Soybean (Glycine max [L.] merrill) as a short-term assay for study of environmental mutagens. A report of the U.S. Environmental Protection Agency Gene-Tox Program

The soybean (Glycine max [L.] Merrill) spot test is suggested as a preliminary screening test for environmental mutagens. This system makes use of various types of spots that originate from the treatment of seeds or seedlings with mutagens. The homozygous dominant y11y11 dark green leaves may show light green and very dark green spots; the heterozygous y11y11 light green leaves may show dark green, yellow or twin spots; and the homozygous recessive y11y11 yellow leaves show light green receptors. The interpretation is that twin spots on the y11y11 leaves originate from somatic crossing-over, and the singles originate primarily from losses or gains of the segments or chromosome carrying the gene y11 or y11. The yellow plants (y11y11) can produce light green sectors if y11 mutates to y11. Studies carried out with a host of chemical and physical agents lend support to the idea that the soybean system can distinguish between several genetic mechanisms underlying the formation of spots. Spots are detected against their native genetic and phenotype background, thus minimizing the effects due to physiological changes. The system is rapid (4-5 weeks per chemical), inexpensive, and involves an eukaryotic organism. It has the advantage of being adaptable for liquid solutions of chemicals, solid wastes, emulsions of chemicals (e.g., in lanolin), and gaseous products.     

Mutagenic effects of nitropyrenes in a soybean test system

The mutagenic activity of 1-nitropyrene (1-NP), 1,3-dinitropyrene (1,3-DNP), 1,6-dinitropyrene (1,6-DNP) and 1,8-dinitropyrene (1,8-DNP) was assayed in heterozygous soybean plants (Y11y11), based on the appearance of mutational spots (yellow, dark green and twin) on the leaves. 1-NP, 1,3-DNP, 1,6-DNP and 1,8-DNP were direct-acting mutagens in a soybean test system, and mutagenicity was enhanced by addition of pyrene as a precursor. The mutagenicity of dinitropyrenes was enhanced by pretreatment with hepatic microsomal fractions of Aroclor 1254-treated rats. Binary and ternary isomeric mixtures of dinitropyrenes produced synergistic mutational response in the test system. The numbers of yellow and dark green spots per leaf increased by treatment with nitropyrenes. The frequency of twin spots did not change. Nitropyrenes stimulated the induction of forward and reverse mutations in soybeans. The number of light green spots (Y11y11) per leaf on homozygous soybeans (y11y11) increased markedly by treatment with 1-NP, 1,3-DNP, 1,6-DNP, and 1,8-DNP. These nitropyrenes would thus appear to cause point mutation and segmental loss as major effects.     

Somatic Crossing over in GLYCINE MAX (L.) Merrill: Effect of Some Inhibitors of DNA Synthesis on the Induction of Somatic Crossing over and Point Mutations

Glycine max (soybean) is the only known higher plant with a definitely established occurrence of somatic crossing over. This material lends itself to the analysis of somatic crossing over, gross chromosomal aberrations and mutations, all of which may be induced by the same treatment of the mutagen given to seeds. This is made possible because gene Y₁₁ for chlorophyll development in the variety L65-1237 is incompletely dominant over its allele y₁₁, so that twin or double spots composed of a dark green (Y₁₁Y₁₁) and a yellow (y₁₁y₁₁) component can be observed adjacent to and as mirror images of each other on the light green Y₁₁y₁₁ leaves in the areas of complementary exchange for these genes. Lack of growth of either component of this double spot as well as several types of chromosomal disturbances give rise to single spots resembling phenotypes of y₁₁y₁₁ or Y₁₁Y₁₁ leaves. Point mutations can be studied by looking for green sectors originating from Y₁₁y₁₁ genotype on the y₁₁y₁₁ plants. Seeds obtained from heterozygous plants were treated with caffeine, cytosine arabinoside, actinomycin D and 5-fluoro-deoxyuridine, all known inhibitors of DNA synthesis, and puromycin, an inhibitor of synthesis of proteins. The treatments with caffeine and actinomycin D increased the frequency of somatic crossing over as measured by the frequency of double spots on Y₁₁y₁₁ leaves, but cytosine arabinoside, 5-fluorodeoxyuridine and puromycin did not. Thus somatic crossing over was induced only by those chemicals which are known to allow rejoining of chromosomes, thereby suggesting a correlation between the two phenomena. These observations indicate that it is not the mere inhibition of DNA synthesis, but some rather more specific event in DNA repair which is responsible for complementary exchanges. Some of these results differ from studies carried out with fungi. The main effect of all chemicals tested, except caffeine and actinomycin D, was inferred to be the production of deletions in Y₁₁y₁₁ plants which raised the frequency of single (dark green or yellow) spots relative to the doubles. Caffeine was the only chemical which constantly increased the frequency of specific point mutations. In the control material, the great majority of spots are found on the upper surface of the leaf. This picture could not be changed in any of the treated materials, thus indicating uniform resistance of spongy mesophyll tissue to the mutagens applied.     

Studies on the expression of somatic crossing over in Glycine max L.

Summary Variety T219 of Glycine max L. has spontaneous yellow, dark green and double (yellow-dark green) spots on the leaves of plants of genotype Y ₁₁ y ₁₁ but no such spots are found on leaves of Y ₁₁ Y ₁₁ or y₁₁y₁₁ plants. It was suggested (Vig and Paddock, 1968) that the double spots result from somatic crossing over whereas the two types of single spots primarily originate from chromosomal disturbances.Cold shocks disproportionately increased the frequency of double spots, but ethyl methanesulfonate (EMS) did not do so. However, in most cases of each treatment, the frequency of single spots increased. It is suggested that EMS is not very potent in bringing about somatic recombination whereas cold shocks are. Plants from a sample of seeds of variety L65-1237 that had been harvested in 1968 at Urbana, Illinois, did not express the spotting phenomenon, but plants from seeds harvested in 1969 at Reno did have spots. Application of mitomycin C(MC) to the seeds of this variety as well as of T219 increased the incidence of double spots manyfold indicating that MC can reveal the potential for somatic crossing over in a variety which might not otherwise express it. Soaking dry seeds of L65-1237 in aqueous solutions of MC for intervals as short as 2 hours was found effective in increasing the frequency of double spots. The role of MC in relation to DNA synthesis and somatic crossing over is discussed. Application of the chromosome-breaking agent, claunomycin (DM) was ineffective in causing double spots.     

Somatic crossing-over in Glycine max (L.) merrill: activation of dimethyl nitrosoamine by plant seed and comparison with methyl nitrosourea in inducing somatic mosaicism

The soybean system used for detecting environmental mutagens is analyzed for various types of spots on the leaves of heterozygous y11y11 plants and homozygous y11y11's induced by a nitrosoamine (dimethyl nitrosoamine, DMN) and a nitrosoamide (methyl nitrosourea, MNU). It is shown that the nitrosoamine can be "activated" by the seed (is converted to a true mutagen) without the addition of NADPH or S-9 fraction of the liver homogenate as is necessary in animal tissue culture or bacterial studies. Whereas somatic mosaicism in soybean can be induced with a dose as low as 1.25 ppm of DMN, the upper limit in spot production is reached at around 60 ppm concentration, applied for 0--24 h. Such saturation effect may be due to a limited amount of DMN being converted to true mutagen. MNU, on the other hand, does not show such limitations, perhaps because of its property of being a direct mutagen not necessitating an intermediate step required for converting the promutagen DMN. The frequency of twin spots on Y11y11 leaves increases only slightly by either DMN or MNU, suggesting only a small increase in somatic crossing-over induced by the two chemicals. The yellow spots increase the most, perhaps due to segmental losses carrying Y11 or non-complementary segregation of exchanges involving non-homologous chromosomes. Neither chemical is found capable of mutating y11 to Y11 as seen by the general lack of light green sectors on y11Y11 plants. Usefulness of the soybean system in studying mutagenesis is briefly discussed.     

Effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) on somatic mutation in a soybean test system.

The mutagenic activity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was assayed in heterozygous soybean plants (Y11y11), based on the appearance of mutational spots (yellow, dark green and twin) on the leaves. When soybean seeds were treated with IQ at concentrations of 0.01-0.1 microgram/ml, the frequency of mutational spots per leaf increased significantly in proportion to the concentration of IQ. At higher concentrations IQ was toxic. The mutagenicity of IQ was enhanced by pretreatment with the hepatic S9 fraction from Aroclor-induced rats. The numbers of yellow and dark green spots per leaf increased markedly by the treatment with IQ and S9-activated IQ, but the number of twin spots did not increase.     

Somatic crossing over in glycine max (L). Merrill: Possible potentiation of the recombinogenic effect of caffeine by 5-fluorodeoxyuridine and cytosine-β-D-arabinofuranoside

The leaves of variety T219 of Glycine max (soybean) exhibit yellow, dark green and twin or double spots on the two simple leaves and the first compound leaf of heterozygous Y₁₁y₁₁ plants. These spots mimic the phenotypes controlled by Y₁₁y₁₁, Y₁₁Y₁₁ and Y₁₁Y₁₁−y₁₁y₁₁ genotypes. It has been argued that p recombination is responsible for the origin of twin spots. At least some of the single spots appear due to failure of one of the components of the double spots.Treatment of seeds with caffeine solutions increases the frequency of all types of spots, particularly those of doubles. 5-Fluorodeoxyuridine (FUdR) and cytosine-β-D-aranibofuranoside (CA), both inhibitors of DNA synthesis, cause potentiation of the effects of caffeine by increasing all three types of spots. Since the relative frequencies of the different types of spots do not differ much in caffeine plus FUdR (or CA) treated materials from those observed in case of caffeine treatment alone, it is suggested that (1) the increase in the frequency of spots in caffeine plus FUdR (or CA) treated material is not due simply to additive effect of the two chemical, and (2) there is true synergism between caffeine and FUdR (or CA) which somehow leads to enhancement of the phenomenon of compementary reunions.     

The mechanism of secondary nondisjunction in Drosophila melanogaster females

Bridges (1916) observed that X chromosome nondisjunction was much more frequent in XXY females than it was in genetically normal XX females. In addition, virtually all cases of X nondisjunction in XXY females were due to XX <--> Y segregational events in oocytes in which the two X chromosomes had failed to undergo crossing over. He referred to these XX <--> Y segregation events as "secondary nondisjunction." Cooper (1948) proposed that secondary nondisjunction results from the formation of an X-Y-X trivalent, such that the Y chromosome directs the segregation of two achiasmate X chromosomes to opposite poles on the first meiotic spindle. Using in situ hybridization to X and YL chromosomal satellite sequences, we demonstrate that XX <--> Y segregations are indeed presaged by physical associations of the X and Y chromosomal heterochromatin. The physical colocalization of the three sex chromosomes is observed in virtually all oocytes in early prophase and maintained at high frequency until midprophase in all genotypes examined. Although these XXY associations are usually dissolved by late prophase in oocytes that undergo X chromosomal crossing over, they are maintained throughout prophase in oocytes with nonexchange X chromosomes. The persistence of such XXY associations in the absence of exchange presumably facilitates the segregation of the two X chromosomes and the Y chromosome to opposite poles on the developing meiotic spindle. Moreover, the observation that XXY pairings are dissolved at the end of pachytene in oocytes that do undergo X chromosomal crossing over demonstrates that exchanges can alter heterochromatic (and thus presumably centromeric) associations during meiotic prophase.     

The ms1 mutation in soybean: involvement of gametes in crosses with tetraploid soybean

Summary Previous studies indicated that ms1ms1 malesterile female-fertile soybean (Glycine max [L.] Merr.) plants can produce seeds with different ploidy levels. The codominant chlorophyll-deficient mutant y11 was used in attempts to understand the embryo-endosperm relationship in seed production in ms1ms1 plants and to determine the mechanism of gamete formation in the ms1 mutation. Crosses were conducted between yellow-green male-sterile plants (ms1ms1Y11y11) and green fertile tetraploid cultivars (Ms1Ms1Ms1Ms1Y11Y11Y11Y11) in the greenhouse in the summers of 1987 and 1988. A total of 2,007 cross-pollinations were made. Thirty hybrid seeds were obtained, and plants were analyzed for chromosome number, fertility, and color. All the hybrid seedlings were tetraploid and fertile. No triploids were found. Among the 30 F₁ plants, 7 were green (Y11Y11Y11Y11), 17 were green-yellow (Y11Y11Y11y11), and 6 were yellow-green (Y11Y11y11y11). The segregation ratio was close to the expected 1 green: 2 green-yellow: 1 yellow-green (X² = 0.38; 0.90>p>0.75). From the results of this experiment, we conclude that: (1) triploids were not produced by crossing diploid ms1ms1 soybean plants with tetraploid plants; (2) tetraploid progeny can be produced from these crosses by the fusion of 2n ms1 eggs, or fusion of other 2n gametophyte cells in the embryo sac with a 2x sperm from tetraploid plants; (3) the megaspore mother cell of male-sterile plants undergoes meiotic division without cytokinesis after telophase II and forms more than the normal number of gametes, which can fuse with each other to generate tetraploid gametophyte cells.Joint contribution: U.S. Department of Agriculture, Agricultural Research Service, Cereal and Soybean Research Improvement Unit, Midwest Area, and Journal Paper No.-13838 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa     

The site of function of the Y chromosome in Drosophila melanogaster males

Summary The Y chromosome is essential for fertility in D. melanogaster males. An analysis of 126 pal-induced Y chromosome mosaics indicated that its function is only required in the germ line of fertile males. This analysis also showed that approximately 1/4 of all pal-induced Y chromosome mosaics had an XO/XYY constitution and hence that they resulted from somatic nondisjunction. Preliminary evidence suggests that pal-induced somatic nondisjunction can occur at the second or subsequent cleavage divisions.     

Sexual maldevelopment and sex reversal, chromosomal causes

The SRY gene on the Y chromosome is the testis determining factor (TDF). It is therefore the initial male determining factor. However, phenotypic sex determination includes a cascade of genes located on autosomes as well as sex chromosomes. Aberrations of these genes may cause sexual maldevelopment or sex reversal. Abnormalities may include single gene mutations and gene loss or gain-changes may involve only sex organs or may be part of syndromes. These changes may also arise as chromosome abnormalities involving contiguous genes. Eight cases with chromosomal abnormalities involving different causative mechanisms are described herein. The most common cause is nondisjunction, including loss or gain of sex chromosomes. Less common causes are mispairing and crossing over in meiosis, chromosome breaks with repair, nonhomologous pairing due to low copy repeats and crossing over, and translocation (familial or de novo) with segregation. Cases include: [see: text].     

ULTRASTRUCTURAL AND PHYSIOLOGICAL DIFFERENCES IN SOYBEANS WITH GENETICALLY ALTERED LEVELS OF PHOTOSYNTHETIC PIGMENTS

Soybeans displaying incomplete dominance for leaf pigmentation possess chloroplasts with characteristic shape and organization of photosynthetic membranes. The chloroplasts of light green plants lack grana typical of dark green or a field type (Beeson) but normally possess doubled thylakoids. Achlorophyllous lethal yellow plants have thylakoids reduced to single spiralled membranes. The yellow plants lack a waxy cuticle over the leaf surface which is characteristic of all other soybeans examined, and they lack catalase activity in microbodies. Photosynthetic rates in the light green plants are at least as high as in the fully pigmented ones and photorespiration levels are not significantly different. Thus, in light green plants greater efficiency of enzymatic processes in photosynthesis apparently offsets the inhibitory activities associated with photorespiration. Single allele alterations from dark green to light green and light green to lethal yellow appear to alter a variety of structures and functions.     

Five primary trisomics from translocation heterozygote progenies in common bean,Phaseolus vulgaris L.

Summary Twelve distinct phenotypic groups of plants were isolated from nondisjunction progenies of 11 translocation heterozygote stocks. All the plants in these phenotypic groups originated in the light weight seed class. Five of the 12 phenotypic groups of plants have been verified as primary trisomics. They are all phenotypically distinguishable from each other and from disomics. One of the five primary trisomic groups, puckered leaf, was directly recovered as a primary trisomic from the original translocation heterozygote progenies. Three of the five trisomics — weak stem, dark green leaf, and convex leaf — originated first as tertiary trisomics. The related primary trisomics were isolated later from progenies of selfed tertiary trisomics. The fifth group, chlorotic leaf, originated at a low frequency among the progenies of three other trisomics: puckered leaf, convex leaf, and dark green leaf. The chlorotic leaf did not set seed under field conditions. The remaining four groups — puckered leaf, dark green leaf, convex leaf, and weak stem — are fertile, though sensitive to high temperature conditions. The transmission rate of the extra chromosome on selfing ranges from 28% to 41%. Physical identification of the extra chromosome has not been achieved for any of the five trisomic groups. Two trisomic groups, dark green leaf and convex leaf, have produced tetrasomics at low frequency. The phenotypes of these two tetrasomics are similar to the corresponding trisomics but more exaggerated.Fla. Agr. Expt. Stn. Journal Series No. 7137     

Ac-Induced Instability at the Xanthophyllic Locus of Tomato

To detect genomic instability caused by Ac elements in transgenic tomatoes, we used the incompletely dominant mutation Xanthophyllic-1 (Xa-1) as a whole plant marker gene. Xa-1 is located on chromosome 10 and in the heterozygote state causes leaves to be yellow. Transgenic Ac-containing tomato plants which differed in the location and number of their Ac elements were crossed to Xa-1 tester lines and F(1) progeny were scored for aberrant somatic sectoring. Of 800 test and control F(1) progeny screened, only four plants had aberrantly high levels of somatic sectors. Three of the plants had twin sectors consisting of green tissue adjacent to white tissue, and the other had twin sectors comprised of green tissue adjacent to tissue more yellow than the heterozygote background. Sectoring was inherited and the two sectoring phenotypes mapped to opposite homologs of chromosome 10; the green/yellow sectoring phenotype mapped in coupling to Xa-1 while the green/white sectoring phenotype mapped in repulsion. The two sectoring phenotypes cosegregated with different single, non-rearranged Acs, and loss of these Acs from the genome corresponded to the loss of sectoring. Sectoring was still observed after transposition of the Ac to a new site which indicated that sectoring was not limited to a single locus. In both sectored lines, meiotic recombination of the sectoring Ac to the opposite homolog caused the phenotype to switch between the green/yellow and the green/white phenotypes. Thus the two different sectoring phenotypes arose from the same Ac-induced mechanism; the phenotype depended on which chromosome 10 homolog the Ac was on. We believe that the twin sectors resulted from chromosome breakage mediated by a single intact, transposition-competent Ac element.     

Sporophytic nondisjunction of the maize B chromosome at high copy numbers

It has been known for decades that the maize B chromosome undergoes nondisjunction at the second pollen mitosis.Fluorescence in-situ hybridization(FISH)was used to undertake a quantitative study of maize plants with differing numbers of B chromosomes to observe if instability increases by increasing B dosage in root tip tissue.B chromosome nondisjunction was basically absent at low copy number,but increased at higher B numbers.Thus,B nondisjunction rates are dependent on the dosage of B's in the sporophyte.Differences in nondisjunction were also documented between odd and even doses of the B.In plants that have inherited odd humbered doses of the B chromosome,B loss is nearly twice as likely as B gain in a somatic division.When comparing plants with even doses of B's to plants with odd doses of B's,plants with even numbers had a significantly higher chance to increase in number.Therefore,the B's nondisjunctive capacity,previously thought to be primarily restricted to the gametophyte,is present in sporophytic cells.     

Mitomycin C induced leaf mosaicism in Glycine max (L.) Merrill in relation to the post-germination age of the seed

Summary The frequency of mitomycin C induced somatic crossing over in variety L65-1237 of Glycine max is shown to be dependent upon the (physiological) age of the seed during post germination period. Effect of mitomycin C during the first four hr of germination is significantly lower than during later periods. This increase in the frequency of somatic crossing over is observed up to about 20–24 hr and is then followed by a decrease. These changes did not appear to be related to the onset and pattern of synthesis of DNA or/and proteins in the embryonic tissues. However, mitomycin C is effective even when no DNA synthesis is going on.     

From the phenomenon of nondisjunction to the problem of chromosome co-orientation (75th anniversary of Bridges' article]

The concepts of the mechanism of chromosome nondisjunction in Drosophila are described in a historical retrospective. Evidences are given for the appropriateness of the term co-orientation in the traditional sense used by geneticists treating nondisjunction. There are 6 variants of co-operation in Drosophila meiosis depending upon the number and particular chromosomes involved in co-orientation. The classical chromosome nondisjunction is a variant of co-orientation in the bivalent composed of two homologous chromosomes. By comparing the different variants of pairing (pairing in bi- and multivalents) resulting in co-orientation, the elementary events preceding co-orientation may be identified. The author reviews his recent data concerning the similarities of the co-orientation of two homologs and the co-orientation of two nonhomologs in Drosophila meiosis. The concept of the role of pairing in the precentromeric heterochromatic region during chromosome co-orientation is considered, and the hypothesis of delayed pairing in this region during meiotic prophase is put forward. Based on the suggested hypothesis clarified are (i) the relationship of pairing, crossing over, and disjunction of homologous chromosomes (ii) the relationship of crossing over and co-orientation of nonhomologous chromosomes, and (iii) the time when the contact resulting in nonhomolog co-orientation takes place.     

Characterization of X-ray induced increase of mitotic cross-overs in Glycine max

Summary Three types of spots can be identified on the leaves of heterozygous light green, Y/y, Glycine max (L.) Merrill: dark green (D) and aurea (A) single spots (resembling the phenotypes of the homozygotes) and double (Db) spots consisting of adjacent D and A tissue. X-irradiation increased the frequency of each type of spot on simple and first compound leaves. The Db spots, indicative of mitotic crossing-over (MCO), increase linearly with increasing dosage. Moisture content of the seeds was independent of the rate of spot increase. At high dosages morphological alterations were observed, including spots on homozygotes, leaf area reduction, smaller seedlings, and abnormal leaf shapes. The frequency of light green spots on normal dark green, Y/Y, seedlings was tabulated and, as with all other spot types, increased with increasing X-ray dosage. Dormant soybean seeds contain leaf primordia of both simple and first compound leaves. Mature simple leaves contained more spots, reflecting a larger primordial cell number, while first compound leaves had larger spots, since each affected cell underwent more mitoses prior to leaf maturation. Within first compound leaves, the terminal leaflets developed asynchronously in relation to the lateral leaflets. Terminal leaflets were shown to be initiated first, have a larger percentage of the leaflet area covered with spots, and have larger mature leaflet area. The spontaneous rate of MCO, 3.39×10^–5 MCO events per mitosis, was increased 282-fold by 1600 R. We also ascertained that Mitomycin C is more specific for Db spot induction than X-rays. These results are compared with our similar irradiation experiments on tobacco shoot apices.     

Unusual in vivo rearrangements of the Y chromosome with mitotic instability in vitro

Summary An unusual structural rearrangement of the Y chromosome resulting in seven different cell lines was found in a male infant with coronal synostosis as the only major clinical symptom. At least three different events must have occurred to explain the patient's karyotype: nondisjunction, chromosome breakage followed by translocation, and tetraploidization followed by somatic recombination. In addition, the structurally abnormal Y chromosome appeared to be unstable in vitro.