**4. Molecular techniques**

### **4.1. Fluorescent in situ hybridization**

The classical cytogenetic techniques were usually used for detecting the changes in chromosomal number and morphology. However, chromosome staining with the traditional methods such as Feulgen or orcein staining can fail in the analysis of small changes in chromosome structure. The fluorescent in situ hybridization (FISH) allows the detection and a more detailed localization of chromosomal rearrangements, both in interphase and mitotic nuclei, which gives new possibilities to study chromosomal aberrations [61]. Additionally, it helps to reveal the mechanisms of the formation of chromosomal abnormalities in plant mutagenesis. Although there are a few number of DNA probes for particular plant chromosomes, *A. thaliana* is a good example when FISH employing chromosome region-specific DNA probes (e.g., centromere, telomere, rDNA) is helpful in chromosome aberration analysis. The translocations in chromosomes of tetraploid plants of *A. thaliana* have been detected by FISH [62]. The effects of maleic acid hydrazide on hairy root tip meristem cells of *C. capillaris* were studied with FISH using rDNA and telomeric sequences as a probe and spontaneous chromosomal rearrangements were determined [63]. It is also important to analyze the chromosomal rearrangements in interphase cells treated with mutagenic chemicals that may cause a decrease in the frequency of cell divisions. The basic steps of this procedure are the same as the other organisms, but several cytogenetic laboratories modified various techniques for plant cells.

### **4.2. TUNEL test**

**3.3. Sister chromatid exchange**

14 Plant Ecology - Traditional Approaches to Recent Trends

differentiated chromosomes [59, 60].

**4.1. Fluorescent in situ hybridization**

**4. Molecular techniques**

The sister chromatid exchange (SCE) test is developed from the semiconservative DNA replication model which we could see the separation of DNA. The cytogenetic monitoring of exposure to potential mutagens in the environment could be done by SCE which is a highly sensitive cytogenetic tool for detecting DNA damage. It involves firstly the breakage of both DNA strand and then an exchange of whole DNA duplexes. The symmetrical exchange during S phase at one locus between sister chromatids that does not alter chromosome length and genetic information is defined. Taylor was the first scientist who made the SCE test visualized for plant cells, but he used tritium and autoradiography, which provided poor spatial resolution [56]. After Taylor, it was discovered that sister chromatids could be differentiated and revealed SCEs in combination with Hoechst dye 33258 incorporation of the DNA base analog 5′-bromodeoxyuridine (BrdUrd) staining [57]. BrdUrd is a synthetic nucleoside that is an analog of thymidine and is actively incorporated into the newly synthesized DNA during replication process. It is commonly used in the detection of dividing cells in living organisms during the S phase of the cell cycle substituting for thymidine. The standard fluorescence plus Giemsa (FPG) staining method also will enable visualization of SCEs in metaphase spreads of growing cells in medium containing BrdUrd with a light microscope [56]. The frequency of SCEs per chromosome set increases after treatment with genotoxic agents. SCE method was first applied in mammalian cells, and later, it has been shown that it can be applied in plant cells. Especially plant species that have relatively large and a low number of chromosomes such as *A. cepa* and *V. faba* are used for SCE analysis [57, 58]. *Crepis capillaris* (L.) Wallr. is also a good material for analyzing the frequency of SCE with 2n = 6 chromosome number. It allows studying SCE frequency in each chromosome type, since it has three pairs of morphologically

The classical cytogenetic techniques were usually used for detecting the changes in chromosomal number and morphology. However, chromosome staining with the traditional methods such as Feulgen or orcein staining can fail in the analysis of small changes in chromosome structure. The fluorescent in situ hybridization (FISH) allows the detection and a more detailed localization of chromosomal rearrangements, both in interphase and mitotic nuclei, which gives new possibilities to study chromosomal aberrations [61]. Additionally, it helps to reveal the mechanisms of the formation of chromosomal abnormalities in plant mutagenesis. Although there are a few number of DNA probes for particular plant chromosomes, *A. thaliana* is a good example when FISH employing chromosome region-specific DNA probes (e.g., centromere, telomere, rDNA) is helpful in chromosome aberration analysis. The translocations in chromosomes of tetraploid plants of *A. thaliana* have been detected by FISH [62]. The effects of maleic acid hydrazide on hairy root tip meristem cells of *C. capillaris* were studied with FISH Another test used to identify apoptosis that has found application in plant genotoxicity studies is the terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) test [64]. TUNEL assay detects DNA fragmentation by the help of fluorescence microscope. TUNEL test is used to detect DNA damage associated with nonapoptotic events such as necrotic cell death induced by exposure to genotoxic chemicals. It is not limited to the detection of apoptotic cells [65] and has also ability to stain cells going through active DNA repair [66]. The regulated cell death plays an important role during development of plants, and it is also essential for plant-specific responses to biotic and abiotic stress factors. The terminal deoxynucleotidyl transferase catalyzes the polymerization of labeled nucleotides to DNA strand breaks in situ. For TUNEL test, successive hand-cut sections of each axis of embedded plant material are stained with propidium iodide (PI) in order to stain the nuclei of dead cells to red and DAPI (4',6-diamidino-2-phenylindol) which can pass through the normal cell membrane and stains the nuclei to blue. DAPI can be used to stain both live and fixed cells. The detection of DNA breaks at a single nucleus can be achieved with TUNEL test within a short time, and the screening of labeled nuclei is easier than other methods. It is recommended for the preliminary genotoxicity investigation of the new identified chemicals [67].

### **4.3. Single-cell gel electrophoresis (comet assay)**

DNA damage in higher plant cells was evaluated by the frequency of chromosomal aberrations in metaphase chromosomes, abnormal anaphase and telophases, and micronuclei; however, these tests measure unrepaired genome damage in cells which have reached mitosis. DNA damage may be originated from DNA metabolism spontaneously or from the effects of environmental factors. There are different kinds and levels of DNA repair mechanisms in cell nucleus to prevent these damages. When the repair mechanisms are ineffective or there was a heavy DNA damage, it may lead to the inhibition of replication, transcription, or protein synthesis; however, in the long term, chromosomal abnormalities or mutations could be formed. It is a sensitive and fast fluorescent technique, which is used to determine the amount of DNA damage on single cell level. After its introduction as "alkaline comet assay," it has been developed with many modifications for investigating the process of apoptosis and became a workable technique for detecting a variety of DNA damages in plant cells. It allows the determination of double- and single-stranded DNA breaks in a single cell and also helps to measure the level of the migration of DNA by using horizontal gel electrophoresis system [68]. The length of the tail and the amount of the DNA in the head and in the tail are measured to assess the toxicity in a computerized image analysis system. The tail moment (TM) can be calculated to show DNA damage [69]. The comet assay allows fast detection of DNA damage, shortly after the injury, before DNA is repaired, and without any need to wait for progression into mitosis [70]. The presence of a cell wall and the absence of free cells in plant tissues cause technical difficulties for performing the comet assay. Over the past few years, many scientists have improved the methodology for the comet assay on plant cells. Navarrete et al. [70]. developed a simple and efficient mechanical extraction to isolate cell nuclei to overcome these problems. This technique was then improved by Gichner. The different internal parameters such as nucleus isolation methods, filtration and lysis steps, agarose concentration, and the external parameters such as room temperature and light intensity were evaluated during these studies [71].

### **4.4. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique**

RAPD-PCR is a PCR-based and quite reproducible technique that yields information on a large number of markers without having to obtain DNA sequence information for primer design [72]. Many scientists used RAPD-PCR technique commonly for a variety of purposes such as cultivar identification, genetic diversity assessment, and the construction of phylogenetic relationships [73], and it has been successfully utilized in genotoxicity identification of toxic chemicals. A number of selective and sensitive assays for DNA analysis in ecotoxicology have been developed with the improvement of recent molecular biology techniques. DNA-based techniques such as RFLP, QTL, RAPD, AFLP, SSR, and VNTR are being used to investigate the variations at the DNA sequence level. RAPD-PCR can be used to detect genotoxicity, and differences in RAPD profiles can clearly be shown when comparing DNA fingerprints from untreated and treated individuals to genotoxic agents [74]. Many studies support the view that the RAPD analysis is a highly sensitive method for the detection of DNA damage induced by environmental pollutants like toxic chemicals. RAPD markers are at this moment low valuable markers due to the lack of repeatability. A few work is usually published at this moment using this kind of markers. This kind of study using other DNA markers will be of much more interest.

### **4.5. Real-time polymerase chain reaction (RT-PCR) technique**

Plants have risk of DNA damage due to continuous exposure to environmental mutagens, and thus a variety of repair mechanisms should operate to maintain genome integrity. *A. thaliana* is a mostly studied plant for the repair mechanisms after exposure to several mutagens such as UV-B radiation [75], heavy metal contamination [76], and wound stress [77]. In the first step of the DNA damage response, DNA lesions or replication inhibition must be detected. The DNA damage response is controlled by the activation of several regulatory kinases and also checkpoint proteins that lead to specific cell cycle arrests as well as changes in the chromatin structure at the site of DNA damage. The transcriptional regulation of the genes could be determined by RT-PCR in order to evaluate the mechanism of plant response to genotoxic agents. To investigate effects of mutagens on the transcript levels of some gene-encoding antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), they were studied in Ref. [78]. The mutants of *A. thaliana* that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated to investigate the respond of plants and its pathways to UV radiation. UVR2 and UVR3 products were previously identified as photolyases that remove UV-induced pyrimidine dimers in the presence of visible light [76]. Hu et al. (2007) investigated the role of calmodulin (CaM) and the relationship between CaM and hydrogen peroxide (H2 O2 ) in abscisic acid (ABA)–induced antioxidant defense in leaves of *Z. mays* [78].

### **5. Flow cytometry**

without any need to wait for progression into mitosis [70]. The presence of a cell wall and the absence of free cells in plant tissues cause technical difficulties for performing the comet assay. Over the past few years, many scientists have improved the methodology for the comet assay on plant cells. Navarrete et al. [70]. developed a simple and efficient mechanical extraction to isolate cell nuclei to overcome these problems. This technique was then improved by Gichner. The different internal parameters such as nucleus isolation methods, filtration and lysis steps, agarose concentration, and the external parameters such as room temperature and light intensity were

**4.4. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR)** 

markers. This kind of study using other DNA markers will be of much more interest.

Plants have risk of DNA damage due to continuous exposure to environmental mutagens, and thus a variety of repair mechanisms should operate to maintain genome integrity. *A. thaliana* is a mostly studied plant for the repair mechanisms after exposure to several mutagens such as UV-B radiation [75], heavy metal contamination [76], and wound stress [77]. In the first step of the DNA damage response, DNA lesions or replication inhibition must be detected. The DNA damage response is controlled by the activation of several regulatory kinases and also checkpoint proteins that lead to specific cell cycle arrests as well as changes in the chromatin structure at the site of DNA damage. The transcriptional regulation of the genes could be determined by RT-PCR in order to evaluate the mechanism of plant response to genotoxic agents. To investigate effects of mutagens on the transcript levels of some gene-encoding antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), they were studied in Ref. [78]. The mutants of *A. thaliana* that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated to investigate the respond of plants and its pathways to UV radiation. UVR2 and UVR3 products were previously identified as photolyases that remove UV-induced pyrimidine dimers in the presence of visible light [76]. Hu et al. (2007)

**4.5. Real-time polymerase chain reaction (RT-PCR) technique**

RAPD-PCR is a PCR-based and quite reproducible technique that yields information on a large number of markers without having to obtain DNA sequence information for primer design [72]. Many scientists used RAPD-PCR technique commonly for a variety of purposes such as cultivar identification, genetic diversity assessment, and the construction of phylogenetic relationships [73], and it has been successfully utilized in genotoxicity identification of toxic chemicals. A number of selective and sensitive assays for DNA analysis in ecotoxicology have been developed with the improvement of recent molecular biology techniques. DNA-based techniques such as RFLP, QTL, RAPD, AFLP, SSR, and VNTR are being used to investigate the variations at the DNA sequence level. RAPD-PCR can be used to detect genotoxicity, and differences in RAPD profiles can clearly be shown when comparing DNA fingerprints from untreated and treated individuals to genotoxic agents [74]. Many studies support the view that the RAPD analysis is a highly sensitive method for the detection of DNA damage induced by environmental pollutants like toxic chemicals. RAPD markers are at this moment low valuable markers due to the lack of repeatability. A few work is usually published at this moment using this kind of

evaluated during these studies [71].

16 Plant Ecology - Traditional Approaches to Recent Trends

**technique**

Flow cytometry (FCM) is a rapid and multiparametric technique that theoretically has the potential to detect minute variations in nuclear DNA (nDNA) content, as well as chromosomal damage, in exposed organisms. It can also provide information on polyploidization and evaluate cell cycle dynamics in plants. Pfosser et al. [79]. evaluate the sensitivity of FCM by detecting the variations in DNA content as small as 1% in aneuploid wheat-rye lines. Relatively to DNA damage, Rayburn and Wetzel correlated the coefficient of variation of the G0/G1 peak with chromosomal aberration in aluminum-exposed plants, as this parameter is able to detect broken and rearranged chromosomes in daughter cells [80]. Monteiro et al. also detected an increase in the full peak coefficient of variation (FPCV) of the G0/G1 peak of lettuce plants exposed to Cd [81].

### **6. Conclusion**

Hundreds of new industrial chemicals have been continuously produced to facilitate our lives, and we are not able to be aware of their damage before we investigate their effects on organisms. Plant bioassays serve as a tool to demonstrate the cytotoxic and genotoxic effects of environmental pollutants by means of clear-cut evidence of chromosome damage and gene mutation. These studies could also be useful to establish a database for environmental conditions in the various regions of the world. Some of these simple and clear-cut indicators revealed by plant bioassays could also be used to demonstrate the genotoxic effects of environmental pollution to the general public. The kind of education that is required is not only about teaching people how to detect and eliminate pollutants but also to educate the general public on the root cause of pollution problems. Pollution is related to every facet of human life, and it is life itself that generates pollution. Regulations and guidelines are essential to cure the symptoms of pollution. Plant bioassay studies deserve to be included by the enforcement agencies, particularly of the developing countries, for their regular monitoring of pollution sites.

### **Author details**

### Özlem Aksoy

Address all correspondence to: ozlem.aksoy@kocaeli.edu.tr, odalgic@yahoo.com

Department of Biology, Faculty of Science and Literature, Kocaeli University, İzmit, Kocaeli, Turkey
