Application of a wheat seedling assay for detecting aneuploidy induced by N-ethyl-N-nitrosourea and 4-nitroquinoline-1-oxide.

N-Ethyl-N-nitrosourea (ENU) and 4-nitroquinoline-1-oxide (4NQO) were evaluated in the allohexapolyploid wheat seedling assay developed by Redei and Sandhu (1988), for its ability to induce aneuploidy and/or small chromosome deletions. The wheat strain used (Neatby's virescent) is homozygous for a pair of recessive alleles (v1) present on chromosome 3B and produces virescent seedlings grown at temperatures below 26 degrees C. When the developing embryos are treated with a test chemical, loss of chromosome 3B or its segment bearing the v1 allele in a progenitor cell produces a green sector in the leaf, whereas a gain of this chromosome induces a white sector. ENU and 4NQO induced dose-dependent increases in the frequency of leaf sectors at concentrations ranging from 0.128 to 1.280 mM and 0.052 to 0.263 mM, respectively. The assay is very simple and can be employed for evaluating the genetic potential of chemicals in a laboratory as well as for in situ hazards assessment under natural environmental conditions.     

The detection of monosomic colonies produced by mitotic chromosome non-disjunction in the yeast Saccharomyces cerevisiae

A diploid yeast strain, D6, is described which monitors mitotic non-disjunction by the phenotypic expression of a set of coupled and recessive markers flanking the centromere of chromosome VII. These markers are not expressed in the heterozygous condition prevailing in D6. The left arm of chromosome VII carries a tightly centromere linked marker, leu1 (leucine requirement), distal to leu1 in this order: trp5 (trytophan requirement), cyh2 (recessive resistance to cycloheximide) and met 13 (requirement for methionine). The right arm is marked with ade3 (simultaneous requirement for adenine and histidine). D6 is homozygous for ade2 and consequently, forms red rather than the normally white colonies. It shows no requirement for the above amino acids and it is sensitive to cycloheximide.Unmasking of all the markers on chromosome VII leads to colonies that are white because ade3 sets a block preceding the ade2 block (which causes the accumulation of a precursor of the red pigment), they require leucine, tryptophan and methionine, and grow on media with cycloheximide. Cells are plated on a cycloheximide medium where red and white colonies are formed. Colonies of spontaneous origin were tested. The majority of the white colonies expressed all the recessive markers whereas only few of the red colonies expressed all the markers on the left arm of chromosome VII.Basically expression of recessive markers on both sides of the centromere can be explained as a result of two coincident events of mitotic crossing over. However, the frequency of colonies expressing centromere linked leu1 was 14 times higher among the white types than the red ones. This suggested that the white, cycloheximide resistant, leucine requiring colonies arose by mitotic non-disjunction and not only by two coincident mitotic crossing over events.Presumptive spontaneous monosomic segregants were placed on sporulation medium. Only 8 out of 30 isolates sporulated, which showed that these eight segregants were diploid at the time of sporulation. They could have arisen by two coincident crossover events or through restoration of a normal disomic condition after non-disjunction had occurred. The genetic data thus leaves us with only its statistical argument in favour of non-disjunction. Further confirmation of monosomic nature of the white cycloheximide resistant colonies was provided by the estimates of their DNA contents. Compared to the stock wild type diploids the presumptive monosomics showed a reduction in DNA content.We have utilized D6 to investigate the possible induction of mitotic non-disjunction after treatment with gamma rays, heat shock at 52°C and ultraviolet irradiation. In all cases white, cycloheximide resistant colonies were produced at levels significantly higher than that found in untreated cultures. In order to detect the production of monosomic cells, treated cultures were grown for 48 h in non-selective medium after exposure to allow for “expression” of the monosomic condition.     

Detection of the loss of 1 chromosome from pair III in Saccharomyces cerevisiae yeasts

A diploid strain of Saccharomyces cerevisiae, T6 is described which monitors both mitotic crossing over and induction of aneuploidy. The chromosome III carries recessive markers: rgh12 of "rough colony" phenotype closely linked to centromere, the left arm is marked with his4, the right arm is marked both with thr4 and the locus of mating type alpha. Expression of all the markers on chromosome III leads to formation of colonies which are rough, require histidine and threonine, and are of alpha mating type. These colonies arise as a result of the loss of a chromosome during mitosis, which makes the strain allow detection of monosomic cells formation. Chromosome XV carries two phenotypically distinguishable and recessive alleles of the gene ade2: ade2-192 (causes red coloration of colonies) and ade2-G45 (causes pink coloration of colonies). Mitotic crossing over generates two reciprocal products which can be revealed together in colonies as pink and red sectors in double-spotted colonies. Both double-spotted and monosomic colonies have been obtained after treatment with gamma-rays. The frequency of mitotic crossing over after irradiation by 1000-3000 Gray increased up to 2-3.2% (the spontaneous level was 0.006%), the frequency of aneuploidy was 0.12 to 0.57% at plating immediately after irradiation (the spontaneous monosomics were not observed among 1.5 X 10(5) cells scored). Induction of mitotic crossing over and aneuploidy by benomyl was rather slight (up to 0.05 and 0.006%, respectively).     

Production of wheat-barley recombinant chromosomes through induced homoeologous pairing

Summary Sears' phlb mutant was used successfully for the first time to induce pairing and recombination between specific barley chromosomes and their wheat homoeologues. Pairing was induced in specially constructed genetic stocks having 19 pairs of wheat chromosomes and triply monosomic for either barley chromosome arm 6HL or 3HL, a related wheat chromosome, and chromosome 5B of wheat carrying the phlb mutation. Wheat-barley recombinant chromosomes were isolated from among the progeny obtained from self-fertilization of the triple monosomic stocks, by screening for dissociation of biochemical markers on the barley arms. Glutamic oxaloacetic transaminase (GOT), aconitase hydratase (ACO), and dipeptidase (DIP) isozymes were used to select recombinants involving the 6HL arm, and esterase (EST) and malate dehydrogenase (MDH) were used for the 3HL arm. Altogether, six recombinants involving 6HL (1.4%) and six involving 3HL (1.1%) were isolated. These wheat-barley recombinant chromosomes are being used to construct a detailed gene order map of barley based on biochemical and molecular markers.     

Tests which distinguish induced crossing-over and aneuploidy from secondary segregation in Aspergillus treated with chloral hydrate or gamma-rays

A system of tests with the ascomycete Aspergillus nidulans was devised that can detect 3 primary effects of genotoxic agents: (1) increases in mitotic crossing-over; (2) induced aneuploidy; and (3) clastogenic effects which cause chromosomal imbalance. Conidia of a new diploid tester strain, heterozygous for 4 recessive markers which alter conidial color, are treated and plated onto nonselective media. In cases of induced crossing-over, large color segments are found in normal green colonies, frequently adjacent to reciprocal twin segments. In contrast, both malsegregation and chromosome breakage produce unbalanced types which grow poorly and segregate further. Cases with yellow segregants are replated and their secondary diploid sectors tested for markers which are located on both chromosome arms in coupling with yA. Induced aneuploidy can be distinguished from chromosome breakage by the pattern of marker segregation. Any aneuploid type will produce euploid sectors solely by segregation of whole chromosomes; trisomic colonies (yA / yA / +) will show 1:2 ratios for yellow (homozygous yA) to parental green (yA/+) sectors and have characteristic phenotypes. Other induced unbalanced types, if heterozygous for deletions or aberrations may produce yellow diploid sectors by secondary crossing-over as well as by nondisjunction and such cases show unique patterns of genetic segregation and non- predictable phenotypes. As a complementary test, haploid strains are treated and induced abnormally growing types are replated and classified by phenotype. Aneuploids are unstable and produce many normal sectors, and some of these disomic or trisomic types can be visually identified.In contrast, induced deletions are lethal, and duplications or 'morphological' mutants show much more stable abnormal phenotypes. This test system was used to characterize the primary effects of gamma-rays and chloral hydrate. Results and evidence were as follows: (1) A dose-dependent increase of color segments resulting from reciprocal crossing-over was found after treatment of dividing nuclei in germinating diploid conidia with gamma-rays, but not with chloral hydrate. (2) Highly aneuploid and polyploid types were induced in diploid and haploid germinating conidia by chloral hydrate but not to any significant extent by gamma-rays. (3) gamma-Rays caused a dose- dependent increase off abnormally growing colonies when dormant or germinating diploid conidia were treated. These colonies produced secondary euploid sectors by spontaneous nondisjunction and frequently also by crossing-over, which provided evidence for induced semidominant and recessive lethal mutations of many types.     

单体异附加系花药培养创制小麦- 中间偃麦草纯合易位系

利用单体异附加系花药培养细胞工程途径,诱导小麦与中间偃麦草发生染色体易位,通过细胞学分析、荧光原位杂交(F ISH)和SSR鉴定出纯合易位系.研究结果表明,经单体异附加系花药培养创制出1个小麦-中间偃麦草纯合易位系99-803;其花粉母细胞(PM C s)减数分裂中期I染色体构型为18.42个环状二价体 2.57个棒状二价体 0.01个单价体;中间偃麦草的7A i-1染色体与小麦7A或7B染色体发生了非罗伯逊易位,且中间偃麦草易位片段较小;通过该途径获得纯合易位系的频率约为2%.以上结果表明,单体异附加系花药培养是一条向小麦转移异源染色体小片段(基因)的快速高效途径.     

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.     

Radiation-induced mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

A number of genetic systems are described which in yeast may be used to monitor the induction of chromosome aneuploidy during both mitotic and meiotic cell division. Using these systems we have been able to demonstrate the induction of both monosomic and trisomic cells in mitotically dividing cells and disomic spores in meiotically dividing cells after both UV light and X-ray exposure. The frequency of UV-light-induced monosomic colonies were reduced by post-treatment with photoreactivity light and both UV-light- and X-ray-induced monosomic colonies were reduced by liquid holding post-treatment under non-nutrient conditions. Both responses indicate an involvement of DNA-repair mechanisms in the removal of lesions which may lead to monosomy in yeast. This was further confirmed by the response of an excision-defective yeast strain which showed considerably increased sensitivity to the induction of monosomic colonies by UV-light treatment at low doses. Yeast cultures irradiated at different stages of growth showed variation in their responses to both UV-light and X-rays, cells at the exponential phase of growth show maximum sensitivity to the induction of monosomic colonies at low doses whereas stationary phase cultures showed maximum induction of monosomic colonies at high does. The frequencies of X-ray-induced chromosome aneuploidy during meiosis leading to the production of disomic spores was shown to be dependent upon the stage of meiosis at which the yeast cells were exposed to radiation. Cells which had proceeded beyond the DNA synthetic stage of meiosis were shown to produce disomic spores at considerably lower radiation doses than those cells which had only recently been inoculated into sporulation medium. The results obtained suggest that the yeast sustem may be suitable for the study of sensitivities of the various stages of meiotic cell division to the induction of chromosome aneuploidy after radiation exposure.     

Genetic analysis of anther culture response in wheat using aneuploid,chromosome substitution and translocation lines

Summary Marked effects of genotype on wheat anther culture response have been observed. Genetic factors have been recognised to be one of the major contributors to in vitro responses of cultured wheat tissues. In wheat anther culture, embryo induction, plant regeneration and albina/green ratio have been determined to be heritable traits. Using Chinese Spring (CS) monosomic 1D, single chromosome substitution lines of chromosome 5B or chromosome arm 5BL from Chinese Spring into six varieties, and F₁ hybrids heterozygous for the 1B chromosome structure (1BL-1BS/1BL-1RS), the anther culture response was studied: genes on CS1D chromosome and 5BL chromosome arm increases the embryo frequency; gene(s) involved in regeneration ability are located on the 1RS chromosome arm; a gene increasing albina frequency is located on Chinese Spring 5B chromosome. Our results support the fact that without gametic selection, a differential development occurred from the particular classes of microspores carrying genes for higher regeneration ability. Moreover, in some crosses, a few genes with major effects were involved in determination of anther culture response.     

Genetic control of amylose content in wheat endosperm starch and differential effects of three Wx genes

The endosperm starch of the wheat grain is composed of amylose and amylopectin. Genetic manipulation of the ratio of amylose to amylopectin or the amylose content could bring about improved texture and quality of wheat flour. The chromosomal locations of genes affecting amylose content were investigated using a monosomic series of Chinese Spring (CS) and a set of Cheyenne (CNN) chromosome substitution lines in the CS genetic background. Trials over three seasons revealed that a decrease in amylose content occurred in monosomic 4A and an increase in monosomic 7B. Allelic variation between CS and CNN was suggested for the genes on chromosomes 4A and 7B. To examine the effects of three Waxy (Wx) genes which encode a granule-bound starch synthase (Wx protein), the Wx proteins from CS monosomics of interest were analyzed using SDS-PAGE. The amount of the Wx protein coded by the Wx-B1 gene on chromosome arm 4AL was reduced in monosomic 4A, and thus accounted for its decreased amylose content. The amounts of two other Wx proteins coded by the Wx-A1 and Wx-D1 genes on chromosome arms 7AS and 7DS, respectively, showed low levels of protein in the monosomics but no effect on amylose content. The effect of chromosome 7B on the level of amylose suggested the presence of a regulator gene which suppresses the activities of the Wx genes.     

Genetic damage induced by ethyl alcohol in Aspergillus nidulans.

Heterozygous diploid conidia of Aspergillus nidulans were treated during germination with ethyl alcohol in concentrations ranging from 0.25% to 20% (v/v). The diploid strain carried three recessive conidial color mutations, in addition to genetic markers on all eight pairs of linkage groups. It was thereby possible to detect events of crossing over, non-disjunction, and mutation. An increase in the dose of ethanol was associated with a decrease in conidial viability and an increase in the relative and absolute frequencies of formation of (a) normal colonies which produced colored sectors and (b) phenotypically abnormal colonies, the majority of which (83.1%) produced normal sectors. At a concentration of 5% (v/v) ethanol, the survivors included 17.59% of the former and 44.7% of the latter colonies. Genetic analysis of the various segregants suggested that the frequencies of both mitotic crossing over and non-disjunction or the misdistribution of chromosomes were increased by ethanol. Among 133 abnormal colonies which segregated normal clones, 79 (59.4%) were associated with one of these genetic events. A total of 297 haploids and 130 diploids arose as normal segregants from the abnormal colonies. There were 31 recognizable events of non-disjunction and 14 crossing over in linkage groups I and II, where these events could be distinguished. These data suggested that the predominant effect of ethanol was a disruption of chromosome distribution. A cytological examination of ethanol-treated, germinating conidia revealed an interference with the mitotic spindle apparatus. The frequency of detectable spindles decreased more than 3-fold after 8 h exposure to 5% (v/v) ethanol. This finding supported the conclusion that ethanol disrupted chromosome distribution, and suggested the mechanism by which it does so. Human clinical data on alcohol consumption were examined in light of these findings.     

Okadaic acid: chromosomal non-disjunction analysis in human lymphocytes and study of aneugenic pathway in CHO-K1 cells

Okadaic acid (OA) is the main marine toxin implicated in the diarrhetic shellfish poisoning (DSP) in humans after consumption of contaminated bivalve molluscs. We have previously shown that OA was an in vitro aneugenic compound that induced chromosome loss via micronuclei formation in CHO-K1 cells. The aims of this study were to investigate the chromosomal non-disjunction (ND) potential of OA in human lymphocytes and the pathways involved for aneuploidy in CHO-K1 cells. Firstly, we analysed the formation of micronuclei and the non-disjunction for chromosomes 1 and 17 in binucleated human lymphocytes cells with the cytokinesis-blocked micronucleus (CBMN) assay coupled to a fluorescent in situ hybridization (FISH) technique with centromere-specific DNA probes. We showed that OA statistically increased the frequency of micronucleated lymphocytes in the dose range from 20 to 35 nM. However, FISH analysis did not reveal any increase in the non-disjunction for both chromosomes whatever the concentration between 2.5 and 35 nM. However, a significant increase in ND for the chromosome 17 was found at 1 nM. Secondly, in CHO-K1 cells, we investigated the dose and time dependent effects of OA: (i) on cell cycle progression, (ii) on mitotic-phase arrest and (ii) on mitotic spindle and centrosome abnormalities. The results showed that OA induced a progressive accumulation of mitotic CHO-K1 cells in prometaphase, an induction of multipolar mitotic spindle with centrosome amplification and the formation of multinucleated cells. We concluded that OA did not induce chromosome non-disjunction but should more likely induced chromosome loss in human lymphocytes. Moreover, our results obtained in CHO-K1 suggest that OA induced aneuploidy by preventing the chromosome attachment to the mitotic spindle and by amplifying the centrosome. The mode of action of the toxin in relation to its inhibition of protein phosphatases 1 (PP1) and 2A (PP2A) and the mitosis process is discussed.     

Isolation, characterization, and genetic analysis of monosomic, aneuploid mutants of Candida albicans

A white, prototrophic Candida albicans strain, heterozygous for the ADE2 gene (ade2/ADE2), was treated with the antimitotic agent methyl benzimidazole carbamate, and yielded red, adenine-requiring colonies at a rate of 4 x 10(-3), an order of magnitude higher than the spontaneous rate of Ade- colony formation. These red Ade- colonies were small, growing at approximately half the rate of the parent strain, and gave rise to large red colonies spontaneously. When the chromosomes of the small red colonies were separated by pulsed-field gel electrophoresis, the band hybridizing with the ADE2 gene was diminished in staining intensity by half relative to the parent and large red-colony strains. Restriction fragment-length polymorphism analysis and auxotrophic mutant spectra after mutagenesis suggested that the small red Ade- strains were monosomic aneuploids lacking one of a pair of chromosome homologues, while the large red strains had regained a homologue, presumably via a second non-disjunction event. Parasexual genetic analysis of two of the auxotrophs isolated from a putative aneuploid suggested that both mutations were linked to the ADE2 gene. These experiments suggest that targeted chromosome loss and monosomic, aneuploid strains have the potential to extend the scope of genetic analysis in this diploid, asexual organism.     

Importance of detecting numerical versus structural chromosome aberrations

The aim is to review briefly the key questions related to aneuploidy/polyploidy and to compare the advantages and disadvantages of the in vitro micronucleus test to assess aneuploidy/polyploidy in vitro. The key questions that will be addressed, concern the importance of polyploidy for health, and cancer in particular, the mechanisms leading to aneuploidy and polyploidy, and the survival of aneuploid/polyploid cells.The recently recognised contribution of numerical chromosome changes to carcinogenesis triggered the development and the implementation of tests specifically aiming at the detection of aneugens in the test battery for mutagenicity and carcinogenicity. The validation of the in vitro micronucleus test in combination with the identification of in vitro divided cells with the cytokinesis-block methodology and of centromeres with pancentromeric or chromosome specific centromeric probes fluorescence in situ hybridisation (FISH) provides a sensitive, easy to score and powerful test which allows assessment of cell proliferation, the discrimination between chromosome breaks, chromosome loss and chromosome non-disjunction and polyploidy. Moreover, classic histology permits the estimation of necrosis and apoptosis on the same slide. The cytokinesis-blocked micronucleus assay could be considered as a multi-endpoint test for genotoxic responses to clastogens/aneugens. This methodology has also shown to be capable of identifying threshold values for the induction of chromosome loss and/or non-disjunction by microtubule inhibitors, data which are particularly important for risk calculations. Similar approaches were conducted in vivo on bone marrow in mice and rats (except for identification of chromosome non-disjunction), and are in development for gut in mice.     

应用基因组原位杂交及RFLP标记鉴定小麦中的大麦染色体

用生物素（Biotin-6-dUTP)标记的大麦Betzes基因组DNA作探针,以普通小麦中国春总DNA作封阻进行基因组原位杂交（Genomeinsituhybridization,简称GISH）,从13株小麦-大麦杂交后代中鉴定出2个含有3条大麦Betzes2H染色体的材料（2n＝43）;2个2H单体异代换系（2n＝42）;7个2H二体异代换系（2n＝42）。用已定位在小麦第2部分同源群短臂上的探针psr131进行RFLP分析,结果表明大麦Betzes、代换系A5有1条区别于小麦中国春的特异带,A     

A genetic analysis of a tomato (Lycopersicon esculentum) genotype with a high frequency of twin spots

Among pale-green tomato plants heterozygous for the xanthophyllic2 (xa-2) mutation that were transformed with a T-DNA harbouring the NPTII and GUS gene, a plant with a high frequency of green/white twin spots was found. The genetic analysis of this plant indicated that the occurrence of these twin spots was caused by a genetic defect located at the distal end of chromosome 10S, where xa-2 also is located. The genetic analysis of green plants regenerated from leaf expiants of this twin-spot plant revealed that the green sectors derive from non-disjunction of the xa-2 ⁺ allele. In an analysis of mitotic chromosome behaviour bridges were observed in approximately 5% of the anaphases, providing arguments that a breakage-fusion-bridge cycle caused by a tissue culture-induced genomic instability is the most likely cause of this aberrant behaviour of chromosome 10.     

一种小麦蓝粒标记单体代换系4E（3D）的创制

以中国春3D单体和小麦-长穗偃麦草4E二体异附加系为材料,通过杂交、回交结合染色体鉴定等方法,培育出了一种具有蓝粒标记的小麦4E(3D)单体代换系.该小麦4E(3D)单体代换系籽粒为浅蓝色,能够正常生长,自交结实率为36.1%,其自交后代可分离出深蓝籽粒小麦4E(3D)二体代换系、浅蓝籽粒小麦4E(3D)单体代换系和白粒小麦3D缺体.结果表明,长穗偃麦草4E染色体对小麦3D染色体缺失有一定的补偿功能,对以染色体定向代换方式快速创制蓝粒标记小麦单体系统具有一定的参考价值.     

The origin, identification, and plant morphology of five rice monosomics.

Five different monosomics of rice (Oryza sativa L.) were obtained by treatment of pollen with gamma irradiation, as a by-product of attempts to determine the cytological loci of certain marker genes, i.e., mature pollen carrying normal alleles at all loci was given gamma rays and used for pollinating strains that were homozygous for recessive marker genes. The monosomics showed distinguishable morphological features and had complete seed sterility. Cytological studies revealed that one monosomic was tertiary, the others primary. The tertiary monosomic was related to chromosome 10. Two primary monosomics for chromosomes 10 and 11 were identified. At metaphase I, the tertiary monosomic showed the chromosome configurations 1 III + 10 II, 11 II + 1 I, and 10 II + 3 I, and all primary ones showed the configuration 11 II + 1 I. All five monosomics showed very poor crossing ability and were not transmitted to the few progenies observed. A few trisomic plants were found in the progenies of a cross between monosomic and normal pollen in one monosomic. This is the first time that many monosomics in rice have been characterized. This information will be useful in studies of rice aneuploidy and cytogenetics. Key words : rice, monosomics, morphology, cytology, transmission, trisomics.     

The detection and assessment of the aneugenic potential of selected oestrogens, progestins and androgens using the in vitro cytokinesis blocked micronucleus assay

The use of 17-beta-oestradiol, testosterone, progesterone, zearanol, trenbolone acetate and melengesterol acetate in animal feed as growth promoters has been banned in the European Union since 1989. However, the data available on their genotoxicity is limited. To bridge this gap the present study was carried out with the aim of evaluating these hormones for their ability to induce aneuploidy. Aneuploidy has been recently considered sufficiently important to be included in the routine testing of chemicals and radiation. These types of numerical chromosomal aberrations may arise by at least two mechanisms, chromosome loss and non-disjunction. Over the past few years, the cytokinesis blocked micronucleus (CBMN) technique has evolved into a robust assay for the detection of aneuploidy induction. At the present time, it is the only assay which can reliably detect both chromosome loss and non-disjunction when the basic methodology is coupled with appropriate molecular probing techniques such as immunoflourescent labelling of kinetochores and Fluorescence in situ Hybridisation. In this present study, aneuploidy induction by three groups of hormones was studied using CBMN assay coupled with Fluorescence in situ Hybridisation. The results from the present study demonstrate that 17-beta-oestradiol, diethylstilboestrol, progesterone and testosterone are genotoxic and induce aneuploidy by non-disjunctional mechanism, whereas trenbolone is also genotoxic by a clastogenic mechanism. However, melengesterol acetate and zearanol proved to be non-genotoxic in vitro.     

Patterns of DNA and chromosome variation during in vitro growth in various genotypes of potato

Patterns of variation in nuclear DNA content and chromosome number were analysed in a temporal sequence, during in vitro growth of calli and cell suspensions in two monohaploids, a dihaploid and a tetraploid of potato (Solanum tuberosum). The results showed that both polyploidization and aneuploidy occurred during the initial stages of callus induction in all the genotypes. With further growth of callus, the frequency and extent of polyploidy and aneuploidy increased. In addition, the patterns of DNA and chromosome variation in cell suspension cultures revealed continued mitotic activity and transmission of cells with higher ploidy levels and aneuploidy. The results suggest that endoreduplication as well as endomitosis are important mechanisms of polyploidization, and that chromosome lagging and non-disjunction contribute to the production of aneuploidy.The various genotypes cultured under the same in vitro growth conditions differed in genetic instability, as assessed from the rate and degree of polyploidization and aneuploidy. Monohaploids showed more rapid rate of polyploidization than the dihaploid and tetraploid potatoes. It was concluded that the differences in genetic stability were due to different ploidy levels and genetic make-up of the genotypes.