**1. Introduction**

Plants are the essential elements of agriculture and forestry and maintain the healthy environment for the rest of the species by producing oxygen and organic carbon compounds. Higher plants are preeminent indicators of genotoxic effects caused by chemical substances existing in the environment and therefore be utilized for detecting environmental mutagens [1]. They are

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exposed to many stress factors including chemical compounds and radiation affecting their seed germination, seedling growth, and floral and fruit development. These stress factors can adversely affect the quality and quantity of the product with leading to morphological, anatomical, physiological, biochemical, and molecular damage to plants [2]. There are different kinds of methods for examining phytotoxicity and genotoxicity because usually there is no standard national procedure. Therefore, the parameters of these methods vary depending on the test substances, the test plants, or the individual procedures. Because of its simplicity, low cost, and relatively high sensitivity, application of plant bioassays is usually favored over other available systems in discovering adverse effects caused by chemical substances, or pollution, existing in the environment [3]. Despite these benefits described above, there are also some limitations in using plant bioassays, such as the longer life span of plants than *Escherichia coli* T. Escherich, *Salmonella typhimurium* Lignieres, *Saccharomyces cerevisiae* Meyen ex E.C. Hansen, or *Drosophila melanogaster* Meigen; likewise, there are differences between the biochemistry of plants and mammals. Nevertheless, positive correlation results have been observed between plant and mammalian systems in many reports, supporting the preference of plant bioassays in these studies [4]. Hence, plant bioassays are commonly used for screening and monitoring environmental chemicals with mutagenic and carcinogenic potential [5, 6]. The International Program on Chemical Safety (IPCS) makes and supports research programs all around the world and develops methodologies for chemical exposure [4, 7]. Many laboratories from diverse regions of the world have been sponsored by IPCS and participated in evaluating the utility of several plant bioassays for detecting the mutagenicity of environmental chemicals [8]. By means of these studies, many methods were developed to assess toxicity in plants. Some of the recent studies with plant bioassays can be seen in **Table 1**.

Plant bioassays are usually based on the detection of chromosomal abnormalities in mitosis, sister chromatid exchanges (SCEs), and, recently, on the DNA damage analysis. Point mutations


**Table 1.** Some of the recent studies with plant bioassays.

such as chlorophyll mutations in leaves, waxy mutations, or embryo mutations of *Arabidopsis* are the other detection methods [17]. Seed germination, root elongation, EC50 (the concentration that lowers %50 of the root length) determination, mitotic index, chromosomal abnormalities in different phases of mitosis, seedling growth, and enzyme activity during germination are the preliminary investigations for plant bioassays. In this chapter, some of the most frequently and recently used methods for detection of genotoxicity with plant biosystems are reviewed.

### **2. Seed germination and root elongation tests**

exposed to many stress factors including chemical compounds and radiation affecting their seed germination, seedling growth, and floral and fruit development. These stress factors can adversely affect the quality and quantity of the product with leading to morphological, anatomical, physiological, biochemical, and molecular damage to plants [2]. There are different kinds of methods for examining phytotoxicity and genotoxicity because usually there is no standard national procedure. Therefore, the parameters of these methods vary depending on the test substances, the test plants, or the individual procedures. Because of its simplicity, low cost, and relatively high sensitivity, application of plant bioassays is usually favored over other available systems in discovering adverse effects caused by chemical substances, or pollution, existing in the environment [3]. Despite these benefits described above, there are also some limitations in using plant bioassays, such as the longer life span of plants than *Escherichia coli* T. Escherich, *Salmonella typhimurium* Lignieres, *Saccharomyces cerevisiae* Meyen ex E.C. Hansen, or *Drosophila melanogaster* Meigen; likewise, there are differences between the biochemistry of plants and mammals. Nevertheless, positive correlation results have been observed between plant and mammalian systems in many reports, supporting the preference of plant bioassays in these studies [4]. Hence, plant bioassays are commonly used for screening and monitoring environmental chemicals with mutagenic and carcinogenic potential [5, 6]. The International Program on Chemical Safety (IPCS) makes and supports research programs all around the world and develops methodologies for chemical exposure [4, 7]. Many laboratories from diverse regions of the world have been sponsored by IPCS and participated in evaluating the utility of several plant bioassays for detecting the mutagenicity of environmental chemicals [8]. By means of these studies, many methods were developed to assess toxicity in plants.

10 Plant Ecology - Traditional Approaches to Recent Trends

Some of the recent studies with plant bioassays can be seen in **Table 1**.

*Triticum aestivum* L. Aniline Micronucleus, mitotic index, and

Volatile organic compounds

**Table 1.** Some of the recent studies with plant bioassays.

*Tradescantia pallida* (Rose) D.R.Hunt var. *purpurea*

*Oryza sativa* L. var *nipponbare*

*Epipremnum aureum* (Linden & André) G.S.Bunting

*pendulum*

*Capsicum baccatum* L. var.

**Plant species Test substance Method Reference** *Vicia faba* L. Wastewater Micronucleus method Liu et al. [9]

Plant bioassays are usually based on the detection of chromosomal abnormalities in mitosis, sister chromatid exchanges (SCEs), and, recently, on the DNA damage analysis. Point mutations

> Pesticide Micronucleus and stamen hair bioassays

*Vicia faba* L. Insecticide Sister chromatid exchange Quintana et al. [12]

*Acalypha indica* L. Lead stress RAPD-PCR Venkatachalam et al. [16]

chromosomal aberration

Mercury Real-time PCR FISH Zhen et al. [13]

Ionizing radiation TUNEL test Scaldaferro et al. [14]

Comet assay Naroi-et et al. [15]

Fadic et al. [10]

Tao et al. [11]

Many plant species have been recommended for ecotoxicity tests using seed germination and root elongation methods. Among them, cabbage, lettuce, and oats are recommended by the US Environmental Protection Agency (EPA) (1983) [18], the Organization for Economic Cooperation and Development (OECD) (1984) [19], and the Food and Drug Administration (FDA) (1987) [20]. Carrot, cucumber, and tomato are also suggested by the EPA and FDA, wheat is accepted by the FDA and OECD, and rice is also mentioned by the OECD. Although not mentioned in any of these documents, millet has been studied at the Illinois State Water Survey for several years [21]. Most frequently used species are *Allium cepa* L., *Lactuca sativa L.*, *Glycine max* (L.) Merr, *Avena sativa* L., *Hordeum vulgare* L., *Pisum sativum* L., *Tradescantia pallida* (Rose) D.R.Hunt , *Vicia faba* L., and *Zea mays* L. The crucifer *Arabidopsis thaliana* (L.) Heynh. is used only for mutation studies as its chromosomes are very small, and the total genome contains only about 70,000 kb in contrast to over a million kilobases in most other plants. The test substance, test duration, test organisms, the species and number of organisms, concentration of the test substance, replicates, randomization, equipment, reliability, environmental conditions (temperature, humidity, watering, lighting, photoperiod, and nutrients), observations, measurements, and analysis of the test results must be done carefully. The seed germination and seedling growth bioassays are more sensitive to separate plant developmental life stages as they integrate the effects of many environmental stress factors on both germination and seedling growth stages, respectively. The early seedling development is a more sensitive endpoint than the seed germination that depends on the energy reserves in cotyledons. Many researchers also found that the different kinds of species used do not respond similarly to toxic chemicals [22, 23]. Seed germination and plant growth bioassays are the most common techniques used to evaluate the toxicity of pesticides [24–27], heavy metals [6], allelochemicals [28], personal care products [29], compost [30], water samples taken from rivers [31], and industrial waste waters [25, 32]. Different plant species have also been used such as cucumber and cress [33], lettuce and soybean [34], red maple, sugar maple, white pine, and pink oak [35] for phytotoxicity tests.

### **3. Cytogenetic techniques**

The frequency and the type of chromosome abnormalities in different phases of mitosis and the micronuclei frequency of interphase cells are analyzed by cytogenetic tests. The DNA damage caused by the genotoxic agents could either be repaired or otherwise could be lead to the DNA alterations. Chromosome abnormalities are the results of DNA double-strand breaks that were unrepaired or inaccurately repaired. Chromosomes are rearranged since broken chromosome ends become "sticky" and may combine with other broken chromosome ends. After mutagenic treatment, because of the chromosomal rearrangements and acentric fragments, dicentric bridges could be observed in mitotic cells of the first cell cycle. Micronuclei frequency also decreases in the interphase cell in the next cell cycle [36]. The micronucleus (MN) test, *A. cepa* and *V. faba* chromosome aberration test, and the T. MN tests have been recommended as the validated plant bioassays for laboratory testing and in situ monitoring of the genotoxicity of environmental mutagens [7]. Sister chromatid exchange (SCE) test can also be used to detect effects of small doses of pollutants; thus, it is adequate for initial genotoxicity evaluation tests [37]. SCEs result from alterations caused in the gene expression and by the loss of heterozygosity. SCE experiments are traditionally performed and well studied in mammalian cells. For plants, the protocols have been mainly developed in *V. faba* root cells [38].

### **3.1.** *Allium***/***Vicia* **chromosome aberration test**

Several mutagens can be detected cytologically by cellular inhibition; disruption in metaphase; induction of chromosomal aberrations, numerical and structural, ranging from chromosomal fragmentation to the disorganization of the mitotic spindle; and consequently all subsequent dependent mitotic phases. The microscopic analysis includes mitotic index, micronuclei presence in interphase cells, and chromosomal aberrations in late anaphase and early telophase cells score. Approximately 1000 cells from all the stages of dividing cells in mitosis are counted in order to find the mitotic index value. Chromosomal abnormalities can be determined, and then, they are scored in the first 100 cells in different stages of mitotic division. The mostly used method to determine all of the abnormalities is to scan the slides from right to left, up, and down [39]. The *Allium* material is well known and has been used for the study of basic mechanisms as well as for scoring the effects of chemicals. *A. cepa* (the common onion) has proved to be the most useful and has repeatedly been suggested as a standard test material [40]. The use of *A. cepa* as a test system was introduced by Levan [41], when the effects of colchicine were investigated. Since then, the *Allium* test has been frequently used. Genotoxicity, cytotoxicity, and chromosome abnormalities in plant biosystems are mostly determined in *A. cepa* (2n = 16) and *V. faba* (2n = 12). They are efficient test organisms because of their availability throughout the year, ease of handling, and cultivation. They also do not need to be cultivated in sterile conditions; they have large and small number of chromosomes, which makes the observation of chromosomal damages in the mitotic cycle easier [42]. The *Allium* test has high sensitivity and good correlation when compared with the mammalian test systems. Ma and Grant [43] suggested including *Allium* test as a standard test system to determine chromosome damages induced by chemicals after the evaluation of 148 chemicals by the *Allium* test since 76% presented positive results. It was reported that the sensitivity of the *Allium* test was practically similar as the one observed for human lymphocyte and algae test systems. Rank and Nielsen [44] showed that the *Allium* test was more sensitive than the MicroScreen and the Ames tests. They also reported that there was a correlation of 82% of the carcinogenicity test in rodents in relation to the *Allium* test. The *V. faba* MN test has been shown to be sensitive in evaluating chromosomal aberrations and assessing genotoxicity from both organic and inorganic soil contaminants [45], sediment [46], organic material such as sewage sludge or composts [47] and water [48, 49]. Many researchers compared sensitivity of the *V. faba* test with other bioassays, i.e., somatic mutation and recombination test (SMART), that utilizes *D*. *melanogaster* Meigen. and compared with the *V. faba* sister chromatid exchange (SCE) test and MN inductions. Both tests showed 62.5% similarity [38]. Plant genotoxicity assays as the MN test on *V. faba* roots provide quantitative, repeatable, and reliable mutagenic data, and they are sensitive tests to detect new environmental mutagens or combination of different kinds of mutagens [50]. They can be used to develop new techniques for alternative assays in the determination of possible genetic damage caused by environmental pollutants such as pesticides, heavy metals, and more recently personal or health-care products. They can also contribute to an in situ monitoring, which can be carried out on a global scale in media as aqueous biota or soils in relation to human activities [1].

### **3.2. Tradescantia stamen hair mutation and micronucleus analysis**

breaks that were unrepaired or inaccurately repaired. Chromosomes are rearranged since broken chromosome ends become "sticky" and may combine with other broken chromosome ends. After mutagenic treatment, because of the chromosomal rearrangements and acentric fragments, dicentric bridges could be observed in mitotic cells of the first cell cycle. Micronuclei frequency also decreases in the interphase cell in the next cell cycle [36]. The micronucleus (MN) test, *A. cepa* and *V. faba* chromosome aberration test, and the T. MN tests have been recommended as the validated plant bioassays for laboratory testing and in situ monitoring of the genotoxicity of environmental mutagens [7]. Sister chromatid exchange (SCE) test can also be used to detect effects of small doses of pollutants; thus, it is adequate for initial genotoxicity evaluation tests [37]. SCEs result from alterations caused in the gene expression and by the loss of heterozygosity. SCE experiments are traditionally performed and well studied in mammalian cells. For plants, the protocols have been mainly developed

Several mutagens can be detected cytologically by cellular inhibition; disruption in metaphase; induction of chromosomal aberrations, numerical and structural, ranging from chromosomal fragmentation to the disorganization of the mitotic spindle; and consequently all subsequent dependent mitotic phases. The microscopic analysis includes mitotic index, micronuclei presence in interphase cells, and chromosomal aberrations in late anaphase and early telophase cells score. Approximately 1000 cells from all the stages of dividing cells in mitosis are counted in order to find the mitotic index value. Chromosomal abnormalities can be determined, and then, they are scored in the first 100 cells in different stages of mitotic division. The mostly used method to determine all of the abnormalities is to scan the slides from right to left, up, and down [39]. The *Allium* material is well known and has been used for the study of basic mechanisms as well as for scoring the effects of chemicals. *A. cepa* (the common onion) has proved to be the most useful and has repeatedly been suggested as a standard test material [40]. The use of *A. cepa* as a test system was introduced by Levan [41], when the effects of colchicine were investigated. Since then, the *Allium* test has been frequently used. Genotoxicity, cytotoxicity, and chromosome abnormalities in plant biosystems are mostly determined in *A. cepa* (2n = 16) and *V. faba* (2n = 12). They are efficient test organisms because of their availability throughout the year, ease of handling, and cultivation. They also do not need to be cultivated in sterile conditions; they have large and small number of chromosomes, which makes the observation of chromosomal damages in the mitotic cycle easier [42]. The *Allium* test has high sensitivity and good correlation when compared with the mammalian test systems. Ma and Grant [43] suggested including *Allium* test as a standard test system to determine chromosome damages induced by chemicals after the evaluation of 148 chemicals by the *Allium* test since 76% presented positive results. It was reported that the sensitivity of the *Allium* test was practically similar as the one observed for human lymphocyte and algae test systems. Rank and Nielsen [44] showed that the *Allium* test was more sensitive than the MicroScreen and the Ames tests. They also reported that there was a correlation of 82% of the carcinogenicity test in rodents in relation to the *Allium* test. The *V. faba* MN test has been shown to be sensitive in evaluating chromosomal aberrations and assessing genotoxicity from both organic and inorganic soil contaminants [45], sediment [46], organic material such as

in *V. faba* root cells [38].

**3.1.** *Allium***/***Vicia* **chromosome aberration test**

12 Plant Ecology - Traditional Approaches to Recent Trends

The genus *Tradescantia*, from the Commelinaceae family, is a higher plant with more than 500 species. Some of these and their clones are used as genetic bioindicators for mutagenic activity, such as *T*. *pallida* (Rose) D.R.Hunt, for environmental monitoring. It has two assay systems, the *Tradescantia* sp. staminal hair assay and the *Tradescantia* sp. MN assay, developed by Ma [51]. Stamen hair and MN tests have been widely employed for genotoxic effect studies with *Tradescantia* species [43, 52]. Almost all of the parts of the *Tradescantia* species including the root tip and also the pollen tube in development provide the best plant materials for cytogenetic toxicity testing studies. *Tradescantia* species have 12 chromosomes which are easily observable. Sax and Edmonds observed that meiotic chromosomes in pollen development were more easily influenced to breakage than mitotic chromosomes. They especially reported that the dividing chromosomes within the cells at meiosis are approximately ten times more sensitive to breakage than those in the interphase cells [42].

Ma and Grant [43] have prepared a historical perspective, detailing the importance of this plant in mutation studies. Firstly, the heterozygosity for flower color in *Tradescantia* sp. clones was used for these studies, and then, the stamen hairs have been determined to be good indicators of mutations. Clone 4430 is a hybrid of *Tradescantia hirsutiflora* Bush. and *Tradescantia subacaulis* Bush. reproduced only asexually, through cloning. This test uses the stamen hairs of *Tradescantia* sp. inflorescences to evaluate the frequency of somatic mutation, induced for mutagens, through changes in the color of stamen hair cells from blue to pink, due to the expression of a recessive gene of these cells. The frequency of micronuclei in tetrad cells of male meiotic cells in *Tradescantia* induced by the tested mutagen was determined [42]. The *Tradescantia* sp. MN test may be used for in situ exposure conditions to evaluate air or water pollution or under laboratory conditions for testing radioactive or chemical agents [53, 54]. The *Tradescantia* sp. stamen hair mutation (Trad-SH) assay (clone 4430) was evaluated for its efficiency and reliability as a screen for mutagens in an IPCS collaborative study on plant systems. The results of the study confirm that the Trad-SH assay is an unsuspicious system for screening potential environmental mutagens. A survey of the current literature indicates that the Trad-SH assay could be used for in situ monitor of liquid, gaseous, and also radioactive pollutants as well although the study was carried out under laboratory conditions [55].

### **3.3. Sister chromatid exchange**

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 differentiated chromosomes [59, 60].
