seed irradiation dose of proton and carbon beams at 6 days after sowing.

 

 ns=  not significant at p=0.05, ** significant at p=0.01

means with the same letters are not significantly different at Duncan p=0.05

Table 03. Total lengths of roots and hypocotyls of fenugreek seedling with different seed irradiation dose of proton and carbon beams at 6 & 8 days after sowing.

ns=  not significant at p=0.05, * significant at p=0.05, DAS= days after sowing

means with the same letters are not significantly different at Duncan p=0.05

 

Figure 01. Relationship between proton irradiation dose (Gy) and seedling height as a percentage of untreated seedlings at 13 days after emergence.

 

Figure 02. Survival response curve of fenugreek seedling with different proton irradiation dose at 3 weeks after emergence.

 

Figure 03. Different proton  and carbon beams doses and hypocotyls lengths of fenugreek seedlings at 6 days after sowing.

 

Figure 04. Different proton  and carbon beams doses and root lengths of fenugreek at 6 days after sowing.

 

Figure 05. Different proton  and carbon beams doses and total lengths of roots & hypocotyls of fenugreek seedlings at 6 days after sowing.

  

(a)

 (b) 

 

Figure 06. High energy carbon and proton beams transport system to irradiation room at the Wakasa Wan Energy Research Center in Japan (a), Irradiated fenugreek seeds grown in aseptic laminar medium (b). 


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