ReviewChromosome aberration assays in Pisum for the study of environmental mutagens
Introduction
The genus Pisum L. is a member of the tribe Vicieae of the Fabaceae family. The genus is regarded as comprising only two species, the cultivated Pisum sativum and a wild species, Pisum fulvum Sibth. and Sm. [1]. The garden pea, native to Eurasia, has been grown as an agricultural crop in the Near East since early Neolithic times (7500–6000 b.c.) [210]. The pea plant is now the fourth most important seed legume, with an estimated total world production of approximately 20 million tonnes [58]. Several authors have treated historical developments beyond the scope of this paper [25], [40], [110], [184].
P. sativum has been used as a test organism in the Soviet space flight Salyut 4 [49]. Reduced or inhibited cell division has been observed in seedlings of P. sativum grown in space and a depression in the rate of mitoses and an increase in cell elongation. Additional reference to the use of P. sativum in space flights may be found in [82].
Blixt [22], [23] has described the ontogeny of the pea plant from the development of the seed to that of the inflorescence. The pod contains, on average, from 4 to 10 seeds depending on genotype. The plant is a self-fertilizing cleistogamous annual; i.e. pollination takes place in the closed flower buds, permitting very little cross-pollination. As a result, there are numerous true breeding lines with a wide range of morphological variation. Fasciated strains do not have meiotic irregularities and can be genetically inherited in a true breeding form [203]. Hybrids between different lines are well adapted to experimental research as they are fertile and no sterility barriers exist. These attributes of the pea plant were early recognized by Gregor Mendel as highly desirable for carrying out hybridization studies and led Mendel to select the pea plant for his studies [122].
The pea was one of the first plants to have its somatic chromosome number identified (2n=14) [28] and likewise its karyotype analyzed [105], [113]. In both hybrids that Mendel studied no abnormal mitoses were observed [28]. A method for the preparation and isolation of chromosome suspensions from P. sativum root tips has been developed [76]. Chromosome rearrangements are common in pea and have been identified by in situ hybridization with chromosome-specific DNA probes [179]. About 2500 genes have been identified from spontaneous and induced mutants of which 259 have been assigned to one of the seven linkage groups [25]. RFLP mapping of the genome has helped resolve genic relationships [52].
In 1982, the US Environmental Protection Agency Plant Gene-Tox Committee reviewed and summarized the data on a number of plant cytogenetic assays as part of the US EPA’s Gene-Tox Program [35]. A genus that was considered for inclusion in the review was Pisum as one species, P. sativum, had been used in a number of cytogenetic and mutation studies. To meet deadlines with the species under review [35], the analysis planned for Pisum was not carried out at that time.
The papers included in this review were selected on criteria formulated by the Gene-Tox Committee. In brief, only papers dealing with well-defined chemical compounds were included and papers in which control data were not provided were excluded. Selected were a total of 75 papers that reported the cytogenetic effects of 122 chemicals (Table 1). In addition, Table 2 lists 17 phenols and aromatic organophosphates and Table 3 lists 22 surfacants that show antimitotic effects. The results of assays with 38 chemicals and/or radiations in combined treatments, as well as 15 chemicals and three types of radiations that induce somatic mutations are tabulated.
Section snippets
Description of the karyotype
P. sativum was one of the first plants to have its somatic chromosome number identified (n=7; 2n=14) [28] and likewise its karyotype analyzed [105]. According to Blixt [21], the first correct interpretation of the morphology of the chromosomes was that by Kihlman in 1952 [92]. A drawing of the chromosomes of P. sativum is given in Fig. 1. The normal (type) karyotype of P. sativum is represented by seed line WL-110 of the Weibullsholm collection [21] (designated line JI 4 in the John lnnes
The assays
Plants of P. sativum can be used for testing for potential mutagens in both somatic and meiotic cells. Root tips of germinating seed and seedlings of young plants lend themselves to such studies. Bowen and Wilson [26] established the Pisum test which consists of treating primary root tips of young pea seedlings with specific amounts of chemical for specific times with the collection of meristematic material for cytological examination at specific intervals. The meristems are examined for
Seed preparation
Fresh seeds of P. sativum should be used as seeds 4-year-old kept under laboratory conditions have a 75% reduction in germinability and a five- to six-fold increase in chromosomal exchanges [36]. Seeds may be treated with the putative mutagen directly or presoaked in water (12/24 h) prior to treatment. If it is considered necessary to surface sterilize the seeds for mold, this can be done by placing the seed up to 60 min in a 1% sodium hypochlorite solution (or a 20% solution of commercial bleach
Survey of Pisum literature
The chromosomes of P. sativum do not vary greatly in size and are best distinguished by arm-length ratios and the presence of satellites [21], [22]. The two pairs of chromosomes with satellites (IV and VII) may be distinguished on overall size and satellite size (see Fig. 1, Fig. 2). Pisum chromosomes have heterochromatic–euchromatic junctions similar to those in V. faba [43] which are remarkably sensitive to induced isochromatid breaks [92]. In studies too extensive to be reported here, Rosen
Test performance
Vo Hung [200] found a treatment of 0.1% concentration of El for 12 h and a 0.5% concentration of EMS for 12 h induced chromosome aberrations in mitotic cells but not in meiotic cells (Table 1). On the other hand, Nigam and Kumar [142] found a 0.1% for 1.5 h induced chromosome aberrations in both mitosis and meiosis. The discrepancy would appear to be differences in experimental conditions. Likewise, duration of treatment was found to influence results. For example, Hyypio et al. [84] found that
Correlation of clastogenicity, mutagenicity and carcinogenicity
A comparison of clastogenicity for the chemicals with that of the Salmonella and carcinogenicity data is given in Table 11 and the results are as follows (Table 11): for the Salmonella comparison, 17 chemicals positive for clastogenicity are negative in the Salmonella test; seven chemicals positive for clastogenicity are positive in the Salmonella test; one chemical negative for clastogenicity is also negative in the Salmonella test; one chemical (phenol) has been reported positive for
Advantages
P. sativum has been used for studying all the cytological endpoints that follow treatment of chromosomes by chemical and physical agents. These include breaks and exchanges which involve chromatid and chromosome aberrations and rearrangements, fragments, bridges, laggards, spindle disturbances and c-mitotic effects. In addition to somatic cells, P. sativum has the advantage that meiotic chromosome aberrations can be readily studied from both seed treatment and from spraying young plants. The
Recommendations
The Pisum assay readily detects clastogens and mutagens. It is therefore recommended that P. sativum bioassay be used to assess genotoxicity from potential chemical and environmental pollutants. It is considered that the meiotic micronucleus assay for assaying the cytogenetic effects of chemicals and radiations (as presently carried out in Tradescantia) has been overlooked in the Pisum assay [109]. Likewise, the Pisum assay should be investigated for in situ testing and monitoring from
Acknowledgements
The authors acknowledge Dr. Roswitha Haas for assistance in the classification of the chemicals.
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