Abstract
The present study was conducted to evaluate the biological responses of soybean irradiated by gamma-rays. Four elite Korean soybean cultivars, Kwangankong, Shinpaldal-2, Shinhwakong, and Ilmikong, were irradiated with 100–400 Gy of gamma-rays. All cultivars showed significant reductions in morphological parameters. Seedling emergence rates of Kwangankong, Shinpaldal-2, Shinhwakong, and Ilmikong decreased by 46%, 31%, 44%, and 43% at 400 Gy, respectively. Plant height and fresh weight decreased with increasing dose. The median reduction dose (RD50) for plant height ranged from 184 to 278 Gy with an average of 212 Gy. The optimal dose of gamma irradiation for inducing mutation in the four elite soybean cultivars was in the range 200–300 Gy. Contents of malonaldehyde (MDA) in the gamma-ray irradiated plants were higher than those in the controls. With the exception of Kwangankong, activities of ascorbate peroxidase (APX) in other cultivars decreased at 100 Gy and started to increase significantly at 200 Gy and Shinhwakong cultivar had highest APX value observed at 300 Gy. No significant changes in peroxidase (POD) activity were observed in the Kwangankong, Shinpaldal-2, and Shinhwakong, while, POD activity increased with increasing dose in Ilmikong. In addition, gamma-ray treatments elicited a marked reduction in chlorophyll a contents compared to chlorophyll b contents in Kwangankong, Shinpaldal-2, and Ilmikong. Growth characteristics (plant height and fresh weight) were highly negatively (P < 0.01) correlated with the dose of gamma-rays, while the MDA content and APX activities were positively (P < 0.05) correlated with dose.
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Key words: Biological responses, Gamma-ray, Growth characteristics, Soybean, Correlation analysis
INTRODUCTION
Mutation breeding is an alternative method to conventional breeding, especially cross-breeding. Mutation breeding induces new genetic diversity to gene pools using chemical and physical mutagens, which generate mutations that can be develop into new cultivars faster than conventional breeding (
Alikamanoglu et al. 2011). Until now, 3,200 mutant varieties of more than 210 plant species have been produced for commercial use (Mutant Variety Database, Joint FAO/IAEA programme,
http://mvgs.iaea.org/default/aspx).
Ionizing radiation affects plant cells at morphological and biochemical levels. Such responses are very important in evaluating the effects of irradiation (
Celik et al. 2014). Ionizing radiation interacts with atoms and molecules, and produces radicals by water radiolysis (
Kim et al. 2015). The products of water radiolysis are ionized water molecules (H
2O) and radicals, such as hydrogen (H
+) and hydroxyl ion (OH
−). H
+ and OH
− are primary free radicals; other radicals such as hydrogen peroxide (H
2O
2) are generated by secondary reactions (
Esnault et al. 2010). Reactive oxygen species (ROS) are highly reactive with macromolecules in the cell and are harmful to it (
Garg and Manchanda 2009). H
2O
2 is not harmful under normal conditions. However, when concentration of H
2O
2 increases, it can cause cell lethality (
Wi et al. 2007). ROS are also produced by various metabolic pathways, such as photorespiration in chloroplast and mitochondria (
Mittler 2002). However, under normal conditions, the concentrations of ROS are controlled by various antioxidative materials such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), and glutathione reductase (GR) (
Foyer and Noctor 2005;
Garg and Manchanda 2009;
Kim et al. 2015). Accumulation of ROS is determined by the antioxidant system (
Foyer and Noctor 2005;
Kim et al. 2012). The malondialdehyde (MDA) content is often used as an indicator of the extent of lipid peroxidation resulting from oxidative stress (
Gawel et al. 2004;
Im et al. 2017b).
In this study, we tested the radiation sensitivity of four elite soybean cultivars within a range of 100–400 Gy of gamma-rays. We measured plant height, fresh weight, MDA content, and antioxidant enzyme activity to evaluate the effects of gamma-rays.
MATERIALS AND METHODS
Plant material and gamma-ray irradiation
Four elite Korean soybean cultivars including Kwangankong (
Lee 2012), Shinpaldalkong-2 (
Kim et al. 1993), Shinhwakong (
Lee et al. 2015a) and Ilmikong (
Shin et al. 1998) were used in this study. Three hundred seeds of each cultivar were irradiated at 100, 200, 300, and 400 Gy of gamma-rays with gamma irradiator at the Advanced Radiation Technology Institute (ARTI) in Jeongeup, Korea.
To test seedling emergence, 100 seeds for each treatment from control and each dose of radiation were planted in pots filled with potting mix (coco peat, peat moss, zeolite, pearlite, caldolomite, wetting agent, and fertilizer) and cultured in a greenhouse. Each experiment was replicated three times, and the emergence rate (seedlings/number of seeds planted) was recorded at 12 days after planting. Plant height and fresh weight were measured at 5 weeks after planting (
Im et al. 2017a). Each replicate included three plants.
Young leaves from irradiated and non-irradiated plants were harvested at 5 weeks after planting and stored at −80°C for biochemical analyses. Fresh leaf samples (0.2 g) were ground in liquid nitrogen using a mortar and pestle for each experiment.
Measurement of MDA content
MDA content were measured to determine the level of lipid peroxidation using the trichloroacetic acid (TCA) method described by
Zhang et al. (2011). After homogenization with 1.5 mL 10% TCA for 30 minutes at 5°C, samples were vigorously vortexed and centrifuged at 10,000
g for 20 minutes at 4°C. Supernatants were mixed with an equal volume of 0.67% 2-thiobabituric acid followed by heating at 95°C for 30 minutes. After cooling on ice for 5 minutes, the samples were centrifuged at 10,000
g for 5 minutes at 4°C. The MDA concentrations were calculated by measuring the absorbance at 450, 532 and 600 nm using an Epoch microplate spectrophotometer (Bio Tek Instruments Inc., USA).
Samples were homogenized thoroughly in 1.2 mL 0.2 M potassium phosphate buffer (pH 7.8 + 0.1 mM EDTA) and were centrifuged at 10,000 rpm for 20 minutes at 4°C. The total soluble protein contents were determined using the Bradford method (
Bradford 1976).
Chlorophyll was extracted using method as described by
Ni et al. (2009). Samples were homogenized with acetone (80%) in dark at 4°C overnight. The supernatant was measured by an Epoch microplate spectrophotometer (Bio Tek Instruments Inc., USA) to calculate chlorophyll content at wavelengths of 603, 645, and 663 nm.
In this study, three replicates were used to assess each response. The results were subjected to analysis of variance using SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). Separation of means was performed using a DMRT test at
P < 0.05. The median reduction dose (RD
50; the dose at which the growth of the plant decreased by 50%) was calculated as a simple linear regression analysis using plant height (
Ryu et al. 2014). Analysis of correlation analysis was performed using the SPSS Ver. 12 (SPSS Inc., Chicago, IL, USA). Correlation analysis was carried out to determine the Pearson correlation coefficient.
RESULTS
Effect of gamma-ray on seedling emergence and growth characteristics
The seedling emergence rates and growth characteristics of gamma-irradiated seeds from the four soybean cultivars are listed in
Table 1. After irradiation, seedling emergence rates were compared to that of the control. Regardless of cultivars, emergence rate at the highest dosage of 400 Gy showed the lowest values. At 400 Gy, seedling emergence rates of Kwangankong, Shinpaldal-2, Shinhwakong, and Ilmikong were 54%, 69%, 56%, and 57%, respectively. Seedling emergence rates of Kwangankong and Ilmikong decreased with increasing radiation dose. While Kwangankong showed no significant reduction between 100 and 300 Gy, Ilmikong showed significant reduction at 200 Gy. In Shinpaldal-2 and Shinhwakong, gamma-rays did not affect the emergence rate up to 300 Gy but did cause a significant reduction in seedling emergence rate at 400 Gy.
In all cultivars, plant height decreased with an increasing dose of radiation. At 400 Gy, the plant height Kwangankong, Shinpaldal-2, Shinhwakong, and Ilmikong decreased by 85%, 72%, 85%, and 80% compared to control, respectively. Three cultivars, Kwangankong, Shinhwakong, and Ilmikong, showed the largest reduction at 200 Gy, but Shinpaldal-2 showed gradual reduction. Shinpaldal-2 showed the highest plant height among the four cultivars and the lowest reduction at 400 Gy.
Fresh weight of Kwangankong reduced gradually with an increasing dose of radiation. Shinhwakong and Ilmikong had a significant reduction at 400 Gy compared to the control, but the fresh weight between 200 and 400 Gy did not show a significant change. Fresh weight of Shinpaldal-2 significantly increased by 21% at 100 Gy and decreased at and above 300 Gy. Shinpaldal-2 had the lowest plant height at 300 Gy.
The RD
50 of gamma-ray irradiated seeds from the four Korean elite soybean cultivars are shown in
Fig. 1. The RD
50 for Kwangankong, Shinpaldal-2, Shinhwakong, and Ilmikong was 199, 278, 187, and 184 Gy, respectively (
Fig. 1). Based on these results, Shinpaldal-2 had the lowest radiation sensitivity of the four soybean cultivars.
Regardless of cultivars, the MDA contents of all treatments were higher than those of the controls (
Fig. 2). The MDA contents of Kwangankong increased with increasing doses of radiation. At 300 Gy, the MDA content was approximately triple that of the control. The MDA contents of Shinpaldal-2 increased by 61 and 57% at 100 and 200 Gy, respectively. At 300 Gy, the MDA content was not significantly different from control. The MDA content of Shinhwakong increased with an increasing dose and had its highest value at 300 Gy. Ilmikong had a significant increase in MDA content above 100 Gy compared to control and its highest value at 200 Gy.
APX activities showed different trends in Kwangankong, compared with the other cultivars (
Fig. 3). Kwangankong had the value at 200 Gy, an increase of 120% compared to the control, and decreased at 300 Gy. At 100 Gy, Kwangankong showed an insignificant increase of APX activity. The other cultivars revealed a similar tendency. The APX activities of Shinpaldal-2 and Shinhwakong insignificantly decreased at 100 Gy and increased at 200 Gy. Shinpaldal-2, Shinhwakong and Ilmikong cultivars had the highest APX activities at 300 Gy and increased by 39%, 98%, and 34%, respectively. Ilmikong APX activity significantly decreased at 100 Gy.
Each cultivar had different POD activities (
Fig. 4). Only Ilmikong showed significant changes in POD activity with different doses of gamma-ray treatments. The POD activity of Kwangankong was highest at 200 Gy, an increase of 17% compared to control. Shinpaldal-2 and Ilmikong had lower POD activities at 100 Gy than that of the control. Shinpaldal-2 had insignificant fluctuations. The POD activity of Shinpaldal-2 decreased by 10% at 100 Gy and increased 40% at 200 Gy compared to control. Shinhwakong had a gradually increasing POD activity up to 200 Gy and showed a 53% increase at 200 Gy. The POD activity of Ilmikong decreased at 100 Gy and started to increase at 200 Gy.
All cultivar showed that chlorophyll
a was more affected by gamma-rays than chlorophyll
b (
Fig. 5). For all treatments Kwangankong had significantly lower contents than control of chlorophyll
a and chlorophyll
b. Shinpaldal-2 had its highest chlorophyll contents at 100 Gy but did not significant increase compared to the control. Chlorophyll content of Shinpaldal-2 decreased gradually at and above 200 Gy. The chlorophyll content of Shinhwakong had its highest value at 200 Gy. In Ilmikong, chlorophyll
a showed a significant falling then rising trend, but chlorophyll
b showed insignificant change.
The Pearson’s correlation coefficients (based on average quantified values between dose, growth characteristics and biological responses) are listed in
Table 2. The gamma-ray dose was positively correlated with the MDA content (
P < 0.05) and APX activities (
P < 0.05). In contrast, the gamma-ray dose was highly negatively (
P < 0.01) correlated with growth characteristics (plant height and fresh weight).
DISCUSSION
In this study, biological responds to gamma-irradiation were investigated on four elite soybean cultivars, which have a high economic value in Korea. Measuring the growth characteristics was one of the best parameters to study the radiation sensitivity of the plants. In the mutation breeding program, growth parameters, such as lethal dose 50 (LD
50) and RD
50, were used to determine the optimal dose (
Van Harten 1998;
Yamaguchi et al. 2009;
Ryu et al. 2016;
Im et al. 2017a). The seedling emergence rate showed a significant reduction at the highest dose of radiation for all cultivars. Plant height and fresh weight decreased with increasing dosage of gamma-rays. The RD
50 of soybeans irradiated with gamma-rays was between 184 Gy and 199 Gy except for Shinpaldal-2. The RD
50 of Shinpaldal-2 was 278 Gy. These results are similar to the RD
50 values observed by
Lee et al. (2016) in lentil beans (
Lens culinaris) irradiated with gamma-rays, which were 156 to 253 Gy. Our values were lower than those reported in the same soybean cultivars irradiated using a proton beam, which were 350 to 400 Gy (
Im et al. 2017a). In mutation breeding, a survival or germination rate of 40 to 60% and/or the growth of the plant decreasing from 30 to 50% in comparison to the non-irradiation plants is taken to be the standard for the optimal radiation treatment (
Van Harten 1998;
Ryu et al. 2014;
Lee et al. 2016;
Ryu et al. 2016). We find that the optimal dose of gamma irradiation for the four elite soybean cultivars for mutation induction is 200 to 300 Gy in this study.
Biological responses of ionizing radiation have various direct and indirect effects on plant cells (
Kim et al. 2015;
Oladosu et al. 2016;
Im et al. 2017b). The ROS radicals can be produced by energy deposited in water molecules. High concentrations of ROS around DNA can attack DNA and induce breaks resulting in loss-of-function (
Celik et al. 2014). The biological responses to ionizing radiation on the generation of ROS can be screened indirectly by measuring the amount of biological indicators, such as MDA, POD, and APX (
El-Beltagi et al. 2011;
Kim et al. 2015;
Im et al. 2017b). In this study, gamma radiation treatment increased MDA content in all of the cultivars. The highest increase rate was in Kwangankong, and its MDA content at 300 Gy is three times that of the control. The MDA is the final product of lipid peroxidation and normally indicates the level of lipid peroxidation (
Gawel et al. 2004;
Foyer and Noctor 2005;
Celik et al. 2014). Lipid peroxidation is a phenomenon attribute to oxidative damage in plant cells (
Yamaguchi et al. 2009;
Kim et al. 2015). Ionizing radiation was revealed to induce oxidative stress with an oversupply of ROS (
Garg and Manchanda 2009;
Kim et al. 2012). APX contents of soybeans irradiated by gamma-rays in the cultivars except for Kwangankong showed a similar tendency. Kwangankong had a significant increase at and above 200 Gy, while the other cultivars decreased at 100 Gy and showed their highest values at 300 Gy. The POD contents of Kwangankong and Shinhwakong showed no significant increase. Shinpaldal-2 decreased in 100 Gy and began increasing at 200 Gy, but did not show a significant change. These activities can damage or modify important components of plant cells and affect physiological and biochemical processes (
Mittler 2002;
Foyer and Noctor 2005;
Wi et al. 2007). Previous studies have revealed that seeds exposed to gamma-rays disturb protein synthesis, hormone balance, leaf gas exchange, and enzyme activity (
Foyer and Noctor 2005;
Celik et al. 2014;
Kim et al. 2015). The variation in biological response between different cultivars of same species is a common symptom in mutation studies because the resistance depends on leaf size, moisture content, and the time of treatment etc. (
Alikamanoglu et al. 2011;
Marcu et al. 2013;
Oladosu et al. 2016;
Im et al. 2017b).
The chlorophyll content is the most important indicator of mutation and environmental stress (
Alikamanoglu et al. 2011;
Jan et al. 2012;
Kim et al. 2015). All irradiated individuals had lower total chlorophyll values than the untreated plants, except for Shinhwakong irradiated with 200 Gy and Shinpaldal-2 irradiated with 100 Gy. Chlorophyll
a contents were more than those of chlorophyll
b. The percentage change in chlorophyll
a and chlorophyll
b are similar. Kwangankong showed the highest value for its control and the lowest value at 100 Gy. Shinpaldal-2 increased at 100 Gy and started to decrease at 200 Gy. Shinhwakong increased by 40% at 200 Gy. Ilmikong decreased at 100 Gy and increased at 200 Gy but then decreased again.
Alikamanoglu et al. (2011) reported that depending on the gamma-ray dose, the total chlorophyll contents decreased in soybean leaves, and the leaf tissue was significantly damaged by high doses of gamma radiation, causing an acceleration of leaf senescence. However, contents of chlorophyll
a and
b significantly increased in the leaves of gamma-irradiated cowpea (
Vigna unguiculata L.) (
El-Beltagi et al. 2011). In addition,
Gicquel et al. (2012) reported that differential, dose-dependent regulation of chloroplast element genes has been revealed in
Arabidopsis, including up-regulation at 10 Gy and down- regulation at 40 Gy. Therefore, we assumed that gamma-irradiation induces different responses for different chlorophyll pigments, depending on the cultivars and dosage.
We evaluated four indicators (MDA content, APX activates, plant height, and fresh weight) that were significantly correlated with gamma-ray dose. Similarly,
Lee et al. (2015b) reported that the MDA content of
cymbidium is highly correlated with proton beam dose. In addition,
Ryu et al. (2014) reported that growth characteristics (germination rate, survival rate, plant height, number of leaves, root length, and fresh weight) were highly negatively (
P < 0.01) correlated with gamma-ray dose.
In conclusions, the results presented here show that the effects of gamma radiation on Korean elite soybean cultivars depend on the gamma-ray dose. Both the growth characteristics and biological responses decreased with an increasing dose in the gamma-ray treatment groups. The effects of irradiation have been described so differently because different species, cultivars and ecotypes as well as diverse dose and time of irradiation have variable effects. Although radiation induced increases in antioxidants activities and changed chlorophyll ratios, none of these characteristics could explain the difference the in response to radiation in these cultivars. These results may be used to determine the optimal radiation doses and in the planning of mutation studies.
ACKNOWLEDGEMENTS
This study was carried out with the support of the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ012214), Rural Development Administration, Republic of Korea, and the National Research Foundation of Korea (NRF) grant funded by the Korean Government (NRF-2018M2A2A6 A05057264).
Fig. 1RD50 values for growth characteristics of soybean cultivars. (A) Kwangankong (B) Shinpaldal-2 (C) Shinhwakong (D) Ilmikong.
Fig. 2Effect of gamma-ray irradiation on the total MDA content of leaves from soybean cultivars. Mean separation within columns was determined by an LSD test at P = 0.05. (A) Kwnagankong (B) Shinpaldal-2 (C) Shinhwakong (D) Ilmikong.
Fig. 3Effect of gamma-ray irradiation on the total APX activity of leaves from soybean cultivars. Mean values were separated using the DMRT test at P < 0.05. (A) Kwnagankong (B) Shinpaldal-2 (C) Shinhwakong (D) Ilmikong.
Fig. 4Effect of gamma-ray irradiation on the total POD activity of leaves from soybean cultivars. Mean values were separated using the DMRT test at P < 0.05. (A) Kwnagankong (B) Shinpaldal-2 (C) Shinhwakong (D) Ilmikong.
Fig. 5Effect of gamma-ray irradiation on the chlorophyll content of the leaves of soybean cultivars. Mean values were separated using the DMRT test at P < 0.05. (A) Kwangankong (B) Shinpaldal-2 (C) Shinhwakong (D) Ilmikong.
Table 1Growth responses of soybean cultivars irradiated with different gamma-ray doses.
Table 1
|
Dose (Gy) |
Emergence rate (%) |
Plant height (cm) |
Fresh weight (g/plant) |
|
Kwanankong |
|
0 |
85.67 |
az)
|
19.08 |
a |
2.15 |
a |
|
100 |
82.33 |
a |
15.06 |
b |
1.92 |
a |
|
200 |
79.00 |
a |
6.24 |
c |
1.48 |
b |
|
300 |
78.67 |
a |
4.27 |
d |
1.38 |
bc |
|
400 |
54.00 |
b |
2.92 |
d |
1.10 |
c |
|
Shinpaldal-2 |
|
0 |
84.67 |
a |
19.61 |
a |
1.97 |
b |
|
100 |
89.33 |
a |
17.40 |
b |
2.39 |
a |
|
200 |
85.67 |
a |
13.20 |
c |
2.13 |
ab |
|
300 |
85.67 |
a |
7.90 |
d |
1.00 |
c |
|
400 |
68.67 |
b |
5.49 |
e |
1.32 |
c |
|
Shinhwakong |
|
0 |
78.00 |
a |
17.16 |
a |
2.04 |
a |
|
100 |
82.33 |
a |
13.21 |
b |
1.69 |
b |
|
200 |
72.67 |
a |
4.24 |
c |
0.98 |
c |
|
300 |
77.67 |
a |
3.03 |
cd |
1.04 |
c |
|
400 |
56.33 |
b |
2.59 |
d |
0.89 |
c |
|
Ilmikong |
|
0 |
77.00 |
a |
15.27 |
a |
2.26 |
a |
|
100 |
75.67 |
a |
10.81 |
b |
1.98 |
a |
|
200 |
66.67 |
b |
3.53 |
c |
1.32 |
b |
|
300 |
62.33 |
bc |
2.86 |
c |
1.25 |
b |
|
400 |
57.33 |
c |
3.10 |
c |
1.20 |
b |
Table 2Correlation coefficients between dose, growth characteristics, and biological responses in soybean cultivars.
Table 2
|
Trait |
P-value |
R2
|
|
Dose and MDA |
*
|
0.551 |
|
Dose and APX |
*
|
0.599 |
|
Dose and plant height |
**
|
−0.882 |
|
Dose and fresh weight |
**
|
−0.804 |
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