Specific-locus mutation assay in Zea may

Mutanon Research, 99 (1982) 317- 337

317

Elsevier Biomedical Press

Specific-locus mutation assays in Zea mays A report of the U.S. Environmental Protection Agency Gene-Tox Program * Michael J. Plewa Institute for Envtronmental Studws, Umverstty of llhno~s, Urbana, IL 61801 (U.S.A.) (Received 7 April 1982) (Accepted 8 April 1982)

Summary The use of the angiosperm Zea mays in assays for the induction of mutations at specific genetic loci is discussed. The effect of chemical mutagens on 3 genetic end points is evaluated. The end points surveyed were the loss of the phenotype of dominant alleles in heterozygotes, the induction of segregating point mutations in M 2 plants, and the induction of forward and reverse mutations in pollen grains. Maize is sensitive to a wide range of mutagens and has the capacity to activate promutagens. This plant has been used to study mutagenesis under both laboratory and in situ conditions.

Introduction Organism

Indian corn or maize (Zea mays U) is an angiosperm that belongs to the grass family, the Gramineae. Maize is a cultivated grain and a major agricultural product By acceptance of this article the publisher or recipient acknowledges the U.S. Government's right to retain a nonexclusive, royalty-free license in and to any copyright covering the article. * Work Group Report prepared for the Gene-Tox Program (Office of Toxic Substances, Office of Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC). The author is a member of the Gene-Tox Work Group on Higher Plant Genetic and Cytogenetlc Assays. Although the review described in this article has been funded wholly or in part by the United States Environmental Protection Agency through Interagency Agreement DOE 40-1123-80, EPA No. 80-DX0953, to the Oak Ridge National Laboratory, it has not been subjected to the Agency's required peer and policy review and, therefore, does not necessarily reflect the views of the Agency and no official endorsement should be inferred. The protocols stated, suggested use of the assay in a screening program, and research recommended should not be taken to represent Agency policy on these matters. 0165-1110/82/0000-0000/$02.75 © Elsevier Biomedical Press

318 of the United States Oiler, 1978). Although the origin of maize is obscure, good evidence exists that it was derived from a closely related plant, teosinte (Zea rnexicana), in what is now Mexico and Central America (Beadle, 1978; Galinat, 1971. 1978). Presumably the Indians selected mutants that were desirable and thus Z. ma.vs evolved. One can assert that the species is a man-made plant. A strong argument for the use of Z. mays as a test organism for genotoxins (agents that can damage genetic material) is that more genetic information exists for maize than for any other plant species. Maize is a pillar of classical genetics, was used extensively in early studies on induced mutation (Anderson, 1948: Anderson et al.. 1949; Gibson et al., 1950; Stadler, 1928, 1944: Stadler and Sprague, 1936), and continues to play an important role in studies on mutagenesis.

Assays The fundamental characteristic of the tests discussed in this paper is that all are events measured at specific genetic loci. Three genetic end points are evaluated; these include the loss of the phenotype of dominant alleles in heterozygotes, the induction of point mutations that segregate out in M 2 plants, and the induction of forward and reverse mutations in the microgametophytes (pollen grains). Each end point is discussed with the methods of treatment and the structure that is scored as the unit of measurement. These structures include the kernel or endosperm, the seedling, and the pollen grain.

Key references The key references concerning the origin, description, and genetics of Z. mars include Sprague (1977) and Walden (1978). A description of maize mutants commonly used in genetic studies is presented by Neuffer et al. (1968). The literature that discusses the loss of the phenotype of dominant alleles in the endosperm includes Gibson et al. (1950); Kreizinger (1960); Bianchi and Contin (1962); Neuffer and Ficsor (1963); Chatterjee et al. (1965a, b); Amano and Smith (1965); and Regiroli and Gavazzi (1975). The chemical induction of mutation measured in seedlings is discussed in papers by Kreizinger (1960); Bianchi and Contin (1962); Chatterjee et al. (1965b); and Conger and Carabia (1977). The experimental design for measuring the chemical induction of mutants that segregate in the first segregating generation is presented by Morgun and Larchenko (1975). Finally, mutation in pollen grains for fundamental genetic studies is discussed in a series of papers by Nelson (1957, 1959, 1962, 1968, 1975, 1976) and Freeling (1978). The use of specific mutations in pollen grains to monitor environmental mutagens is presented by Plewa (1978).

Criteria for literature selection The assays to be evaluated were selected by the Gene-Tox Committee on Plants. Appropriate references were retrieved from the Environmental Mutagen Information

319 Center data base by author and title. Papers were chosen from the list of titles and each paper was evaluated for its suitability in the Gene-Tox program. Only complete publications that presented original data on chemically induced mutations at specific loci in maize were reviewed. Those publications that presented data from adequate experimental designs (i.e., sufficient population size, acceptable controls, and proper experimental protocol) are included. Not included is the large number of mutation studies conducted for the purpose of improving agronomic characteristics. An omission does not necessarily indicate that its scientific quality is poor but that the committee believes the paper should not be included because of the limitations imposed by the objectives of the Gene-Tox program.

Test description The organism Maize is a useful genetic tool because of its genetic variability and the ease with which controlled crosses can be performed. The characteristics and advantages of maize as an experimental organism have been reviewed by Coe and Neuffer (1977). Maize is suited for studies in mutagenesis because (a) it is a higher life form with a diploid number of 2 n - - 2 0 chromosomes; (b) its chromosomes are suitable for cytogenetic studies, and a large body of information exists on cytogenetics (Carlson, 1977); (c) its 10 linkage groups are well-mapped (Coe and Neuffer, 1977; Neuffer et al., 1968); (d) controlled genetic crosses are easy to perform with hundreds of progeny produced from each cross; (e) its growth requirements are well-known, and it can be cultivated in the field, greenhouse, or plant growth chamber; (f) hundreds of inbreds, hybrids, and varieties with defined genotypes are maintained at the Maize Genetics Cooperation at the University of Illinois, Urbana, IL, and are available to researchers. Finally, an enormous amount of information exists on the development, morphology, physiology, biochemistry, genetics, and breeding of maize. Recent reviews include Jugenheimer (1976), Sprague (1977), and Walden (1978). The life cycle of maize consists of an alternation of generations, a diploid sporophyte and a haploid gametophyte. The sporophyte is monoecious; the male spores or microspores form in the anthers of the tassel, and the female spores or megaspores form in the ovules of the pistillate flowers. Within the. anther the microsporocytes proceed through meiosis and produce a tetrad of microspores. Each haploid microspore undergoes 2 mitotic divisions that result in the formation of 2 sperm nuclei and a tube nucleus. This trinucleate organism is the male gametophyte or pollen grain. Each ovule contains a megasporocyte that undergoes meiosis. Of the 4 meiotic products, only 1 cell enlarges into a haploid megaspore. The megaspore divides mitotically to form a multicellular structure, termed an embryo sac, that contains 4 types of cell, an egg cell, 2 polar nuclei, 2 synergids, and approximately 25 antipodal cells. The fully developed embryo sac is the female gametophyte or megagametophyte. A pollen grain germinates after contacting the style (silk), and a

320 pollen tube proceeds through the style to the ovule. Upon entering the megagametophyte one sperm nucleus fertilizes the egg to form a diploid zygote while the other sperm nucleus fuses with the 2 polar nuclei to form the triploid endosperm. The zygote develops into a new diploid sporophyte generation. A ssavs The fundamental aspect of the assay is the induction of mutation at a specific genetic locus and its measurement by a defined criterion in the plant. No general or gross phenotypic alteration in individuals or families that results from treatment is acceptable. The specific-locus test encompasses a variety of treatment conditions. measurement criteria, and genetic end points. For the purpose of the Gene-Tox program only 3 genetic end points are included. These are the mutation of dominant alleles in heterozygous kernels or plants, the induction of point mutations that appear in the first segregating generation, and the induction of point mutations in pollen grains. Each of these end points is examined in detail with regard to the genetic basis of the assay, general protocol, and interpretation of data. The specificlocus assay is further categorized by the structure scored (i.e.. endosperm, seedling, etc.). Mutation of specific loci in heterozygotes A convenient method to assay for the effect of a genotoxin is to treat individuals that are homozygous for specific dominant alleles and test cross the treated plants by untreated plants that are homozygous recessive for the corresponding alleles. Any progeny that expresses a recessive trait included in the genotype of the test cross is considered exceptional. Exceptional individuals are assumed to result from a mutation of the dominant allele in a gametophyte of the treated organism. This assay is specific and requires a moderate amount of time, depending upon the conditions of treatment and the structure scored. The assay is relatively simple to perform because of the ease in making test crosses and the production of large progeny populations. Generally the endosperm and seedlings are scored for mutations. Tests using endosperm mutations Point mutations that are expressed in the endosperm have been used successfully to evaluate mutagenic properties of various chemicals (Amano and Smith, 1965: Bianchi and Contin, 1962; Chatterjee et al., 1965a, b; Gibson et al., 1950: Kreizinger, 1960; Neuffer and Ficsor, 1963). A number of loci can be scored simultaneously. If the genes are linked, the induction of multilocus deletions can be distinguished from single point mutations or minute deletions. Genetic basis. The selection of known recessive alleles in the test cross defines the genetic specificity of this assay. A number of mapped genes is expressed in the aleurone (the outermost cell layer of the endosperm) or in the composition of the endosperm itself. The endosperm is triploid (3n) due to the fusing of a sperm nucleus with 2 polar nuclei. The most widely used alleles for this test are:

321 TABLE 1 SPECIFIC LOCI FREQUENTLY USED IN MUTATION STUDIES IN MAIZE a Name

Symbol

Chromosome Position

Phenotype

Liguleless

Ig

2

1

Ligule and auricle absent, leaves upright

A nthocyaninless

a

3

111

Colorless aleurone, green or brown plant, brown pericarp with P-RR

Shrunken-2

sh-2

3

111.2

Inflated, transparent, sweet kernels collapse after drying. Angular, brittle kernels

Sugary

su

4

71

Endosperms wrinkled and transparent when dry, sweet at milk stage

Glossy

gl

7

36

Cuticle wax altered, seedling leaf shiny and water adheres in droplets

Yellow-green

yg-2

9

7

Colored aleurone

C

9

26

Colored aleurone

Shrunken

sh

9

29

Inflated endosperm collapsed after drymg forming smoothly indented kernels

Bronze

bz

9

31

Modifies purple aleurone and plant color to pale or reddish brown, anthers fluoresce yellow when exposed to UV light

Wax)'

wx

9

59

Amylopectin replaces amylose in endosperm and pollen

Vtrescent

v

9

66

Yellowish wlute seedling, greens rapidly

Glossy

g1-15

9

69

Cuticle wax altered, leaf shiny and water adheres, expressed after 3rd leaf

Colored

R

10

57

Red or purple color m aleurone and/or anthers, leaf tip, brace roots

Yellowish green plant

a) From Neuffer et al. (1968) and Coe and Neuffer (1977).

a n t h o c y a n i n l e s s , s h r u n k e n - 2 , sugary, colored aleurone, s h r u n k e n , bronze, w a x y , a n d colored. The symbols, location of each allele o n its chromosome, a n d a description of

each p h e n o t y p e are presented in T a b l e 1. Since the treated individuals carry a c o r r e s p o n d i n g d o m i n a n t allele, a m u t a t i o n at a specific locus of a gametophyte will p r e v e n t the expression of the d o m i n a n t allele. After the appropriate test cross an affected locus will be uncovered by the appearance of a recessive p h e n o t y p e in a supposedly heterozygous kernel. The results are easy to interpret if a m u t a t i o n occurs at a specific locus a n d the e n d o s p e r m exhibits a completely recessive phenotype. The loss of the expression of a d o m i n a n t allele in a particular cell lineage results in a sector if cell viability is unaffected. Kreizinger (1960) discovered that the frequency of gene expression in terms of sectors to entire kernels was not influenced by the m e t h o d of treating pollen, the year of treatment, or the stock of maize used in the assay. Kreizinger also

322 discovered that the size of the endosperm sectors formed a normal distribution about the mean value of one-half. Sectoring has been reported after the tassel or pollen has been treated with: ultraviolet radiation (Konzak and Singleton, 1956), mustard gas (Gibson et al.. 1950), diepoxybutane (Bianchi and Contin, 1962; Kreizinger. 1960). ethyl methanesulfonate or ultraviolet radiation (Neuffer and Ficsor, 1963), or ethyl methanesulfonate and y-radiation (Amano and Smith, 1965: Chatterjee et al., 1965a, b). The mosaic pattern of some sectors is a result of the breakage-fusion bridge cycle (McClintock, 1941). Kreizinger (1960) suggested that discrete sectoring may result because a mutagen may affect a segment of a multistranded chromatid, interfere with chromosome replication that results in subsequent chromosome or chromatid breakage, induce an unstable genic state, or produce some effect upon the chromosome matrix. A m a n o and Smith (1965) reported that mutagen treatment of kernels or seedlings usually results in mutations that are expressed in the whole kernel. Treatments of tassels or pollen result in the production of some sectors in the endosperm. This suggests that sectoring is related to chromosome breakage or instability and that a number of cell divisions is required to lose the factors involved in sectoring. Since a large number of marker alleles is expressed m the endosperm (Table 1), tests involving multiple genes, both linked and unlinked, can be used. The advantage of this assay is that point mutations and multilocus deletions can be differentiated, and the action of mutagenic agents can be inferred (Amano and Smith, 1965: Chatterjee et al., 1965a, b; Neuffer and Ficsor, 1963). Suggested protocol. A series of protocols has been described for specific-locus tests involving the endosperm. These protocols are discussed by Kreizinger (1960) and A m a n o and Smith (1965). Treatment protocols include the zygote (Chatterjee et al., 1965a), kernel, seedling (Amano and Smith, 1965), tassel (Amano and Smith, 1965: Kreizinger, 1960), or mature pollen (Chatterjee et al. 1965b: Coe and Neuffer, 1977). The most widely used treatment regimens are those of the kernel, seedling, and tassels or pollen. Each method is presented separately. Treatment of kernels. Kernels should be selected from an inbred line with a known genotype. The use of specific inbreds assures a high level of isogeneity. A list of inbreds and their genotypes is available from the Maize Genetics Cooperation. Sibling kernels from a recent harvest should be used after being inspected for quality. A protocol for treatment of kernels by soaking is presented by A m a n o and Smith (1965). The experimental design defines the conditions of treatment. If the experiment is designed to test a chemical on cells that are not undergoing D N A synthesis, dormant kernels should be used. The majority of these cells is in the G I stage of the D N A cycle. If it is desirable to test the agent with cells undergoing D N A synthesis, the kernels should be soaked for 72 h at 20°C in aerated water. The majority of these cells is in the S, G 2, and M stages of the cell cycle (Conger and Carabia, 1977). Thus it is important to define the stage at which the kernels are treated. The kernels are then placed in various concentrations of the test agent for a defined period of time. The physical characteristics of each test chemical, such as p H effects, solubility in water or other solvents, and photosensitivity, will determine the treatment protocol. It is essential to include each aspect of the treatment conditions in appropriate controls.

323 The test chemical should be dissolved in an appropriate solvent. All solutions should be prepared immediately prior to treatment and adjusted to pH 7 by using a 0.01-0.1 M phosphate buffer. If the test agent is pH-dependent, the protocol should be adjusted accordingly. Approximately 2-5 ml of test solution should be used per kernel per treatment, and the treatment groups and controls should be aerated constantly. The environmental conditions of the control and treatment groups should be held constant. It is important to record the concentration of mutagen, volume, solvent, pH, buffer, number of kernels in the control and treatment groups, temperature, and the length of time of the treatment. Usually the treatment times range from 4 to 12 h. After treatment, the kernels should be rinsed 2 or 3 times with water and the rinse water retained for proper and safe disposal. A final rinse under cold running tap water for 0.5-4 h is recommended to ensure that all of the mutagen solution is removed. The kernels can then be planted. After the plants mature they can be test crossed with untreated plants of the appropriate genotype. The resulting kernels are scored for mutation of specific dominant alleles. Treatment of seedlings. The treatment of maize seedlings is described by Amano and Smith (1965). Kernels are selected as described above and germinated on slanted moist filter paper. After the roots reach 2-5 cm in length, the tips are cut to ensure the rapid uptake of the chemical solution. The seedlings are placed in glass vials so the roots are immersed in the test solution with the endosperms and shoots in the air for normal respiration. The vials should be placed in a water bath to control temperature. The chemical solutions should be prepared as mentioned; the treatment time can be extended to 24 h. In all cases the controls should be handled in the same manner as the treatment groups except for exposure to the chemical under study. After treatment the roots should be rinsed thoroughly in water and the seedlings planted. After the plant matures it can be test crossed with untreated plants that carry the corresponding recessive alleles. The resulting kernels are assayed for mutation of specific dominant alleles. Treatment of pollen. There are two general approaches for exposing pollen to chemicals. One method is to treat the tassel and allow the chemical to enter the anther and pollen grains. Another approach is to harvest mature pollen, treat the pollen, and make the appropriate genetic crosses. The tassel can be treated while it is on the plant by injecting an agent into a pad or wick adjacent to the tassel. The ontogenetic stage at which the tassel is treated is important. If the chemical is injected during the microsporocyte stage, the pollen mother cells will be undergoing meiosis. If the treatment is conducted on emerged tassels, the pollen grains are in various stages of maturity. The methods of treating tassels are discussed by Kreizinger (1960), Neuffer and Ficsor (1963), Amano and Smith (1965) and Regirofi and Gavazzi (1975). The procedure involves the insertion of a cotton or fiber glass wick adjacent to the immature tassel and the application of the chemical solution to the wick. Approximately 5 ml of the test solution is introduced into the plant, and pollen is collected during anthesis. The pollen from the treated plants is applied to silks of plants used as females that carry the corresponding recessive alleles. The resulting kernels are scored for the loss of the phenotype of the dominant alleles.

324 In another method, young extended tassels are harvested prior to anthesis and the cut end is placed into a flask containing the test solution. After 18-24 h of treatment the pollen is collected and used in appropriate crosses (Kreizinger, 1960). In the above procedures the tassels are treated in an effort to induce mutations in pollen grains. It is difficult to control the access of the chemical to the pollen grain. These procedures have inherent problems in quantification, and therefore dose-related effects are difficult to assess. The direct exposure of mature pollen to a chemical mutagen is a rapid and simple approach for inducing mutation. Pollen grains are fragile and most attempts to use treated pollen in genetic crosses have failed. However. Coe (1966) and Coe and Neuffer (1977) developed a method that uses paraffin oil to suspend the pollen grains in a mutagen. The chemical agent is suspended in light paraffin oil by vigorous stirring for at least 1 h. The suspension is mixed with fresh pollen in a capped vial using at least 15 volumes of oil suspension per volume of pollen, and then the pollen-oil mixture must be agitated periodically for 50 min and applied to fresh silks. For use in the endosperm assay, pollen from plants that carry the dominant marker alleles is treated. After the treatment the mixture is applied to the silks of plants that carry the corresponding homozygous recessive alleles. A series of doses covering orders of magnitude is recommended to test for a dose-related response. Data from the mutagen-treated gametophytes are compared with data from controls. Mutation of dominant alleles in the endosperm is scored. This method is useful because it allows for the direct treatment of pollen, is relatively fast, and the concentration of an agent exposed to the pollen is controlled.

Tests usmg seedling mutations Two assays may be used in specific-locus tests for mutations expressed in the seedling. One involves crossing treated plants with untreated ones that carry the corresponding recessive alleles at the loci under study. If endosperm and seedling marker genes are incorporated in the genotype of the test cross plants, the resulting kernels can be scored for mutations in the endosperm, and when planted, mutations can be scored in the seedlings. This approach increases the number of loci that can be assayed simultaneously. A second approach is to score for the loss of the phenotype of the dominant allele in leaf tissue of plants that are heterozygous for yellow-green-2 (Table 1). This assay is relatively fast since the kernels are treated and the seedling leaves are scored in 20-25 days. Genettc basis. The alleles commonly used in specific-locus tests on seedlings include liguleless, glossy yellow-green-2, vtrescent, and glossy-15 (Table 1). The genetic basis of the assay is defined by the genotype of the test-cross plants or in the use of heterozygous yg-2 plants. The unit of measurement is the mutation of a specific dominant allele and the subsequent expression of the recessive phenotype. Suggested protocol. The protocol for assaying mutation in maize seedlings is discussed by Bianchi and Contin (1962), Amano and Smith (1965) and Chatterjee et al. (1965a, b). The treatment of kernels, seedlings, or pollen is identical to the protocol discussed for uncovering marker genes in the endosperm. After harvest, kernels are planted and grown for approximately 2 weeks to the 3-leaf stage.

325 Exceptional seedlings (i.e., those that express the recessive phenotypes) are scored. The number of single mutations and multiple gene mutations is counted and their frequency is calculated. The use of heterozygous yg-2 plants in mutation studies is discussed by Conger (1975, 1976) and Conger and Carabia (1977). Dormant kernels or kernels soaked in water for 72 h at 20°C may be used. Approximately 30 kernels per treatment group are exposed to various concentrations of the test agent. Doses may be determined by conducting germination tests over a wide range of concentrations that covers orders of magnitude. The toxicity of the agent is the limiting factor. Treatment conditions are the same as for the specific-locus test using genes expressed in the endosperm. After treatment the plants are cultivated in a plant growth chamber with a 16-h photoperiod of fluorescent and incandescent light. A temperature of 20°C is maintained to enhance the expression of the yg-2 allele. The 4th and 5th leaves are scored for the frequency of yellow-green sectors after approximately 25 days. Appropriate controls are included with each experiment, and the frequency of yellow-green sectors per leaf is scored at the same time.

Detection of mutations in the first segregating generation A second general assay for the induction of point mutations in maize is to score for recessive alleles that appear in the first generation following treatment (M 2 generation). This method has not been widely used because of the length of time required.

Genetic basis The genetic basis of this assay is the segregation of recessive alleles (induced by treatment) in the first segregating generation. To ensure that a phenotype is due to the expression of a mutant gene, two criteria must be met. The expected phenotypic frequencies of mutant seedlings must appear in the segregating generation, and plants that express the exceptional phenotype should be self-crossed and the trait tested for monogenicity.

Suggested protocol A general protocol for this assay is outlined by Morgun and Larchenko (1975). Kernels, seedlings, or pollen are treated as previously described and sibling crosses are made within each treatment group. The progeny kernels are planted and self-crossed. The resulting kernels are checked for recessive endosperm mutations. A sample, usually 40-100 kernels, from each ear is planted in a sandbench and the seedlings are inspected for mutants. A monogenic trait should segregate 3 normal to 1 exceptional plant. Finally, several exceptional plants should be self-crossed to test whether they are homozygous for the mutant allele. If the mutation is a seedling lethal, several normal siblings should be self-crossed to test whether the trait will segregate in the progeny of presumptive heterozygotes. The number of mutants that occur within families from the first generation is tabulated and a mutant frequency of mutations is calculated based on the total

326 number of M 2 families. Plants from appropriate control families will furnish the spontaneous mutant frequencies. Mutation of specific loci expressed in pollen

Since the microgametophyte is a functional haploid, studies that measure the frequency of mutant pollen grains can be conducted with ease. The test requires only a moderate amount of time because no crosses are required. The major advantage of this assay is that the pollen grain is the unit of measurement. Thus large numbers can be analyzed, providing the assay with a high degree of genetic resolution. Most of the work in this area has been done with the waxy locus: however, other loci are available (Freeling, 1978). Genetic basis

Near the beginning of this century an endosperm trait in maize was introduced from China that was distinctly different from the American varieties of floury, sweet, flint, or pop. This novel variety was named "waxy" because the endosperm had the appearance of hard wax (Collins, 1909). Genetic studies confirmed that the waxy allele is recessive to starchy ( W x ) , and wx segregates in the F2 generation as a Mendelian monohybrid (Collins and Dempton, 1909). In waxy kernels, the starch of the endosperm contains only amylopectin, while in kernels carrying the dominant allele, Wx, the endosperms contain starch composed of a mixture of amylopectin and amylose (Sprague et al., 1943; Weatherwax, 1922). Because of the presence of amylose, the endosperms of kernels carrying the Wx allele stain a dark blue-black color when reacted with iodine. When the endosperms of w x / w x kernels are reacted with an iodine solution, a red color is produced. It was soon discovered that the wx phenotype could be detected in pollen grains by the iodine test. Pollen grains are functional haploids, and in a heterozygous plant both alleles segregate according to Mendel's first law (Brink and MacGillivray, 1924; Demerrc, 1924). Furthermore, the data indicate a single gene and its alleles can be similarly expressed in both the sporophytic and gametophytic generations. The starch type of a pollen grain is controlled by the genetic constitution of that pollen grain, not by the parental sporophyte. Thus, a genetic reversion of wx to W x can be detected by scoring for pollen grains from plants that are homozygous wx that stain a dark blue-black color when subjected to an iodine test (Bianchk 1966; Nelson, 1959, 1962; Plewa, 1978; Plewa and Gentile, 1976a). Conversely, a forward mutation of W x to wx can be detected by scoring for pollen grains that stain a reddish-tan color from plants that are homozygous W x (Amano, 1968; 1972; Eriksson, 1963, 1966, 1969; Eriksson and Tavrin, 1965). The use of pollen grains as indicator organisms for genotoxins is feasible, because a specific genetic alteration can be identified within a large sample population. Mutation studies that used ionizing radiation with acute or chronic treatments clearly indicated the assay was responsive to mutagens. However, a mutant pollen grain does not necessarily result from a discrete and individual mutational event. Since pollen grains are meiotic products, mutant pollen grains may arise from a

327 mutated germ-line cell. The earlier a mutagenic event occurs, the greater the number of mutant pollen grains. Lindgren (1975) analyzed maize and barley plants thecum by thecum or anther by anther for w a x y pollen grains. He discovered the frequency of single mutant pollen grains was higher than expected, and he suggested a single mutant pollen grain may be due to the induction of mutation in a single strand of D N A one cell cycle prior to meiosis, in a chromatid during meiosis, or in both chromatids in the first mitotic division within the developing pollen grain. A cluster of mutant pollen grains within a maize thecum may be due to several independent mutational events, mutational events that involve both strands of DNA one cell cycle prior to meiosis, or mitotic mutational events in germ-line cells. The high frequency of single mutant pollen grains per thecum indicates that the mutation rate during meiosis is higher than it is during mitosis (Eriksson and Tavrin, 1965; Lindgren, 1975). Nevertheless, the data clearly demonstrate that mutations assayed in maize pollen after acute or chronic mutagenic treatment have a dose-dependent response (Bianchi, 1966; Eriksson, 1963, 1966, 1969; Eriksson and Tavrin, 1965; Plewa and Gentile, 1976a). Apparently, compensation exists between the mutagenic sensitivities of mitotic and meiotic cells. Perhaps a portion of the observed linear kinetics in the induction of w a x y pollen grains is a result of the number of germ-line cells present at various ontogenetic stages of the plant. A young plant has fewer germ-line cells; therefore, the probability of recovering a mutation in the meiotic cells of the tassel is low. However, if a germ-line cell is mutated, the number of mutant pollen grains derived from that cell may be high because a large sector or cluster will result. Conversely, if an older plant is exposed to a mutagen, the number of "target" germ-line cells is greater and the probability of recovering a mutation in the meiotic cells is increased. Because of ontogeny, the number of mutant pollen grains derived from such a germ-line cell will be reduced. S u g g e s t e d protocol

The assay is conducted by treating kernels or plants. Acute or chronic treatment conditions can be used. When the tassels reach early anthesis, they are harvested, labeled, and stored in 70% ethanol. Tassels can be stored indefinitely under these conditions. In the forward-mutation test, homozygous W x plants are exposed to a test agent. The end point is the mutation that renders a pollen grain incapable of synthesizing the carbohydrate amylose. This event is broadly defined as a forward mutation. The specific genetic alteration may be a point mutation within the w x locus, a deletion of the w x locus, or a chromosome aberration that results in a deficiency that includes the w x locus. The remote possibility exists that a mutation may be induced in a regulatory gene. Although the forward-mutation assay is not as specific as a reversion test, it provides a measure of genetic damage in the broad sense in that it involves a single locus. In the forward-mutation test, the w x mutants are detected by the gelatin-iodine staining technique. Pollen grains carrying the W x allele incorporate amylose in their starch. Iodine combines with the amylose and forms a blue-black complex. Pollen grains that do not contain amylose stain tan in the presence of iodine and are scored

328 as w x mutants. Each tassel is removed from its storage jar and agitated in clean 70% ethanol to remove contaminant pollen grains from the surface. A few randomly chosen unopened florets are removed and placed in a petri dish filled with 70% ethanol and agitated. 10-20 anthers are dissected from these unopened florets and are placed in a stainless steel cup of a VirTis microhomogenizer containing 0.5 ml of the gelatin-iodine stain. The preparation of the stain is described by Nelson (1962). The anthers are minced with scissors and homogenized for 30 sec. The homogenate is strained through cheesecloth onto the surface of a large glass microscope slide and covered with a glass coverslip. After the suspension solidifies, the slide is examined under a dissecting microscope at a magnification of 40 X . Pollen grains which have not undergone mutation at the w x locus carry the W x allele and stain black. Plump tan-staining wx mutants are counted and the total number of full (viable) pollen grains on the slide is estimated. This estimate is determined by adding the number of pollen grains within 20 randomly chosen l - m m 2 areas and multiplying by an appropriate factor. After a number of slides are analyzed, the frequency of forward mutant pollen grains is calculated for each plant by dividing the total number of wx mutants by the estimated number of viable pollen grains. The reversion test is conducted in a fashion similar to that for the forward-mutation test except that plants homoallelic for a specific w x heteroallele are treated. Most of the work done in reverse-mutation assays has involved either the w x - C or w x - 9 0 heteroalleles. Pollen grains that contain the w x allele stain a reddish tan when reacted with the gelatin-iodine stain. However, if a reverse mutation occurs at the w x locus to the dominant allele, W x , starchy pollen grains will result. These pollen grains contain amylose and stain black when reacted with the gelatin-iodine stain. The slides are scored as in the forward-mutation test. and the frequency of mutant pollen grains is recorded. Approximately 250000 pollen grains per tassel should be analyzed in either test. The frequency of mutant pollen grains for each plant must be calculated. 5 - 1 0 plants per treatment group should be used. The mean frequency of mutants and the standard error for each treatment group should be calculated and compared with those of the control.

Interpretation of data This section presents a general outline that defines the criteria for the interpretation and presentation of data. The data in a large number of papers could not be incorporated in the Gene-Tox program because it could not be interpreted. The following suggestions are only general, and it remains for the individual scientist to construct experiments of good design and to present adequate data to allow independent evaluation. P r e s e n t a t i o n o f data

Data for each treatment group should be presented in tabular form so the reader can reconstruct the experiment and determine the mutation frequencies mentioned

329 TABLE 2 PRESENTATION OF DATA FOR SPECIFIC-LOCUS TESTS IN KERNELS OR SEEDLINGS a Treatment

Number analyzed

Number of loci uncovered Complete

Negative control Positive control Treatment 1 Treatment 2 Treatment 3

Sectoral

Mutant frequency Total

> 2 500 > 2 500 >2500 > 2 500 > 2 500

a Chemical concentrations and treatment times should be given. Statistical tests and confidence levels should be defined. Significant responses should be identified at an a level.

i n the text. The p r e s e n t a t i o n of data in various specific-locus tests is illustrated in Tables 2, 3 a n d 4. The tables illustrate the traditional m e t h o d s of data p r e s e n t a t i o n a n d are d r a w n from papers cited in previous sections. The description should i n c l u d e a m e a n frequency of m u t a t i o n for each control a n d t r e a t m e n t group. Variability should be described in units of s t a n d a r d deviation. The data for negative a n d positive controls a n d each treatment group should be expressed in identical units. If there is a d o s e - d e p e n d e n t response, it would be helpful to present the i n f o r m a t i o n graphically. T o be acceptable data must be derived from an adequate experimental design. T w o p r o b l e m s became a p p a r e n t in a review of the literature: appropriate control p o p u l a t i o n s were either n o t used or not reported a n d the size of the p o p u l a t i o n assayed was often too small. I n Tables 2, 3 a n d 4 the p o p u l a t i o n sizes are to be considered as the m i n i m u m a n d the n u m b e r s are those f o u n d in adequate experim e n t a l designs described in the literature.

TABLE 3 PRESENTATION OF DATA FOR THE yg-2 ASSAY a Treatment

Negative control Positive control Treatment 1 Treatment 2 Treatment 3

Number of leaves assayed

Total number of yg-2 sectors uncovered

Mean yg-2 frequency per leaf -+,S.D. or S.E.

Leaf 4

Leaf 5

Leaf 4

Leaf 4

> 30 > 30 >30 > 30 > 30

> 30 > 30 >30 > 30 > 30

Leaf 5

Leaf 5

a Chemical concentrations and treatment times should be given. Statistical tests and confidence levels should be defined. Significant responses should be identified at an a level.

330

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Z

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z

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331

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332

Statistical evaluation A complete and informative discussion on the use of statistical analysis of data generated by mutagen assays is presented by Ehrenberg (1977). It is important to consider the statistics when designing an experimental protocol. Aspects such as population size, confidence limits, and power of the statistical test should be addressed prior to conducting the experiment. The data in tabular form should be accompanied by some descriptive statistics. The most common are the mean values for each group and the standard deviation or the standard error of the mean. In the various specific-locus tests discussed, the chi-square statistic for testing the null hypothesis is used to determine if the observed number of exceptional individuals is significantly different from the expected values derived from the controls. Other commonly used parametric tests are the t statistic, when 2 groups are being compared, or the analysis of variance, when more than 2 groups are evaluated. In the tests involving pollen grains significant differences among means may be evaluated with the analysis-of-variance test. The mean frequency of mutant pollen grains is calculated on the basis of the frequencies observed for individual plants within the group. If there are equal numbers of plants in all groups, a randomized block design may be used; if the number of plants differs within each group, a one-way analysis of variance may be used. If an F test indicates significance, appropriate follow-up tests can be used to determine which groups are different. As described by Ehrenberg (1977), a complete statistical analysis of data derived from a mutagen assay should include values of the level of significance of the statistical test, the level of confidence of a positive result, and the power of the confidence interval in a negative result. A new statistic (q~) has been developed by Katz (1979) to determine if a treatment induces a significant increase in the frequency of mutant pollen grains. He adapted the ~ statistic for use in the analysis of mutation data from pollen grains. The test is defined as ,/,= n ( M - - 0.5) - N ( m + 0.5)

tnN( m + M ) where m is the observed number of mutant pollen grains in the control group, M is the observed number of mutant pollen grains in the experimental group, n is the estimated number of pollen grains analyzed in the control group, and N is the estimated number of pollen grains analyzed in the experimental group. If a one tailed test is performed and if the level of significance is set at 0.05, a mutagenic response is demonstrated if ~ > 1.64. Problems arise in determining the best approximation of the true mutation rate a m o n g gametes when premeiotic mutational or recombinational events occur. Such events may result in a distribution of exceptional gametes or individuals among the progeny that is "clustered" as opposed to binomial. A series of equations has been developed by Engels (1979) to estimate the mutation rate among gametes when premeiotic events are involved. The true mutation rate among gametes (#) can be

333

estimated as a weighted average (pw) or as an unweighted average (pu). The best estimator is the average with the least variance. Engels provides the following equations as unbiased estimators for the mutation rates among gametes and their estimators of variance. The weighted average mutation rate Pw. is expressed as Pw = m / n

where m is the observed number of mutants and n is the population of individuals scored. The estimate of its variance (17"w) is expressed as 2

2

2

17w = Y m, -p,,, En, n 2 _ X n ~,

The weighted average mutation rate describes a distribution of mutants that are believed to be meiotic in nature. A second estimator of the mutation rate among gametes is the unweighted average mutation rate, Pu, which is expressed as

Pu----(1)[E(mJn,)] where k is the number of families involved in an experiment. The estimate of the variance of p~(l?u) is expressed as

1

l;'~-k(k-1)

~(m, 12 ,n,!

1

k 2 ( k - 1 ) ( y'm'12"n,,

Engels states that when the effect of clustering is pronounced, the unweighted average mutation rate provides a better approximation of the true mutation rate among gametes.

Hazard evaluation The specific-locus tests of maize, as well as other plant-based mutagen assays, should be used in the evaluation of environmental hazards. Plants offer unique advantages in evaluating the mutagenic properties of environmental pollutants and should be included in any battery of tests (Constantin, 1978; de Serres, 1978; Nilan, 1978). In addition to routine tests, plant assays are useful in studies that monitor chronic exposure to test agents. Aspects such as in situ environmental monitoring of specific areas can be studied best by using plant systems (Heslop-Harrison, 1978; Klekowski, 1978; Lower et al., 1978; Plewa, 1978; Schairer et al., 1978). With the advent of plant-microbe mutagen assays, the data indicate that green plants can activate promutagens into mutagens (Barnes and Klekowski, 1978; Benigni et al., 1979; Gentile et al., 1977; Owais et al., 1978; Plewa, 1978; Plewa and Gentile, 1976b, Scott et al., 1978; Veleminsky et al., 1979). Maize is being used to test the mutagenic properties of the major pesticides used in its commercial production (Plewa et al., 1979). The awareness of plant-mediated activation should be incorporated in the process of detecting environmental mutagens and of evaluating their hazard to public health.

334

Test performance A summary of chemical agents that have been adequately tested for mutagenic properties is presented in Table 5. It would be premature to evaluate maize for its sensitivity to various chemical classes.

Conclusions Strengths and weaknesses of the assay The strengths of the specific-locus tests in Z. mays are many. The organism is a higher eukaryote with well-defined genetics. The genetic end points of the tests are specific and the data generated are quantitative. There exists a long history in which the plant has been used in studies on induced mutagenesis. The action of mutagens can be inferred in some of the assays. Both somatic and germinal cells can be used to detect mutagens and to monitor for acute or chronic exposure of test agents. The sample size per treatment group of the structures analyzed ranges from hundreds in the yg-2 seedling test to thousands in tests that measure the mutation of dominant alleles in kernels or seedlings to millions in tests that measure mutation in pollen grains. Finally, the use of the plant for in situ environmental monitoring and in studies on the activation of promutagens has demonstrated its versatility as a genetic indicator organism. The weaknesses of the maize assay are that it is difficult to extrapolate the data to man, and 1 or 2 generations are required in conducting a test.

Use in mutagenicity programs Maize would be very useful as a tier 2 test organism or as a higher eukaryote assay in phase 2 of a phased testing strategy. The phased approach developed by the U.S. Environmental Protection Agency (Waters, 1977) is a testing strategy in which the extent of testing a chemical is determined by the results of the previous phase and by the degree of potential human hazard. In the phased approach, phase l tests primarily involve microbial assays and determine if a mutagen hazard exists. Phase 2 tests include genetic assays based on m a m m a h a n cells, plants, and insects. These assays are used to confirm the results of the phase 1 tests and to further characterize the genotoxic properties of the chemical. Those chemicals that yield positive responses in phases 1 and 2 are then analyzed in phase 3. Phase 3 tests involve whole animal studies and are the final validation of a hazard and provide a quantitative basis for risk assessment. The rapid yg-2 heterozygote test is an attractive phase 2 assay. The high genetic resolution of these assays makes them a logical choice in screening procedures. The advantages offered by the usage of plants in such studies should not be overlooked (Nilan, 1978).

335

Recommendations for research Research is needed to improve the utility of the maize assays. The long generation time may be alleviated by developing rapidly maturing lines that incorporate a series of genetic end points. Assays that detect non-disjunction of chromosomes and chromosome aberrations should be developed. Finally, a series of tests is needed that measures mutation or dysfunction of regulatory genes as opposed to structural genes (Freeling, 1978). Students of maize genetics have an opportunity to make a contribution to the field of environmental mutagenesis, and no doubt progress will be forthcoming.

References Amano, E. (1968) Comparison of ethyl methanesulfonate and radiation induced waxy mutants m maize, Mutation Res., 5, 41-46. Amano, E. (1972) Genetic fine structure analysis of mutants induced by ethyl methanesulfonate, Gamma Field Symp., I l, 43-59. Amano, E., and H.H. Smith (1965) Mutations induced by ethyl methanesulfonate in maize, Mutation Res., 2, 344-351. Anderson, E.G. (1948) On the frequency and transmitted chromosome alterations and gene mutations induced by atomic bomb radiauons in maize, Proc. Natl. Acad. Sci. (U.S.A.), 34, 386-390. Anderson, E.G., A.E. Longley, C.H. LI and K.L. Retherford (1949) Heredity effects produced in maize by radiations from the Bikini atomic bomb, I. Studies on seedlings and pollen of the exposed generation, Genetics, 39, 639-646. Barnes, W.S., and E.J. Klekowski (1978) Testing the environment for dispersed mutagens, Use of plant bioconcentrators coupled with microbial mutagen assays, Environ. Health Perspect., 27, 61-68. Beadle, G.W. (1978) Teosinte and the origin of maize, in: D.B. Walden (Ed.) Maize Breeding and Genetics. Wiley, New York, pp. 113-128. Benigni, R., M. Bignami, I. Camoni, A. Carere, P. Conti, R. lachetta, B. Morpurgo, and V.A. Ortali (1979) A new in vitro method for testing plant metabolism in mutagenicity studies, J. Tox. Environ. Health, 5, 809-819. Bianchi, A. (1966) Some aspects of mutagenesis in maaze, Proc. Symp. Mutational Process, Prague, 1965. pp. 30-37. Bianchi, A., and M. Contin (1962) Mutagenic activity of isomeric forms of diepoxybutane in maize, J. Hered., 53. 277-281. Brink, R.A.. and J.H. MacGillivray (1924) Segregation for the waxy character in maize pollen and differential development of the male gametophyte, Am. J. Bot., 11,465-469. Carlson, W.R. (1977) The cytogenetics of corn, in: G.F. Sprague (Ed.). Corn and Corn Improvement. Am. Soc. Agron. Inc., Madison, WI, pp. 225-304. Chatterjee, N.K., A.L. Caspar and W.R. Singleton (1965a) Genetic changes in maize induced by ethyl methanesulfonate, J. Hered., 56, 276-277. Chatterjee, N.K., A.L. Caspar and W.R. Singleton (1965b) Genetic effects of ethyl methanesulfonate and gamma ray treatment of the proembryo in maize, Genetics, 52, 1101-1111. Coe, E.H. (1966) Liquid media suitable for suspending maize pollen before pollination, Proc. Mo. Acad. Sci., 3, 7-8. Coe, E.H., and M.G. Neuffer (1977) The genetics of corn, in: G.F. Sprague (Ed.), Corn and Corn Improvement, Am. Soc. Agron. Inc., Madison, WI, pp. 111-223. Collins, G.N. (1909) A new type of Indian corn from China, Bull. Bureau Plant Ind. U.S. Dept. Agr., 161, 544. Collins, G.N., and J.H. Dempton (1909) Inheritance of waxy endosperms in hybrids of Chinese maxze, Bull. Burea Plant Ind. U.S. Dept. Agr., 161, 547.

336 Conger. B.V. (1975) Maize r.b.e, of fission neutrons for Induction of somatic mutations Jn maize, Int J. Radlat. Biol., 27. 271-281. Conger. B.V. (1976) Effecuveness of fission neutrons versus g a m m a ra&at~on for inducing somatic mutations in presoaked seeds of maize. Mutation Res., 34, 223-232. Conger, B.V., and J.V. Carabia (1977) Mutagenic effectiveness and efficiency of s o d m m az~de versus ethyl methanesulfonate m maize: mduct~on of somatic mutations at the vg-2 locus by treatment of seeds different in metabolic state and cell population. Mutation Res., 46. 285-296. Constantln. M.J. (1978) Utihty of specific locus systems in higher plants to m o m t o r for mutagens, Environ. Health Perspect., 27, 69-75. Demer6c, M. (1924) A case of pollen dimorphism in maize, Am. J. Bot.. 11. 461-464. de Serres, F.J. (1978) Introduction. utilization of higher plant systems as monitors of environmental mutagens. Environ. Health Perspect.. 27, 3-6. Ehrenberg, L. (1977) Aspects of statistical inference in testing for genetic toxicity, m" B.J. Kalbey (Ed.). Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 420-459. Engels, W.R. (1979) The estimation of m u t a u o n rates when premelotlc events are involved, Environ. Mutagen., 1, 37-43. Eriksson. G. (1963) Induction of waxy mutants in maize by acute and chromc g a m m a irradiation. Hereditas. 50, 161-178. Eriksson, G. (1966) Variations in radiosensitwity during meiosis of pollen mother cells m maize and barley, Proc. Symp. Mutational Process, Prague, 1965. pp. 47-52, Eriksson. G. (1969) The waxy character, Heredltas, 63, 180-204. Enksson, G, and E. Tavrin (1965) Variations in radlosensitivlty during meiosis of pollen mother cells of maize, Hereditas, 54, 156-169. Freeling, M. (1978) Maize Adh-1 as a momtor of environmental mutagens, Environ. Health Perspect. 27, 91-97. Galinat, W.C. (1971) Origin of maize, Am. Rev. Genet., 5, 447-478. Galinat, W.C. (1978) The inheritance of some traits essential to maize and teosinte, in: D.B. Walden (Ed.), Maize Breeding and Genetics, Wiley, New York. pp. 93-109. Gentile, J.M., E.D. Wagner and M.J. Plewa (1977) The detection of weak recombinogemc activities in the herbicides alachlor and propachlor using a plant-activation bioassay, Mutation Res., 48, 113-116. Gibson. P.B., R.A. Brink and M.A. Stahmann (1950) The mutagemc action of mustard gas on Zea mars, J. Hered., 41,232-238, Heslop-Harrison, J. (1978) Summary and perspectives, Environ. Health Perspect., 27. 197-206. Jiler, H. (Ed.) (1978) Commodity Year Book 1978. Commodity Research Bureau, New York. JugenheLmer, R.W. (1976) Corn: Improvement, Seed Production, and Uses, Wiley, New York, 670 pp. Katz. A.J. (1979) Design and analysis of experiments on mutagenicity, II. Assays involving microorganisms. Mutation Res., 64, 61-77. Klekowski. E.J. (1978) Screening aquatic ecosystems for mutagens w~th fern bioassays, Environ. Health Perspect., 27, 99-102. Konzak, C.F., and W.R. Singleton (1956) The mutation of hnked maize endosperm 1ocl induced by thermal neutron, X-, gamma, and ultraviolet radiation, Proc. Natl. Acad. Sci. (U.S.A.), 42, 239-245. Kreizmger, J.D. (1960) Diepoxybutane as a chemical mutagen in Zea mays, Genetics, 45, 143-154. Lmdgren, D. (1975) Sensitivity of premeiotic and meiotic stages to spontaneous and induced mutations in barley and maize, Hereditas, 79, 227-238. Lower, W . R . P . S . Rose and V.K. Drobney (1978) In SltU mutagemc and other effects associated with lead smelting, Mutation Res., 54, 83-93. McClintock, B (1941) The association of mutations with homozygous deficiencies in Zea mays, Genetics, 26, 542-571. Morgun, V.V., and E.A. Larchenko (1975) Genetic activity of new nltroso compounds in mmze, Genetika, 11, 13-18. Nelson, O.E. (1957) The feasibility of investigating "genetic fine structure" in higher plants. Am. Nat., 91, 331-332. Nelson, O.E. (1959) lntraclstron recombination in the W x / w x region in maize, Science, 130, 794-795.

337 Nelson, O.E. (1962) The waxy locus in maize, I. Intralocus recombination frequency estimates by pollen and by conventional analyses, Genetics, 47, 737-742. Nelson, O.E. (1968) The waxy locus in maize, If. The location of the controlling element alleles, Genetics, 60, 507-524. Nelson, O.E. (1975) The waxy locus in maize, llI. Effect of structural heterozygosity on intragenic recombination and flanking marker assortment, Genetics, 79, 31-44. Nelson, O.E. (1976) Previously unreported wx heteroalleles, Maize Genet. Coop. Newslett, 50, 109-113. Neuffer. M.G., and G. Ficsor (1963) Mutagenic action of ethyl methanesulfonate in maize, Science. 139, 1296-1297. Neuffer, M.G., L. Jones and M.S. Zuber (1968) The Mutants of Maize, Crop. ScL Soc. Am., Madison, WI, 74 pp. Nilan, R.A. (1978) Potential of plant genetic systems for monitoring and screening mutagens, Environ. Health Perspect., 27, 181-196. Owais. W.M., M.A. Zarowitz, R.A. Gunovich, A.L. Hodgdon, A. Kleinhofs and R.A. Nilan (1978) A mutagenic in vivo metabohte of sodium azide, Mutation Res., 53, 355-358. Plewa, M.J. (1978) Activation of chemicals into mutagens by green plants: a preliminary discussion, Environ. Health Perspect., 27, 45-50. Plewa, M.J.. and J.M. Gentile (1976a) Plant activation of herbicides into environmental mutagens: the waxy reversion bioassay, Maize Genet. Coop. Newslett., 50, 44. Plewa, M.J.. and J.M. Gentile (1976b) Mutagenicity of atrazine: a maize-microbe bioassay, Mutation Res., 38, 287-292. Plewa, M.J., E.D. Wagner and J.M. Gentile (1979) Analysis of the mutagenic properties of pesticides incorporating animal and plant activation, Environ. Mutagen., 1, 142. Regiroli, G., and G. Gavazzi (1975) Chemical mutagenesis at the R locus in maize, Maydica, 20, 57-66. Schairer, L.A., J. Van't Hof, C.G. Hayes, R.M. Burton and F.J. de Serres (1978) Exploratory monitoring of air poUutants for mutagenicity activity with the Tradescantia stamen hair system, Environ. Health Perspect., 27, 51-60. Scott, B.R., A.H. Sparrow, S.S. Schwemmer and L.A. Schairer (1978) Plant metabolic activation of 1, 2-dibromoethane (EDB) to a mutagen of greater potency, Mutation Res., 49, 203-212. Sprague, G.F. (Ed.) (1977) Corn and Corn Improvement, Am. Soc. Agron. Inc., Madison, WI 774 pp. Sprague, G.F., B. Brimhall and R.M. Hixon (1943) Some effects of the waxy gene m corn on properties of the endosperm starch, J. Am. Soc. Agron., 35, 817-822. Stadler, L.J. (1928) Genetic effects of X-rays in maize, Proc. Natl. Acad. Sci. (U.S.A.), 14, 69-75. Stadler, L.J. (1944) The effect of X-rays upon dominant mutation in maize, Proc. Natl. Acad. SCi. (U.S.A.), 30, 123-128. Stadler, L.J., and G.F. Sprague (1936) Genetic effects of ultra-violet radiation in maize, I. Unfiltered radiation, Proc. Natl. Acad. Sci. (U.S.A.), 22, 572-578. Veleminsky, J., L. Silhankova, V. Smiovska and T. Gichner (1979) Mutagenesis of Saccharomyces cerevzsiae by sodium azide activated in barley, Mutation Res., 61, 197-205. Walden, D.B. (Ed.) (1978) Maize Breeding and Genetics, Wiley, New York, 794 pp. Waters, M.D. (1977) Monitoring the environment, in: Toxicity Testing In Vitro, Academic Press, New York. Weatherwax, P. (1922) A rare carbohydrate in waxy maize, Genetics, 7, 568-572.