EFFECTS OF ULTRAVIOLET RADIATION ON CYTOLOGICAL AND MOLECULAR ASPECTS OF TOMATO

omato (Lycopersicon esculentum L.) is considered as an important vegetable crop of special economic importance in the horticultural industry worldwide (Wang et. al., 2005). Tomato consumption is known to have health benefits due to its high content of phytonutrients. Recent studies suggest that high intake of fresh or processed tomato lowers the risk of prostate cancer (Campbell et al., 2004).

Giza, Egypt omato (Lycopersicon esculentum L.) is considered as an important vegetable crop of special economic importance in the horticultural industry worldwide (Wang et. al., 2005). Tomato consumption is known to have health benefits due to its high content of phytonutrients. Recent studies suggest that high intake of fresh or processed tomato lowers the risk of prostate cancer (Campbell et al., 2004).
Ultraviolet radiation (UV) is a kind of radiation that falls in a part of nonionizing electromagnetic spectrum region which represents 8-9% of the total solar radiation (Frederic, 1993). UV is traditionally divided into three wavelength ranges: UV-A (320-400 nm), UV-B (280-320 nm) and . UV-C is harmful to living organisms and can induce a variety of damaging effects in plants, while UV-A is the less hazardous part of UV radiation (Hollosy, 2002).
During the past two decades, increased level of solar UV radiation have been measured at temperate and Polar Regions of the Earth surface as a consequence of the progressive reduction in the stratospheric ozone layer (Madronich et al., 1998;Hollosy, 2002).
Usually UV-C and most of the UV-B are reflected through stratospheric ozone, so only UV-A and a little of the UV-B reach the earth (Steeger et al., 2001). Organisms on earth are adapted to UV-A, but may not be adapted to UV-B. Now and due to the reduction in atmospheric ozone, a great deal of attention has been focused on UV-B induced photo damage organisms. A 10% decline in the total stratospheric ozone would raise the amount of UV-B reaching the earth's surface by 20% (Misra et al., 2002).
Increased UV-B radiation caused by reduced stratospheric ozone is expected to continue into the 21 st century (Madronich et al., 1998). A large number of studies have been conducted to evaluate the potential consequences of an increase in UV-B radiation on many plants (Zheng et al., 2003). However, most of these studies addressed the effects of UV-B on the whole plant level. Less information on the UV-induced damage at the DNA level are found. Owing to its high energy level, the T impact of radiation on metabolic process can be very harmful. DNA, protein and photosynthetic apparatus are considered primary potential targets of UV radiation on plants (Hollosy, 2002;Santina et al., 2008).
Cell irradiation with both UV-B and UV-C radiations results in various DNA photoproducts. These products may cause mutation during replication (Jiang and Taylor, 1993). The most common DNA photoproducts are cyclobutane-type pyrimidine dimmers (CPDs) and the pyrimidine (6, 4) pyrimidone dimmer. In addition, DNA protein cross-links, DNA strand breaks and deletion or insertion of base pairs can also be induced by UV-B exposure. Quaite et al. (1992) found that (CPDs) represent approximately 75% of total DNA damage.
The production of reactive oxygen species (ROS) by UV has been also demonstrated (Mackerness and John, 2001) and the induction of antioxidative enzymes constitutes a defense response to these molecules (Santos et al., 2004). The accumulation of flavonoids is also considered a defense mechanism against UV, as they protect the mesophyll tissue through epidermal screening of this radiation (Santina et al., 2008).
Recently, molecular markers have been used to investigate DNA sequence variation(s) in and among the crop species and create new sources of genetic variation by introducing new and favorable traits from landraces and related crop species.
Among molecular markers, Randomly Amplified Polymorphic DNA (RAPD) is generally favored because of its sensitivity, simplicity and costeffectiveness, coupled with the fact that DNA sequence information is not required for primer design, no radioisotope labeling is needed for sample detection, and only small amount of template DNA is required. In addition, this technique is reliable when assaying considerable number of primers and selecting few repeatable RAPD fragments (Nayak et al., 2003;Lin et al., 2009). RAPD is very useful in the study of biodiversity, hybridization, gene mapping and genetic map construction (Elham et al., 2010) Inter-simple sequence repeats (ISSRs) molecular markers amplify regions between microsatellite loci. This class of markers does not require any prior knowledge about the sequences to be amplified and shows high polymorphism in the material, being very useful in studies of genetic diversity, phylogeny, genomics and evolutionary biology (Reddy et al., 2002). This type of markers provides highly effective plant fingerprinting (Joshi et al., 2000;Adawy et al., 2004).
The start codon targeted (SCoT) polymorphism is a novel, simple and reliable gene targeted marker technique based on the translation start codon (Collard and Mackill, 2009). Primers for SCoT marker analysis were designed based on the conserved region surrounding the translation initiation codon, ATG. Using a single 18mer primer as a forward and reverse pri-mer in the PCR, Collard and Mackill (2009) designed thirty-six primers that were used successfully for cultivar identification and genetic diversity analysis in many crops.
Therefore, the objectives of the present investigation are to detect the effects of the UV-B radiation on the mitotic division as well as on the genomic DNA of tomato using RAPD, ISSR and SCoT assays.

Plant Material
Seeds of tomato (variety Castle Rock) were germinated in greenhouse at AGERI. At age of 3 weeks, plants were subjected to UV-B radiation for periods of 20, 40 and 60 minutes. Seeds of tomato (variety Castle Rock) were germinated in greenhouse at AGERI with 16 hr illumination per day. At age of 3 weeks, plants were subjected to UV-B radiation for periods of 20, 40 and 60 minutes.

UV-B irradiation
Seeds of Lycopersicon esculentum L. were germinated on moistened filter paper in Petri dishes with 16 hour illumination per day for three weeks till the shoots reached 1.5-2 cm long. The source of UV radiation (SunStudio) was placed at a distance of 30 cm from the plants. Irradiation in the UV-B wavelength (mainly 290-320 nm) was measured using a calibrated spectroradiator located at the Biophysics unit, Physics department of the Faculty of Science, Ain Shams University. The level of UV-B radiation utilized approaches natural conditions found in certain regions like Italy (Meloni et al., 2000). The radiation supplied a weighted irradiance of 6.3 KJ m 2 of biologically effective enhanced UV-B. Three different exposure periods of 20, 40 and 60 minutes were employed.

Cytological studies
Root tips (1-2 cm) were collected in bulk and fixed in Carnoy solution made of absolute ethanol:glacial acetic acid (3:1). After 24 hours root tips were transferred to 70% ethyl alcohol and stored in 14C. The aceto-carmine staining method was used to stain the root tip cells as described by Fayed et al. (1985) and Sayed-Ahmmed (1985). The fixed root-tips were washed thoroughly with distilled water and macerated by 1 N HCl at 60C for 3 minutes and then washed with distilled water. The root tips were squashed on dry clean slides and stained with a small drop of fresh stain. Root tips were warmed gently in aceto-carmine for a short time. The cover slips were removed by the dry-ice method and the slides were immersed in a series of ethyl alcohol (50, 70 and 96 absolute alcohol: xylol (1:1) and then xylol for 5 minutes in each and then mounted in Canada balsam and dried. At least twelve prepared slides were used to determine the mitotic index and chromosomal aberrations.
The effects of UV-B radiation on the frequencies of chromosomal aberra-tions and micronuclei were analyzed. All examinations were done with a light microscope at 300x and 400x magnifications. The mitotic index represented the percentage of dividing cells divided by the total cells examined. The numbers of chromosomal aberrations were estimated in dividing cells.

Total DNA extraction from irradiated tomato plants
Leaves of three weeks old irradiated plants were pooled in bulk and used to extract DNA with DNeasy Plant Mini Kit supplied from Qiagen Hilden Germany catalog number 69104. Tissues were ground using liquid nitrogen and 400 µl of AP1 buffer and 4 µl of RNase stock solution (100 mg/ml) were added to a maximum of 100 mg of ground tissue, the mixture was incubated for 10 min at 65C. The tube was mixed 2-3 times during incubation, then 130 µl of AP2 buffer was added to the lysate, mixed and incubated for 5 min on ice. The lysate was applied to mini spin column (pink) and centrifuged for 2 min at 20,000 xg then the supernatant was transferred to a new tube and 1.5 volume of AP3 buffer was added and mixed by pipetting. The supernatant was transferred to mini spin column (white) and centrifuged for 1 min at 6000 xg then the column placed in a new 2 ml collection tube. A volume of 500 µl AW buffer was added to the column, centrifuged for 1 min at 6000 xg and the flow through was discarded then this step was repeated. The column was transferred to a new tube and 100 µl of AE buffer were added di-rectly on the column then incubated for 5 min at room temp. The column was centrifuged for 1 min at 6000 xg to elute the DNA, which was stored at -20C till further investigation

Molecular Marker Analysis
RAPD amplification was performed as described by Williams et al. (1990), using eleven 10-mer random primers (Table 2). ISSR assays were performed as described by Adawy et al. (2004) and Hussein et al. (2006). Nineteen ISSR primers (Table 4) were selected from different published papers to be employed in ISSR analysis. SCoT amplification was performed as described by Collard and Mackill (2009), using fifteen 18mer primers (Table 6). These primers were selected from published papers (Collard and Mackill, 2009;Xiong et al., 2011).

Data Analysis
The banding patterns generated by RAPD, ISSR and SCoT were examined to determine the level of polymorphism and the genetic relatedness among the four tested treatments and the control. The amplified fragments were scored as present (1) or absent (0). The genetic similarity among the control and three treatments was estimated according to Dice coefficient. Cluster analysis was based on similarity matrix obtained within-weighted pair group method using arithmetic average (UPGMA), and the relationships between the treatments were displayed as dendrogram.

RESULTS AND DISCUSSION
Tomato plants were subjected to both cytological studies and fingerprinting analysis using RAPD, ISSR and SCoT where five plants were used for each treatment.

I-Cytological Studies
Mitotic index (MI) was determined as a parameter of mitotic activity. The result obtained after exposure of tomato plants to UV-B radiation showed a slight effect on MI. This result shows that UV-B non-ionizing radiation with the chosen exposure times had no harmful effect on cell division. The mean values of summary data are shown in Table (1). UV-B radiation induced both clastogenic and aneugenic effects. The frequency of mitotic abnormalities in response to UV-B treatment increased in a time-dependent manner. An increase in the frequency of mitotic abnormalities was observed after 20, 40 and 60 min, these periods were in accordance with Mostafaa et al. Mitotic abnormalities included lagging chromosomes, chromosome fragments and micronuclei (Fig. 1). Chromosome bridges and C-Metaphase have been noticed in very small numbers in the root tips ( Fig. 1e and 1h). The most pronounced increase in frequency of laggards was observed after one hour of exposure to UV (0.59%) as compared with control (0.03%) ( Table 1 and Fig. 1). Laggards may be explained on the basis of abnormal spindle formation and failure of the spindle to carry the respective chromosomes to the poles. It has previously been reported that unsynchronized laggards might be due to the discrepancies in spindle formation. Laggards at anaphase can be attributed to the delayed terminalization or perhaps to stickiness of chromosomal ends. Behavior of laggard chromosomes is characteristic in that they generally lead to micronuclei formation (Pesnya and Romanovsky, 2013). These data indicate that UV-B radiation may damage the formation of spindle microtubules.
Effect of UV-B radiation on DNA is well represented by the production of chromosomal fragments/breaks. The frequency of chromosome fragments in the control group was 0.05% and the frequency of micronuclei was 0.22%. Exposure to UV-B radiation increased the frequencies of fragments and micronuclei in the root tips. The most increase in fragment frequency (0.42%) was recorded after one hour of exposure to UV-B radiation (Table 1). The highest increase in micronuclei frequency (0.61%) was observed after 40 min of exposure to UV (Table 1). Micronuclei were observed in previous studies at telophase I/II following gamma radiation and may be due to the association of fragments and/or lagging chromosomes which failed to reach the poles and got included in the daughter nuclei (Jafri et al., 2011). Micronuclei derived from laggard chromosomes have a higher probability to survive and undergo condensation in synchrony with the main nuclei than micronuclei derived from chromosome fragments. Micronuclei are mutagenic aspects which may lead to the loss of genetic material (Eckert et al., 1997) Our results are thus in agreement with previous reports and show that UV-B radiation can induce mitotic and chromosomal abnormalities in plant cells. We also concluded that an increase in radiation exposure time leads to an increase in the percentage of mitotic abnormalities, in accordance with Kravetz (2009) with a slight effect on mitotic index.

RAPD analysis
The amplification profiles of the four tomato treatments (Control and three durations treatments) produced by the 11 selected primers revealed a total of 65 polymorphic bands out of 119 reproducible products (Table 2 and Fig. 2). This corresponds to a level polymorphism of 54.6%. The number of amplicons/primer ranged from 6 (OP-D20) to 15 (OP-A18) whereas the number of polymorphic bands per primer ranged from 1 (OP-D20) to 9 (OP-A18 and OP-A20).
The genetic similarity among the three tomato treatments and the control ranged from 71.7 to 83.3 (Table 3). The lowest genetic similarity (71.7) was be-tween control and treatment No. 2 irradiated with UV-B for a period of 40 minutes. On the other hand, the highest genetic similarity (83.3) was between treatment No. 2 and treatment No. 3 irradiated with UV-B for periods of 60 minutes.
The dendrogram of genetic relationships from RAPD data separated the three tomato treatments and the control into two main clusters (Fig. 3). The first cluster comprised two treatments (Control and treatment No. 1) and the second cluster comprised the other two treatments (treatment No. 2 and treatment No. 3) irradiated with 40 min and 60 min, respectivily.
The total polymorphic band between each treament and control was 29, 47 and 33 in treatments 1, 2 and 3, respectively. These result revealed that the highest level of genetic variation was observed between the control and treatment no. 2 (40 min), which refelected the highest genomic changes. While, the lowest level of genetic variation was observed between the control and treatment no. 1 (20 min).
In this respect, El Sherif et al. (2011) used twenty-eight RAPD primers to investigate the effect of different doses of gamma irradiation on growth, yield, calyx extract and quality as well as molecular changes of Roselle plants. Six primers out of twenty-eight RAPD primers successfully amplified DNA fragments from Hibiscus sabdariffa L. DNA samples. The results indicated the occurrence of structural changes in treatments. Fifty-four fragments were visualized across the six primers with percentage of polymorphism (38.4, 70, 50, 77.3, 54.5 and 38.4%). The result of RAPD analysis indicated that increasing doses of gamma irradiation caused severe effects on the plant development.
Moreover, Danylchenko and Sorochinsky (2005) investigated the possible mutation changes in alfalfa seedlings DNA resulting from exposure to UV-B and γ-ray using the RAPD technique. The authors concluded that the appearance of polymorphic bands between control and irradiated plants can be explained as the result of different DNA structural changes (breaks, transpositions, deletions etc). They also showed that single nucleotide substitutions in 10-mer primers indeed can be reflected on the amplicons profiles. Cuadra et al. (2004) revealed that treating Jaborosa magellanica with UV-B radiation caused changes in plant growth, plant chemistry and by using random primers and PCR amplification procedure, a high degree of polymorphism was detect-ed when treated plants were compared to non-irradiated plants. These biochemical changes may be interpreted as plant response to UV-B radiation stress and as an indicator of DNA damage. Hegazi and Hamid Eldin (2010) studied the effect of different gamma irradiation doses and water soaking on seeds of two okra varieties (Sabahia and Balady) using RAPD and SDS-PAGE. The RAPD results showed variation in DNA profile in response to gamma irradiation treatments. The percentage of polymorphism observed was 47.37% in var. Sabahia while, it was 50% in var. Balady. The relatively high doses of gamma irradiation (400 and 500 Gy) induced more changes in genomic DNA pattern than the low dose (300 Gy). In addition, the authors mentioned that these results are in good correspondence with Wendt et al. (2001) who used the RAPD markers to study the effect of gamma radiation on potato.

ISSR analysis
The 19 ISSR primers used in the present study produced good reproducible and scorable patterns (Fig. 4). As shown in Table (4), a total of 130 fragments were generated by the 19 primers with an average of 6.9 fragments/primer. Primer H2 yielded the highest number of products (13 amplicons), while primers (H5, 7, 9, 11 and 13) revealed the lowest number (4 amplicons). The number of polymorphic markers also varied between primers, ranging from 0 in primers (H5, 6, 7, 9 and 11) to 9 in primers (H2 and H4). This corresponds with a level polymorphism of 35.4%. The average number of polymorphic fragments/primer among the UV treatments and control was 2.4. (5), the estimated similarities among the control and 3 UV-B treatment durations ranged from 83.8 to 93.8. The highest genetic similarity (93.8) was between treatment No. 1 and treatment No. 3. While, the lowest genetic similarity (83.8) was between control and treatment No. 2. These results are in good agreement with our expected arrangement of these treatments.

As shown in Table
The dendrogram of genetic relationships from ISSR data clearly separated the control in a separate cluster while, all treated treatments (treatment No. 1, 2 and 3) were separated in the second cluster (Fig. 5).
The total polymorphic bands between each treament and control was 31, 35 and 24 in treatments 1, 2 and 3, respectively. These results revealed that the highest level of genetic variation was observed between the control and treatment No. 2 (40 min), reflecting the highest genomic changes. While, the lowest level of genetic variation was observed between the control and treatment 3 (60 min).
In this respect, Cuadra et al. (2010) analyzed the effects of UV-B radiation on Gnaphalium's DNA and on UV-absorbing compounds using five ISSR primers. Among these primers; the four ISSR primers selected for this analysis generated a total of 189 fragments. The dendrograms obtained using these markers efficiently separated plants from different treatments. The authors observed linear relationship between UV-B dose and percentage of dissimilarity which may be related to DNA damage caused by the different UV-B treatments.
On the other hand, Pestana et al.
(2011) evaluated the genetic variability in putative banana mutants irradiated with gamma ray, using a set of agronomical and ISSR markers. The ISSR analysis using 19 primers yielded a total of 186 polymorphic bands, where, 74 were monomorphic with an average of 9.8 total bands and 5.8 polymorphic bands, respectively. The authors concluded that these results can be used successfully in the genetic breeding program of banana aiming to develop new varieties in a short time.
Meanwhile, Khatri et al. (2011) used 11 ISSR primers to study genetic variation among the radiated population in banana. ISSR primers produced 37 bands in all the clones studied (two monomorphic and 35 polymorphic bands). The number of fragments produced by the primers ranged from 2-5, with an average of 3.45 fragments. Genetically most similar genotypes were G-215MP3 and G-215MP4 (89.8%) while, most dissimilar genotypes were Basrai and G-215MP2 (38.5%). Based on similarity matrix, clones were divided into three clusters and two groups.
In addition, Mudibu et al. (2011) aimed to determine the effect of irradia-tion with gamma rays on the genetic variability using ISSR markers in different soybean varieties. The results showed that the level of polymorphic loci among the soybean varieties varied from 70 to 90%. The authors noticed that the increasing of gamma rays dose consequently increased the level of polymorphism in progenies by 10%.

SCoT analysis
In the present investigation, eighteen SCoT primers were employed to investigate the genetic polymorphism among the three tomato treatments and the control. As shown in Table (6) and Fig.  (6), all of the 18 primers detected polymorphic patterns and revealed a total number of 182 amplicons with 43.0% level of polymorphism. The number of amplicons per primer ranged from 6 to 18 with an average of 12.1 amplicons per primer. The number of monomorphic amplicons varied from 3 to 12 with an average of 6.9 monomorphic amplicons per primer. However, the number of polymorphic amplicons varied from 1 to 12 with an average of 5.2 polymorphic amplicons per primer.
As shown in Table (7), the estimated similarities among the three tomato treatments ranged from 79.8 to 89.9. The highest genetic similarity (89.9) was be- The dendrogram of genetic relationships from SCoT data clearly separated the control in a separate cluster from all treatments (Sample No. 1, 2 and 3) which are placed in a second cluster (Fig. 7).
In this respect, since SCoT marker was recently innovated by Collard and Mackill (2009), till now, up to our knowledge, there is no published paper reporting the use of SCoT marker to assess the genetic changes that happened after plant irradiation.
However, many studies employed SCoT as a novel molecular marker technique to evaluate and study the genetic relationships between closely related genotypes. In this manner, Xiong et al. (2011) used start codon targeted polymorphism technique to study genetic diversity and relatedness among 20 accessions of four major botanical varieties of peanut. Out of 36 primers screened, 18 primers could produce unambiguous and reproducible bands. All 18 primers generated a total of 157 fragments, with a mean of 8.72 ranging from 4 to 17 per primer. Of 157 bands, 60 (38.22%) were polymorphic. One to seven polymorphic bands were amplified per primer, with 3.33 polymorphic bands on average. Polymorphism per primer ranged from 14.29 to 66.67%, with an average of 36.76%.
On the other hand, Luo et al. (2011) employed two molecular marker systems (SCoT and ISSR) in order to identify and detect the genetic relatedness between 23 of mango germplasms. Using 18 selected SCoT primers, 158 bands were generated, of which 104 (65.82%) were polymorphic.

Genetic relationships among the different tomato treatment plants as revealed by combined data
The scoring data derived from the three marker types (RAPD, ISSR and SCoT) were combined and computed to generate more accurate relatedness among the three tomato treatments. The range of pair similarity coefficient between them ranged from 79.5 to 89.5 (Table 8). The lowest similarity coefficient (79.5) was observed between control and sample No. The cluster analysis using the different marker systems revealed three dendrograms (Figs. 3, 5 and 7). All dendrograms clearly discriminated between the control and irradiated treatments except RAPD dendrogram. Therefore, to obtain a more accurate cluster analysis based on large genome coverage, a dendrogram was constructed using the combined data obtained from the different types of markers (RAPD, ISSR and SCoT). As shown in Fig. (8), this dendrogram, based on the combined data, clearly clustered the control and studied tomato treatments into two main clusters. The first cluster comprised only the control while, the second cluster comprised the rest of the three irradiated durations. This could be attributed to the higher level of genome coverage achieved by combining data of three types of markers than when using each marker separately. Similar conclusion was drawn by Adawy et al. (2004), Agrama and Tuinstra (2003) and Hussein et al. (2006) who indicated that the combined dendrogram can clearly discriminate among close genotypes. Thus, combined analysis of RAPD, ISSR, and SCoT data provide a quick, reliable and highly informative system for DNA fingerprinting and also allows establishing genetic relationships in many crops. In conclusion, induced mutations using physical mutagens is one way to create genetic variation resulting in new varieties with better characteristics. The genetic variability caused by mutations has been used in plant improvement programs that enable plant breeders to select new genotypes with improved characteristics such as abiotic and biotic tolerance, grain yield and quality. In order to understand the significance of change due to increased UV-B radiation, future research on plant effects should focus on basic research including biochemical and physiological mechanisms conducted in the laboratory, also it is advisable to stop man made contamination of the stratospheric ozone to keep living organisms safe on earth.

SUMMARY
Measurement of ozone level proved that stratospheric ozone layer is being depleted as a result of contamination with man-made chloroflurocarbon. One component of global climate change is the loss of stratospheric ozone which protects the earth from UV radiation. UV is traditionally divided into three wavelength ranges: UV-A, UV-B and UV-C. UV-C radiation is completely absorbed by atmospheric gases. UV-B is additionally absorbed by stratospheric ozone and thus only a very small proportion is transmitted to the earth's surface, whereas UV-A radiation is hardly absorbed by ozone.
Organisms on earth are adapted to UV-A, but may not be adapted to UV-B. Now and due to the reduction in atmospheric ozone, a great deal of attention has been focused on UV-B induced photo damage organisms. A 10% decline in the total stratospheric ozone would raise the amount of UV-B reaching the earth's surface by 20%.
UV radiation has been shown to be harmful to living organisms, damaging DNA, lipids, protein and membrane. Plants, which use sunlight for photosynthesis, cannot avoid exposure to increased levels of UV radiation so they are at higher risk. In order to understand the significance of change of climate to increased UV-B radiation. Our work has demonstrated the severe effect of UV on tomato cytological parameters as well as on the level of DNA using different molecular marker assays.
Tomato seeds (Castle Rock variety) were germinated, then at age of three weeks; they were exposed to UV-B radiation during 0, 20, 40 and 60 min periods. Mitotic abnormalities and mitotic index were analyzed. Exposure to UV-B radiation slightly affected the mitotic index but induced both clastogenic and aneugenic effects. UV-B radiation increased the frequency of mitotic abnormalities in a timedependent manner. Changes in DNA fingerprint was monitored in UV irradiated seedlings using RAPD, ISSR and SCoT molecular marker assays.        7): Dendrogram for the three treatments of tomato constructed from the SCoT data using Un-weighed Pair-group Arithmetic average (UPGMA) and similarity matrices computed according to Dice coefficient. Where C represents the control and 1, 2 and 3 represent 20 min, 40 min and 60 min. exposure of plants to UV-B, respectively.