In recent years, there has been a great deal of attention toward free radicals, reactive oxygen species (ROS) generated by exposure of crop plant cells to physical radiations. Henceforth, the current study was planned to compare oxidative stress and mutagenic potential of different irradiation doses of fast neutron (FN) and UV-B on meiotic-pollen mother cells (PMCs), pollen grains (PGs) and seeds yielded from irradiated faba beans seedlings. On the cytogenetic level, each irradiation type had special interference with DNA of PMC and exhibited wide range of mutagenic action on the frequency and type of chromosomal anomalies, fertility of PGs and seed yield productivity based on the irradiation exposure dose and radiation sensitivity of faba bean plants compared with un-irradiated ones. On the molecular level, SDS-PAGE and RPAD-PCR analyses of seeds yielded from irradiated seedlings exhibited distinctive polymorphisms based on size, intensity, appearance, and disappearance of polypeptides bands compared with un-irradiated ones. The total values of protein and DNA polymorphisms reached 88% and 90.80% respectively. The neutron fluency (2.3 × 106 n/cm2) and UV-B dose for 1 hr were recorded as bio-positive effects. The present study proved that genetic variations revealed by cytogenetic test could be supported by gene expression (alterations in RAPD and protein profiles).
It has been known for many years that exposure of crop plant cells under natural conditions of growth and development to physical radiations such as ionizing FN and nonionizing UV-B resulted in excessive production of free radicals ROS [
Despite the ROS destructive activity, their production in plant tissues is controlled by the very efficient enzymatic and nonenzymatic antioxidant defense systems which serve to keep down the levels of free radicals, permitting them to perform useful biological functions without too much damage and act as a cooperative network employing a series of redox reactions [
Biologically, FN differs from UV-B radiation in the way in which energy is distributed in irradiated tissues and their biological effects in the living cell [
It is important for detection of oxidative stress and mutagenic potential of various types of radiations on crop plants, to understand their biological consequences and their molecular action on chromosome, protein, and DNA of plant cell by introducing cytogenetic and molecular assays. Cytogenetic tests are considered to be indicator of cytotoxicity, genotoxicity, genetic variability, and estimation of the mutagen potency in meiotic PMCs and PGs [
In light of the previously mentioned, the main goal of this study is to evaluate and compare the effects of various levels of ROS generated by different irradiation doses of FN and UV-B on PMCs, PGs, and seeds yield of irradiated
Faba bean seeds (
The seedling of group (A) is packed regularly in polyethylene high-density bags and irradiated by fission neutrons from Cf252 point source using four fluencies (
Irradiated and un-irradiated seedlings were transferred immediately to soil and sown in rows under field conditions. A spacing of 30 cm row to row and 15 cm plant to plant were maintained. At maturity, ten flower buds from ten plants for each irradiation dose in addition to un-irradiated ones were collected, fixed immediately in Carnoy’s fixative (3 : 1) absolute ethyl alcohol : glacial acetic acid for 24 hours and then stored in refrigerator in 70% ethyl alcohol and finally stained using acido-carmin smear method [
Quantitative parameters of six plants irradiated by the various irradiation doses of FN and UV-B radiation mentioned previously were measured as mean number of pods/plant, mean number of seeds/plant, and mean dry weight of 100 seeds compared with those of un-irradiated one. Seeds yielded from irradiated and un-irradiated plants were harvested and analyzed on molecular level using SDS-PAGE analysis of seed storage proteins and RAPD analysis of seed DNA via the polymerase chain reaction (PCR).
Seeds yielded from all irradiated faba beans seedlings by FN and UV-B were used for SDS-PAGE analysis.
Sterilized seeds were milled and defatted according to [
The protein extraction technique was employed according to [
Gel photographing and documentation were carried out using Bio-Rad gel documentation system. The number of bands revealed in each gel lane were counted and compared with each other’s using Gel Pro-Analyzer. Quantitative variations in band number as well as band concentration were estimated using BIO-RAD Video densitometer, Model Gel Doc 2000.
Seeds yielded from irradiated faba beans seedlings by stimulatory FN fluency
Fifty grams of dried seeds of both two doses of FN and UV-B was crushed in a mill and powdered by using a domestic grinder. The powder was sieved using thin mesh and only finely ground powder was kept in refrigerator until DNA extraction. One gram of finely sieved seed powder was taken, and genomic DNA was isolated using Hexadecyl trimethyl ammonium bromide (CTAB) as described by [
The yield of DNA per gram of seed material extracted was measured by using UV spectrophotometer (Perkin Elmer) at 260 nm and A280 nm. Thepurity of DNA was determined by calculating the ratio of absorbanceat 260/280 nm. For quality and yield assessments, electrophoresis was done of all DNA samples on 0.8% agarose gel, stained with ethidium bromide, and bands were observed in gel documentation system (Alpha Innotech) and compared with the known standard Lambda DNA marker. The gels were visualized and photographed under UV light (Gel documentation system, Bio-Rad).
Briefly, the PCR reaction mixture contains 2.5
Gels were visualized with Photo Print (Vilber Lourmat, France) imaging system, and analysis of RAPD bands was performed by BioOne D++ software (Vilber Lourmat, France). The RAPD bands (markers) were scored as 1 if present and 0 if absent.
The ability of fast neutron and UV-B radiation to exert genotoxic action on PMCs DNA in 1st and 2nd meiotic divisions was observed in spite of long period of recovery (Table
Number, frequencies of abnormal PMCs in 1st and 2nd meiotic divisions, frequencies of meiotic abnormalities, types of abnormal meiotic PMCs, and percentages of fertile PGs after irradiation of
Irradiation types | Doses |
1st meiotic |
2nd meiotic |
Total no. of PMCs | Total no. of abnormal PMCs | % of abnormal PMCs ± SE | Types and frequency of meiotic abnormalities of PMCs | PGs | ||||||||||
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Total no. of PMCs | % of abnormal Cells | Total no. of PMCs | % of abnormal Cells | Stickiness | Disturbed | Unorient. | Micronuclei + fragments | Lagging + free | Bridge | Tripolar | Multipolar | No. of PGs | Mean % of fertility | |||||
Un- irradiated | 0.00 | 835 | 0.00 | 1050 | 0.00 | 1885 | 0 | 0 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 600 |
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FN fluencies (Φ) n/cm² | 2.5 × 105 | 541 | 38.26 | 339 | 47.79 | 880 | 369 |
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19.56 | 30.37 | 14.43 | 13.90 | 8.52 | 2.64 | 9.13 | 1.60 | 600 |
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2.3 × 106 | 446 | 15.47 | 505 | 14.85 | 951 | 145 |
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25.06 | 37.95 | 12.51 | 5.82 | 6.71 | 6.26 | 3.58 | 1.12 | 600 |
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3.0 × 107 | 515 | 58.64 | 440 | 42.50 | 955 | 498 |
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25.56 | 27.37 | 10.30 | 13.73 | 4.61 | 8.40 | 7.59 | 2.44 | 600 |
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1.5 × 108 | 412 | 66.50 | 336 | 65.18 | 748 | 493 |
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41.92 | 12.47 | 11.86 | 15.75 | 6.95 | 3.48 | 4.94 | 2.69 | 600 |
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UV-B doses |
1/2 h | 526 | 33.08 | 435 | 38.39 | 955 | 341 |
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12.00 | 7.84 | 20.85 | 42.22 | 3.82 | 10.04 | 0.88 | 2.35 | 600 |
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1 h | 459 | 21.70 | 435 | 19.06 | 1158 | 236 |
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22.40 | 5.91 | 8.93 | 33.20 | 4.48 | 19.55 | 3.26 | 3.26 | 600 |
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2 h | 424 | 49.06 | 450 | 43.55 | 874 | 404 |
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31.68 | 4.70 | 8.17 | 25.57 | 6.44 | 20.96 | 1.49 | 1.00 | 600 |
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3 h | 680 | 49.71 | 421 | 49.64 | 1101 | 607 |
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37.50 | 8.25 | 10.51 | 31.16 | 3.44 | 6.34 | 1.27 | 1.74 | 600 |
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The most common meiotic chromosomal aberrations induced by fast neutron ((1)–(4)) and UV-B radiation ((5)–(8)). (1) Telophase I with micronucleus, (2) disturbed anaphase, (3) sticky metaphase I, (4) sticky anaphase II with two lagging chromosomes, (5) metaphase I with two unoriented chromosomes, (6) sticky anaphase I with bridges, (7) metaphase II with sticky fragment, (8) four micronuclei with sticky telophase II, (9) (a) sterile Pollen grains (colorless), and (b) fertile one (color).
The different parameters of seeds yield productivity of irradiated faba beans seedlings that were represented in the mean number of pods/plant, number of seeds/pod, and average weight of 100 seeds/gm were dose-dependent as evident from their reduction by most irradiation doses of FN and UV-B radiations expect FN fluency (
The productivity parameters of seeds yielded from irradiated
Irradiation types | Doses | Seed yield parameters | ||
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No. of pods/plant | No. of seeds/plant | Average wt. of 100 seeds/gm | ||
FN fluencies (Φ) n/cm² | 0.00 |
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2.5 × 105 |
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2.3 × 106 |
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3.0 × 107 |
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1.5 × 108 |
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UV-B doses |
0.00 |
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1/2 h |
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1 h |
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2 h |
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3 h |
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Each irradiation dose of FN and UV-B used in the current study exhibited distinctive quantitative and qualitative alterations in electrophoretic banding pattern of total seed proteins yielded from irradiated faba bean seedlings compared to un-irradiated ones. These protein alterations are based on changes in bands molecular weights (MWs), bands intensities, fractionation of some bands, appearance of new bands (unique bands), and disappearance of some bands (polymorphic bands) as shown in (Table
Electrophoretic banding analysis of seeds storage proteins yielded from irradiated
No. | MW/KDa | Protein marker | Fluencies of FN radiation (n/cm²) |
Un-irritated |
Various doses of UV-B radiation ( |
Polymorphism types | |||||||||||||||||
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2.5 × 105 | 2.3 × 106 | 3.0 × 107 | 1.5 × 108 | 1/2 h | 1 h | 2 h | 3 h | ||||||||||||||||
Lane M | Lane 1 | Lane 2 | Lane 3 | Lane 4 | Lane 5 | Lane 6 | Lane 7 | Lane 8 | Lane 9 | ||||||||||||||
KDa | % | KDa | % | KDa | % | KDa | % | KDa | % | KDa | % | KDa | % | KDa | % | KDa | % | KDa | % | ||||
1 | 315 | + | + | + | 32.77 | + | 106.58 | + | 106.84 | + | 66.82 | + | 87.95 | + | 90.73 | + | 101.3 | + | 96.49 | + | 52.91 | M | |
2 | 181 | − | − | − | − | − | − | − | − | + | 32.69 | − | − | − | − | − | − | − | − | P | |||
3 | 175 | − | − | − | − | + | 17.83 | − | − | − | − | − | − | − | − | − | − | − | − | − | − | U | |
4 | 158 | − | − | − | − | − | − | + | 16.35 | − | − | − | − | + | 11.28 | + | 17.83 | + | 12.57 | − | − | P | |
5 | 145 | − | − | − | − | − | − | − | − | + | 14.3 | − | − | − | − | + | 10.37 | − | − | − | − | P | |
6 | 128 | − | − | − | − | + | 17.83 | − | − | − | − | + | 15.39 | − | − | − | − | − | − | − | − | P | |
7 | 120 | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | 9.43 | − | − | − | − | U | |
8 | 116 | 116 | 8.86 | − | − | + | 10.54 | + | 15.16 | − | − | − | − | + | 11.28 | + | 12.48 | − | − | − | − | P | |
9 | 97 | 97 | 29.84 | − | − | − | − | − | − | + | 9.23 | − | − | − | − | − | − | + | 9.43 | − | − | P | |
10 | 86 | − | − | + | 106.00 | + | 125.86 | + | 5.34 | + | 99.46 | − | − | − | − | − | − | − | − | P | |||
11 | 75 | − | − | + | 81.51 | + | 103.47 | − | − | − | − | + | 99.23 | + | 114.7 | + | 118.96 | − | − | + | 9.22 | P | |
12 | 66 | 66 | 33.00 | + | 77.69 | + | 118.97 | + | 106.00 | − | − | + | 105.23 | + | 111.6 | + | 118.96 | − | − | + | − | P | |
13 | 60 | − | − | − | − | − | − | − | − | − | − | + | 128.02 | − | − | − | − | − | − | − | 7.22 | p | |
14 | 54 | − | − | + | 77.69 | + | 95.21 | − | − | − | − | + | 126.51 | − | − | + | − | − | − | − | − | P | |
15 | 50 | − | − | + | 91.41 | + | 94.02 | + | 86.79 | − | − | + | 117.37 | − | − | + | 126.51 | − | − | − | − | P | |
16 | 45 | − | − | + | 52.69 | + | 101.95 | + | 125.54 | + | 100.21 | + | 138.29 | + | 109.63 | + | 77.41 | + | 58.55 | + | 14.84 | M | |
17 | 40 | − | − | + | 14.84 | − | − | + | 38.65 | + | 73.35 | + | 56.23 | + | 55.71 | + | 77.19 | − | − | + | 28.59 | P | |
18 | 38 | 38 | 32.88 | + | 21.36 | + | 14.57 | − | − | − | − | − | − | − | − | − | − | − | − | − | − | P | |
19 | 29 | − | − | − | − | + | 11.62 | − | − | − | − | − | − | − | − | − | − | − | − | − | − | U | |
20 | 25 | − | − | + | 225.17 | + | 232.77 | + | 266.37 | + | 256.43 | + | 267.16 | + | 261.93 | + | 237.79 | + | 231.78 | + | − | p | |
21 | 23 | − | − | − | − | + | 19.42 | + | 41.76 | + | 77.98 | − | − | + | 78.44 | + | 35.64 | + | 36.11 | − | 118.23 | P | |
22 | 18 | 18 | 43.44 | + | 41.76 | + | 56.82 | + | 94.7 | + | 44.88 | − | − | + | 61.21 | − | − | + | 29.95 | − | − | P | |
23 | 16 | − | − | + | 16.23 | + | 26.58 | + | 17.85 | + | 17.34 | + | 9.28 | + | 12.48 | + | 10.46 | + | 29.95 | − | − | P | |
24 | 14 | − | − | − | − | + | 26.58 | − | − | − | − | − | − | − | − | + | 11.07 | − | − | − | − | P | |
25 | 12 | 12 | 103.15 | + | 134.29 | + | 173.34 | + | 188.58 | + | 174.19 | + | 177.85 | + | 179.34 | + | 137.12 | + | 146.44 | + | 128.09 | M | |
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Total bands of each lane | 12 | 18 | 13 | 11 | 14 | 12 | 15 | 9 | 7 | ||||||||||||||
% Total bands | 10.81 | 16.22 | 11.71 | 9.91 | 12.61 | 10.81 | 13.51 | 8.11 | 6.31 | ||||||||||||||
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Polymorphic bands | Unique bands | Monomorphic bands | % of polymorphism | ||||||||||||||||||||
Total protein bands of all lanes = 111 | No. of bands | % | No. of bands | % | No. of bands | % | |||||||||||||||||
88 | |||||||||||||||||||||||
22 | 19.82 | 3 | 2.70 | 3 | 2.70 |
RAPD-PCR amplification products of DNA extracted from seeds yielded from irradiated
Total no. of scorable bands | Polymorphic bands | ||||||||||||||||
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Primers | Sequence of primers from 5′→3′ | Amplicon lengths (pb) | Fluencies of fast neutrons radiation (n/cm²) | Control |
Various doses of UV-B radiation ( |
Total no. | % of total bands | No. of unique bands | % of unique bands | No. of nonunique bands | % of nonunique bands | % of polymorphic bands | No. of monomorphic bands | % of monomorphic | % of Total polymorphism | ||
1.5 × 108 | 2.3 × 106 | 1 h | 3 h | ||||||||||||||
Lane 1 | Lane 2 | Lane 3 | Lane 4 | Lane 5 | |||||||||||||
A-02 | TGCCGAGCTG | 1500–50 | 12 | 13 | 10 | 10 | 12 | 57 | 22.98 | 4 | 7.02 | 9 | 15.79 | 17.54 | 4 | 7.02 | 58.82 |
A-03 | AGTCAGCCAC | 1300–50 | 11 | 15 | 13 | 11 | 10 | 61 | 24.60 | 6 | 9.83 | 15 | 24.59 | 34.43 | 100.00 | ||
A-10 | GTGATCGCAG | 550–50 | 6 | 5 | 4 | 9 | 6 | 30 | 12.10 | 3 | 10.00 | 4 | 13.33 | 20.00 | 1 | 3.33 | 75.00 |
A-12 | TCGGCGATAG | 900–65 | 5 | 8 | 7 | 7 | 9 | 35 | 14.11 | 4 | 11.43 | 7 | 20.00 | 31.43 | 1 | 2.86 | 91.67 |
A-15 | TTCCGAACCC | 700–75 | 8 | 8 | 7 | 6 | 5 | 34 | 13.71 | 5 | 14.71 | 5 | 14.71 | 23.53 | 1 | 2.94 | 72.73 |
A-17 | GACCGCTTGT | 800–65 | 5 | 11 | 5 | 7 | 3 | 31 | 12.50 | 5 | 16.13 | 9 | 29.03 | 45.16 | 1 | 3.23 | 93.33 |
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Overall total DNA bands |
47 |
60 |
46 |
50 |
45 |
248 | 27 | 10.89 | 49 | 19.78 | 30.65 | 8 | 3.23 | 90.80 |
SDS-PAGE banding patterns of storage protein in seeds yielded from irradiated
RAPD analysis was employed in the present study to evaluate the extent of the DNA alterations in seeds yielded from irradiated faba beans seedlings by the two bio-positive doses (FN fluency (
(a) and (b) DNA banding pattern of RAPD analysis of seeds yielded from irradiated
Cytogenetic test is considered to be indicator of oxidative potential, genotoxicity, and estimation of the mutagen potency in meiotic PMCs and PGs [
The present study observed that SDS-PAGE analysis exhibited distinctive qualitative and quantitative alterations in electrophoretic SDS-proteins stored in seeds yielded from irradiated faba beans seedlings. These alterations are based on variations in molecular weights and intensities of polypeptides bands as well as gain or loss of protein bands that led to highly levels of protein polymorphism. Electrophoretic analysis of protein provides information concerning the structural genes and their regulatory systems that control the biosynthetic pathways of that protein. Each polypeptide band represents the final products of a transcriptional and translational events occurring due to active structural genes [
The appearance of new bands (unique bands) may be explained on the basis of mutational events at the regulatory system of unexpected gene(s) or on the basis of band subfractionation which could be attributed to the cytological anomalies in PMCs leading to gene duplication followed by the occurrence of point mutation that encoded the fractionated band [
The result obtained in this study indicated that the UV-B doses for (2 and 3 h) may cause highly oxidative protein cross-links due to alteration of the expression of several genes that leading to proteins denaturation and disappearance of numerous bands due to aggregation or cross linking of individual polypeptide chains [
RAPD assay used in the current study showed that various irradiation doses of the FN and UV-B exhibited distinctive qualitative and quantitative alterations in the RAPD profiles based on gene products, the amplified DNA sizes, their intensities, and appearance or disappearance of DNA bands that led to generation highly levels of DNA polymorphism. Variations in the characteristic DNA banding pattern generated by RAPD analysis may be caused by rearrangements of the genomic DNA, base pair deletions, mutations, inversions, translocations, and transpositions within base pair sequences of DNA which result in the loss or gain of DNA bands resulting in different DNA lengths and consequently highly level polymorphisms [
The current study investigated that DNA alterations in RAPD profile could be explained on the basis of the biological way by which the radiation type interacts with DNA, by producing their own ROS through the direct and/or indirect effect in the irradiated cells. In relation to ROS generated in faba bean seedlings by bio-negative fluencies of FN can induce DNA deletions in nucleus and chromosome that range in size from a few base pairs to several megabases [
The present study observed that the cytogenetic analysis of meiotic PMCs and PGs in addition to SDS-PAGE and RAPD analyses of proteins and DNA of seeds yielded from irradiated faba beans seedlings by neutron fluency (
The authors would like to extend their sincere appreciation to the deanship of Scientific Research at King Saud University for funding this research through the Research Group Project no. RGP-VPP-231. They also wish to express their deep thanks to Professor Magda Hanafy, Biophysics Department, Science College, Zagazig University, for her effort in performing the fast neutron irradiation and estimation of FN fluencies.