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Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Consumption of meat and fish and risk of lung cancer: results from the European Prospective Investigation into Cancer and Nutrition
    Linseisen, J. ; Rohrmann, S. ; Bueno-de-Mesquita, B. ; Büchner, F.L. ; Boshuizen, H.C. ; Agudo, A. ; Gram, I.T. ; Dahm, C.C. ; Overvad, K. ; Egeberg, R. ; Tjonneland, A. ; Boeing, H. ; Steffen, A. ; Kaaks, R. ; Lukanova, A. ; Berrino, F. ; Palli, D. ; Panico, S. ; Tumino, R. ; Ardanaz, E. ; Dorronsoro, M. ; Huerta, J.M. ; Rodríguez, L. ; Sánchez, M.J. ; Rasmuson, T. ; Hallmans, G. ; Manjer, J. ; Wirfält, E. ; Engeset, D. ; Skeie, G. ; Katsoulis, M. ; Oikonomou, E. ; Trichopoulou, A. ; Peeters, P.H. ; Khaw, K.T. ; Wareham, N. ; Allen, N. ; Key, T. ; Brennan, P. ; Romieu, I. ; Slimani, N. ; Vergnaud, A.C. ; Xun, W.W. ; Vineis, P. ; Riboli, E. - \ 2011
    Cancer Causes and Control 22 (2011)6. - ISSN 0957-5243 - p. 909 - 918.
    heterocyclic amines - dietary habits - heme iron - women - calibration - cohort - recalls - mortality - mutagens - fat
    Evidence from case–control studies, but less so from cohort studies, suggests a positive association between meat intake and risk of lung cancer. Therefore, this association was evaluated in the frame of the European Prospective Investigation into Cancer and Nutrition, EPIC. Data from 478,021 participants, recruited from 10 European countries, who completed a dietary questionnaire in 1992–2000 were evaluated; 1,822 incident primary lung cancer cases were included in the present evaluation. Relative risk estimates were calculated for categories of meat intake using multi-variably adjusted Cox proportional hazard models. In addition, the continuous intake variables were calibrated by means of 24-h diet recall data to account for part of the measurement error. There were no consistent associations between meat consumption and the risk of lung cancer. Neither red meat (RR = 1.06, 95% CI 0.89–1.27 per 50 g intake/day; calibrated model) nor processed meat (RR = 1.13, 95% CI 0.95–1.34 per 50 g/day; calibrated model) was significantly related to an increased risk of lung cancer. Also, consumption of white meat and fish was not associated with the risk of lung cancer. These findings do not support the hypothesis that a high intake of red and processed meat is a risk factor for lung cancer
    Classical mutagenesis in higher plants
    Koornneef, M. - \ 2002
    In: Molecular Plant Biology / Gilmartin, P.M., Bowler, C., Oxford, GB : Oxford University Press - p. 1 - 10.
    genetica - mutagenese - mutanten - plantenveredeling - mutagenen - genetics - mutagenesis - mutants - plant breeding - mutagens
    For a long time, mutagenesis research in plants focused on crop improvement and, especially for crop plants, opimised protocols were developed with barley being one of the favourite species. However, the interest in mutagenesis has shifted to basic plant research in the last 20 years, when the power of mutant approaches in combination with molecular techniques to investigate the molecular nature of the genes became fully appreciated
    Genetic analysis of seed development in Arabidopsis thaliana = [Genetische analyse van de zaadontwikkeling in Arabidopsis thaliana]
    Leon - Kloosterziel, K. - \ 1997
    Agricultural University. Promotor(en): M. Koornneef. - S.l. : Leon-Kloosterziel - ISBN 9789054857709 - 119
    genen - genomen - mutaties - mutagenese - mutagenen - plantenfysiologie - plantenontwikkeling - vruchten - rijp worden - brassicaceae - zaadzetting - zaden - formatie - kieming - zaadkieming - kiemrust - genes - genomes - mutations - mutagenesis - mutagens - plant physiology - plant development - fruits - ripening - brassicaceae - seed set - seeds - formation - germination - seed germination - seed dormancy

    This thesis deals with the genetic aspects of seed development in Arabidopsisthaliana. Mutants affected in several aspects of seed development and, more specifically, in seed maturation have been isolated by various selection procedures. The mutants have been analyzed genetically, physiologically, and morphologically. Some of the mutants are impaired in the biosynthesis or sensitivity to the plant hormone, abscisic acid (ABA). All ABA-related mutants show reduced seed dormancy, indicating the important role of this hormone in the establishment of dormancy. In a direct screen for reduced dormancy, two mutants (rdo) with reduced dormancy were found. These were not ABA-deficient and showed the same sensitivity to ABA, ethylene, auxin, and cytokinin as the wild-type. In contrast to this embryo-determined reduced dormancy, reduced dormancy can also originate in an altered seed coat (testa), like in the altered testa shape ( ats ) mutant. Here, the altered testa shape is caused by a defect in the development of the integuments. Extreme ABA-insensitive mutants ( abi3 ) have green seeds that fail to complete many other aspects of seed maturation, including the induction of dormancy and desiccation tolerance, and the accumulation of seed storage proteins and lipids. In addition to abi3 mutants, lec and fus mutants exhibit such a severely disturbed seed maturation as well, with dark purple seeds due to anthocyanin accumulation. The fus3 mutant shows normal ABA-sensitivity. These various seed maturation mutants indicate that specific genes, some acting dependently and some acting independently from ABA, are responsible for seed maturation programs. The seed maturation mutants were subjected to a physiological and biochemical analysis. A GA-deficient mutant was combined with these mutants. Analysis of these double mutants indicated that seeds of the abi3 and lec mutants did not require GA for germination, in contrast to fus3 seeds. This correlates with ABA-sensitivity for germination. The composition of storage proteins and carbohydrates in abi3 , lec, and fus3 mutant seeds has been compared. The abi3 , lec, and fus3 mutants all showed severely reduced storage proteins. The desiccation intolerance of these seed maturation mutants was not correlated with the lack of specific carbohydrates. Furthermore, the mutants had a higher total content of carbohydrates. This is probably a consequence of the lower levels of storage lipids and proteins.
    Desynapsis and FDR 2N-megaspore formation in diploid potato : potentials and limitations for breeding and for the induction of diplosporic apomixis
    Jongedijk, E. - \ 1991
    Agricultural University. Promotor(en): J.G.T. Hermsen; J. Sybenga. - S.l. : Jongedijk - 111
    solanum tuberosum - aardappelen - cytogenetica - meiose - mutaties - mutagenese - mutagenen - apomixis - parthenogenese - solanum tuberosum - potatoes - cytogenetics - meiosis - mutations - mutagenesis - mutagens - apomixis - parthenogenesis - cum laude

    The cultivated potato, Solanum tuberosum L., is a highly heterozygous autotetraploid (2n=4x=48) plant species, which after its introduction into Europe in the 16th century has become one of the world's major food crops. The potato has traditionally been grown from tubers. However, as tubers are an excellent substrate for many pathogens and parasites, it is extremely difficult and expensive to produce healthy seed tubers. Most developing countries lack both the knowledge and infrastructure required for the efficient production of healthy seed tubers and the currency for its importation and distribution. As a consequence small farmers in developing countries are generally forced to use diseased tubers from a previous harvest, which may result in dramatic yield losses. In response to the urgent need of cheap but healthy plant material the International Potato Center (CIP) in Peru has, since the Planning Conference on this subject in 1979, propagated the new technology of growing potatoes from true seeds, True potato seeds are relatively easy and cheap to produce and even when harvested from heavily diseased plants generally do not carry any diseases. However one of the major problems in breeding potato varieties that can be maintained and grown from true seeds is the lack of uniformity. True seed progeny of existing varieties or inter-varietal hybrids is mostly highly heterogeneous due to the extreme heterozygosity of the potato.

    Several methods to synthesize sufficiently uniform true potato seed varieties have been proposed. One of these methods takes advantage of the frequent occurrence of numerically unreduced (2n) gametes in wild and cultivated diploid potato species, which enables the production of hybrid tetraploid progeny from tetraploid-diploid (unilateral sexual polyploidization) or diploid-diploid (bilateral sexual polyploidization) matings. The vigour and uniformity of tetraploid populations produced in this way, largely depends on the mode of 2n-gamete formation in the selected diploid parents. Depending on the genetic consequences of meiotic abnormalities that result into 2n-gamete formation, two distinct modes, viz. first division restitution (FDR) and second division restitution (SDR), can be distinguished. In general FDR is considered superior to SDR because of its abilitity to preserve a relatively large amount of favourable parental heterozygosity, including complex types of epistasis. In this respect the combination of FDR 2n-gamete formation with mutant synaptic genes that substantially reduce gene recombination, is of particular significance as it would provide a means to enhance the ability of FDR 2n-gametes to pick up the genetic constitution of the selected diploid parents with a minimum amount of reassortment. Using synaptic mutants with a virtually complete lack of gene recombination maximum performance and nearly complete uniformity may thus be expected from 2xFDR-2xFDR crosses.

    Complete or nearly complete uniformity might also be achieved by the induction of apomictic seed formation. Apomixis sensu stricto is the asexual development of maternal embryo's and seeds and thus combines the advantages of both vegetative propagation (uniformity) and generative propagation (disease free plant material). Apomictic embryos may arise either directly from somatic cells outside the embryosac (adventitious embryony) or from unreduced and unfertilized (parthenogenesis) cells, usually egg cells, of the embryosac (gametophytic apomixis). In the latter case unreduced embryosacs are formed that may be of either aposporic or diplosporic origin. In apospory it develops directly from a somatic, mostly nucellar cell of the ovule. In diplospory the unreduced embryosac derives from a generative archesporial cell of the ovule, either directly by omission of meiosis or indirectly by modified meiosis in which neither reduction in chromosome number nor (substantial) gene recombination takes place. Fertilization of the secondary embryosac: nucleus may or may not be required as a stimulus for endosperm formation and subsequent parthenogenetic development of the unreduced egg cells into mature embryos and seeds (pseudogamous and autonomous apomixis respectively).

    Although apomictic seed formation has never been observed in Solanaceae its importance for growing potatoes from true seeds justified an attempt to breed for apomictic reproduction in potato. The research described in this thesis focussed on the perspectives for inducing gametophytic apomixis, in particular pseudogamous diplosporic apomixis, because:
    1. The manipulation of gametophytic apomixis (i.e., apospory and diplospory) is to be preferred to adventitious embryony as the presence of embryosacs; enclosing apomictic embryos is an efficient barrier against most viruses and thus greatly facilitates the production of virus free true potato seeds.
    2. Apospory has never been detected in potato and thus would require an extensive mutation breeding programme. In view of the scanty and in many cases contradictory knowledge about its genetic control (monogenic-polygenic; dominant-recessive) and the lack of efficient screening methods for identifying 'positive' mutations, the outcome of such a trial and error procedure would be highly uncertain.
    3. It has been suggested that apomixis consists of a number of distinct and genetically controlled elements that lie within the reproductive potentialities of sexual plants. In case of diplosporic apomixis the elements that can roughly be distinguished are a strongly reduced chromosome pairing and/or reduced gene recombination (asynapsis/desynapsis), the formation of unreduced megaspores and embryosacs through FDR, and parthenogenetic development of unreduced egg cells into mature embryos and seeds. As some of these elements - viz. recessive genes for asynapsis/desynapsis, genes for (SDR) 2n-megaspore formation and genes for parthenogenesis along with pseudogamy - are available in potato, their combination in a single genotype may be feasible.
    4. An attempt to induce diplospory requires detailed studies on the, as yet unkown, effects of mutant synaptic genes in female meiosis and the largely obscure causes and mechanisms of 2n- megaspore formation. Such studies might thus provide vital 'spin off' results to be used in pursuing the as yet utopian production of vigorous and uniform tetraploid true potato seed varieties from 2xFDR-2xFDR crosses.

    An accurate knowledge of the normal pattern of female meiosis and embryosac formation is essential for deciding whether or not an unreduced embryosac is of diplosporic origin and for recognizing abnormalities in female meiosis that are associated with the expression of mutant synaptic genes and the formation of 2n-eggs. Therefore, normal meiosis and embryosac formation were studied in several diploid potato clones (Chapter 1). In contrast to results reported in the literature, this study indicated that the archesporium of potato cannot be delimited to a single cell. A surplus of archespores sometimes developed into normal sexual embryosacs. So the occurrence of multiple embryosacs within a single ovule need not necessarily be due to apospory as had previously been suggested by some potato researchers. On the basis of the normal sequence of female meiosis it was inferred that 2n-megaspores, if formed by meiotic abnormalities in normal synaptic plants, are likely to be of exclusive SDR origin, whereas meiotic abnormalities resulting in consistent FDR 2n-megaspore formation, and thus the induction of diplospory, would actually require mutant synaptic conditions. Subsequent research was therefore primarily focussed on (1) the identification and characterization of mutant synaptic genes that express in female meiosis, (ii) the identification of synaptic mutants with consistent FDR 2n-megaspore formation and (iii) the elucidation of the cytological mechanisms of FDR 2n-megaspore formation.

    For this purpose, first quick routine methods allowing for large scale screening and detailed studies of female meiosis had to be developed, because conventional embedding-sectioning techniques are very laborious and hamper quantitative interpretation of meiotic processes. Two techniques were developed. One enabled the screening of female meiosis and embryosac development in intact methyl salicylate cleared ovules and permitted bulk preparation of fixed ovaries within 2 hours (Chapter 2). The other, a 30 minute enzyme-squash procedure, allowed for detailed studies on the effect of mutant synaptic genes and genes for 2n-megaspore formation on chromosome behaviour (Chapter 3).

    Using these techniques it was established that (at least) three of the six mutant synaptic genes that had been reported for potato were allelic and similarly expressed in both male and female meiosis, whereas a mutant that had been claimed to express in female meiosis only proved to be non- existent. The former three mutants were characterized by normal chromosome behaviour throughout pachytene and a falling apart of bivalent chromosomes at diakinesis and thus displayed a typical desynaptic behaviour. They were therefore reassigned the gene symbol ds -1 (Chapter 4). Asynaptic mutants with a virtually complete absence of chromosome pairing and hence gene recombination are the most attractive candidates for engineering diplosporic apomixis. However, as cogent cases of asynapsis have not yet been reported for potato, desynaptic mutants are the best alternative that is currently available. Therefore the following questions were raised:
    1. Is it possible to select for consistent 2n-megaspore formation in ds -1 mutants?
    2. If so, are these 2n-megaspores of exclusive FDR origin as was predicted?
    3. To what extent does crossing-over and hence gene recombination occur?

    In chapter 6 the first two questions are addressed. The level of 2n-megaspore formation was determined in 126 ds -1 mutants using seed set from 2x.4x and 2x.2xFDR testcrosses as a criterion. Although the majority formed on the average less than 5 seeds/fruit, 14% of all ds-1 mutants produced 2n-megaspores in frequencies that resulted in consistent seed set within the 5-25 seeds/fruit range and allowed for routine production of nearly exclusive tetraploid progeny from 2x.2xFDR crosses. Subsequent cytological analyses revealed that 2n-megaspore formation in ds-1 mutants resulted from a direct equational division of univalent chromosomes in the first meiotic division (pseudo-homotypic division), an FDR mechanism that had previously been reported to occur in some of the diplosporic apomictic plant species. Additional data on SDR 2n-megaspore formation in full-sib normal synaptic plants indicated that both SDR and FDR 2n-megaspore formation are likely to be caused by common genes for precocious chromosome division. Depending on the relative timing of cell cycle and chromosome division this precocious chromosome division may impose post-reductional (SDR) or pre-reductional (FDR) 'restitution' of the somatic chromosome number under normal and mutant synaptic conditions respectively.

    Simply inherited marker traits are required for the analysis of genetic recombination in ds -1 mutants and had to be identified first. Some of the genetic markers used are characterized in chapter 8. On the basis of extensive analysis of chiasma formation and gene-centromere mapping of a number of simply inherited marker genes in normal synaptic plants and desynaptic mutants it could be concluded that the ds -1 gene substantially reduced the overall frequency of crossing-over and thus gene recombination in both male and female meiosis (Chapters 5 and 7). In addition, the genetic analyses revealed that FDR 2n-megaspores and FDR 2n-pollen from ds-1 mutants preserve approximately 94.1 % of the overall parental heterozygosity as opposed to 79.5 % that is preserved by FDR 2n-pollen from normal synaptic plants. The ds -1 gene was further demonstrated to particularly enhance the ability of FDR 2n-megaspores, and 2n-pollen to pick up the genetic constitution of the parental clone, including complex types of favourable epistasis, with a minimum amount of reassortment.

    Summarizing, it may be stated that the identification of diploid desynaptic mutants with consistent FDR 2n-megaspore formation extends the opportunities for direct transfer of enhanced diploid germplasm to tetraploids by means of sexual polyploidization and, since FDR 2n-megaspores and 2n-pollen from ds -1 mutants are relatively efficient in preserving the genetic constitution of selected diploid parents particularly demonstrates the feasibility of 2x( ds -1;FDR)-2x( ds -1;FDR) crosses for the production of relatively uniform and vigorous true potato seed varieties.

    Because of the potential for limited genetic recombination the use of the ds -1 gene in the development of diplosporic apomixis seems less obvious. However, as genes for asynapsis have not been identified in potato so far, it may be considered the best alternative that is currently available. Moreover some genetic diversity in apomictic progeny of diplosporic plant species as a consequence of autosegregation is quite common. The finding that some desynaptic clones formed FDR 2n-eggs through pseudo-homotypic division (≈diplospory) strongly supports the hypothesis that gametophytic apomixis consists of a number of distinct and genetically controlled elements which may be combined to attain approximately identical reproduction in largely sexual plant species. The application of this approach to produce completely uniform true potato seed varieties obviously requires breeding for increased levels of FDR 2n-egg formation in synaptic mutants completely suppressing genetic recombination, and in addition either introduction of genes for pseudogamous seed development in such clones or the development of an efficient system for pseudogamous seed production.

    Finally, it should be recognized that mutant synaptic genes may impose certain limitations. Because they are generally expressed in both male and female meiosis and thus are either largely sterile or produce only functional FDR 2n-gametes; resulting in polyploidization upon crossing, they have to be manipulated in heterozygous condition. Breeding schemes that consist of (i) introducing mutant synaptic genes and genes for FDR 2n-gamete formation in advanced diploids through backcrossing and (ii) subsequent selection of improved mutant synaptic segregants with FDR 2n-gamete formation following intercrossing of advanced heterozygotes, would be appropriate but laborious. As to FDR 2n-megaspore formation it should moreover be realized that mutant synaptic conditions are actually required. Since heterozygous diploid clones are normal synaptic and thus at best form SDR 2n-megaspores the question remains how to predict whether or not such clones carry genes that bring about substantial FDR 2n-megaspore formation in derived synaptic mutants. If the hypothesis holds true, that SDR and FDR 2n-megaspore formation are caused by common genes for division precocity (Chapter 6), the occurrence of SDR 2n-megaspore formation through postreductional precocious chromosome division in normal synaptic heterozygotes, might be a helpful criterion.

    Occurrence of indole compounds in some vegetables : toxicological implications of nitrosation with emphasis on mutagenicity
    Tiedink, H.G.M. - \ 1991
    Agricultural University. Promotor(en): J.H. Koeman; L.W. van Broekhoven; W.M.F. Jongen. - S.l. : Tiedink - 157
    groenten - groenteteelt - carcinogenese - carcinogenen - toxicologie - mutaties - mutagenese - mutagenen - vegetables - vegetable growing - carcinogenesis - carcinogens - toxicology - mutations - mutagenesis - mutagens

    From the introduction to the chemistry, occurrence and formation of N-nitroso compounds (NOC) (Chapter 1), it can be concluded that human exposure to these potent carcinogenic compounds is mainly through the endogenous nitrosation of dietary precursors. Vegetables are the major source of nitrosating compounds, while also nitrosatable substrates can occur in vegetables. Therefore the first aim of the present study was to screen Dutch vegetables for the presence of nitrosatable compounds by measuring their potential to form directly mutagenic NOC upon nitrite treatment. The second aim was to study the identity and mutagenic properties of the NOC formed. Since it was not feasible to cover all vegetables in connection to the second aim of the study, the efforts were concentrated on two of them, brassicas and fava beans. The latter were chosen because they were already known to contain precursors of directly mutagenic NOC and consequently the study was divided into two parts: Part 1 dealing with brassicas and Part 2 with fava beans.

    Part 1

    In Chapter 4 experiments are described in which extracts of 31 Dutch vegetables were screened for their ability to form directly mutagenic NOC upon nitrite treatment, irrespective of their nitrate content. All vegetables tested formed NOC upon nitrosation and induced Salmonella ( S. ) typhimurium revertants; Brassica vegetables were high responders on both of these parameters. Moreover, a significant correlation was found between their glucosinolate content (both alkyl/aryland indolylglucosinolate) and the amounts of NOC formed in extracts of these vegetables upon nitrosation. This suggests that glucosinolates are involved in the formation of NOC. Therefore purified glucosinolates were tested for their potential to form NOC (Chapter 6). Only indolylglucosinolates and their hydrolysis products formed NOC upon nitrosation. Mutagenicity was restricted to the nitrosated hydrolysis products. Since upon hydrolysis of indolylglucosinolates indole compounds are formed, which are nitrosatable substrates, the kinetics of the formation of NOC from indole compounds were investigated, as well as the stability of the nitrosated products formed (Chapter 5). Indole-3-acetonitrile (I 3 A), indole-3-carbinol (I 3 C) and indole, the hydrolysis products of the most commonly indoyglucosinolate, glucobrassicin, immediately reacted with nitrite to form directly mutagenic NOC and after an incubation time of about 15 min. maximal amounts of NOC were formed. The formed NOC were stable at both pH 2 and 8, but only when nitrite was present.

    In order to determine the contribution of indole compounds to the total mutagenicity of nitrite treated brassicas, the presence of several known indole compounds in green cabbage was investigated (Chapter 6). Only indole-3-carboxaldehyde and I 3 A could be detected. Both were not found to be important precursors of directly mutagenic NOC in green cabbage. The former did not form NOC at all and the second, although it occurred in considerable amounts (12 mg/kg fresh weight), only contributed for 2% to the total mutagenicity of nitrite treated green cabbage.
    From these results it can be concluded that:
    1. The correlation found between the amounts of glucosinolates in brassicas and the levels of NOC formed in extracts of these vegetables upon nitrite treatment is not based on a causal relationship.
    2. In brassicas both glucosinolates and indole compounds should not be considered as important precursors of NOC.
    3. By testing compounds out of their normal matrix, results can be obtained which differ considerably from those obtained under more realistic situations; (I 3 A), when tested pure, was found to be a potent precursor of NOC, while in green cabbage its contribution to the total mutagenicity was marginal.
    Although (I 3 A), was not considered an important precursor of NOC and contributed only marginally to the mutagenicity of nitrite treated brassicas, it was found to be a potential genotoxic and tumour promoting agent after nitrosation (Chapter 9). Recently it was shown in a Japanese study that nitrosated I 3 A is able to induce tumorous lesions in the fore stomach of rats. Therefore the possible risk involved in the consumption of brassicas due to the endogenous formation of nitrosated I 3 A was assessed (Chapter 7). For different reasons it seems most unlikely that the endogenous formation of nitrosated I 3 A out of brassicas should be considered as a realistic threat to human health: (1) The nitrosation rate of I 3 A in vivo is expected to be low. (2) In animal studies nitrosated I 3 A was administered pure, in very high concentrations. (3) Nitrosated I 3 A will only be stable in the presence of nitrite.

    Since in brassicas other precursors of directly mutagenic NOC occur, further studies are recommended to elucidate their identity and the implications of their potential endogenous nitrosation.

    Part 2

    4-Chloro-6-methoxyindole (4C6MI), the naturally occurring indole compound in fava beans, was evaluated for its genotoxic and tumour promoting potential after nitrite treatment (Chapter g). Remarkably, nitrosated 4C6MI appeared to have both genotoxic and tumour promoting potentials. The initiation effects were measured in bacteria and mammalian cells, the tumour promoting effects were measured by inhibition of gap junctional intercellular communication of V79 Chinese hamster cells. Both effects were observed at concentrations, which were in the same order of magnitude as the estimated daily intake of 4C6MI in a Colombian population. Hence, the results support the model for gastric cancer etiology, proposed by Correa et al. (1976, 1983). In this model the formation of NOC out of fava beans was supposed to be a causative factor of gastric cancer of the intestinal type, endemic in Colombia.

    In the study described in Chapter 10 the occurrence of 4C6MI in Dutch fava beans was investigated. An improved purification method was developed because the procedures described in the literature proved to be inadequate. Although the new purification method requires further improvement, a reasonable estimate of the levels of 4C6MI could be made. The levels ranged from about 3 to 7.5 mg/kg dry weight, which is one to two orders of magnitude higher than those reported for Colombian beans. However, in Chapter 12 It was assumed that the levels of 4C6MI in Colombian beans may be much higher than those reported. Further studies are required to investigate this aspect. From the results described in Chapter 10 it was concluded that almost all mutagenicity of nitrite treated Dutch fava beans can be attributed to 4C6MI.

    Although in previous studies a difference was observed in the mutagenicity of nitrite treated white and brown cooking cultivars after nitrosation, this was not found in the present study (Chapter 10 & 11). Moreover, all cultivars induced much more S. typhimurium revertants after nitrosation than in previous studies. No explanation can be given for these equivocal results and therefore studies are recommended to investigate the influence of environmental factors on the levels of 4C6MI in fava beans (Chapter 10).

    The mutagenicity of nitrite treated fava beans could be inhibited for 80-100% by addition of casein, indicating binding of nitrosated 4C6MI to casein (Chapter 11). This binding is independent of pH, in a range of pH 2-6 and appeared to be reversible, since mutagens could be released from the casein. It was estimated that about 25% of nitrosated 4C6MI formed in nitrite treated fava beans binds to proteins present in fava beans. This binding is reversible too. Fava bean mutagens also bind to wheat bran. Although the binding efficiency to wheat bran is less than to casein and the reversibility of this binding has not been studied, the binding of fava bean mutagens to wheat bran can be an important observation. Wheat bran fibres will not be digested and therefore might serve as vehicles for mutagens to leave the body without harmful effects. Further investigation of this is recommended.

    The results of the present study are further support for the hypothesis that the consumption of fava beans is causally related to the etiology of gastric cancer in Colombia. But the risks involved in the consumption of fava beans due to the endogenous nitrosation of 4C6MI for the population in The Netherlands is considered to be low, because of: (1) The low consumption of fava beans. (2) The lower levels of nitrate in Dutch drinking water. (3) Differences in food patterns between the Colombian and the Dutch population (the levels of consumption of vegetables and dairy products in particular). However, it can not be excluded that there are groups in The Netherlands with consumption habits deviating from that of the average population, who can in principle be assigned as persons at risk.

    It is recommended to investigate the in vivo nitrosation of 4C6MI and the carcinogenicity of its nitrosated product in further detail by using an appropriate animal study.

    Indoor and outdoor airborne particles : an in vitro study on mutagenic potential and toxicological implications
    Houdt, J.J. van - \ 1988
    Agricultural University. Promotor(en): J.H. Koeman; J.S.M. Boleij. - S.l. : van Houdt - 120
    toxicologie - luchtverontreiniging - stof - blootstelling - atmosfeer - aërosolen - samenstelling - luchtkwaliteit - mutaties - mutagenese - mutagenen - toxicology - air pollution - dust - exposure - atmosphere - aerosols - composition - air quality - mutations - mutagenesis - mutagens
    Introduction

    Air pollution components are present as gases and as particulate matter. As particle deposition takes place in various parts of the respiratory system particulate matter may have other toxicological implications than gaseous pollutants, which all may penetrate in the lower part of the respiratory tract. In addition, suspended particulate matter represents a group of pollutants of variable physical as well as chemical composition. Therefore airborne particulate matter cannot be regarded as a single, pure pollutant.
    This study deals with the mutagenic potency of airborne particulate matter bound organics and some of its toxicological implications.
    The assessment of health hazards is not easily possible without knowledge of the chemical character of the particles. Risk assessment through a toxicological consideration of the individual constituents has serious drawbacks because of the large number of chemicals involved and the complexity of the mixture. Since only 30-40% of the organic compounds on airborne particles have been identified, the contribution of unidentified compounds to the toxicological risk may be significant. Therefore the assessment of the overall mutagenic or carcinogenic activity in air samples may provide a more realistic basis for the evaluation of the possible risks, than an evaluation on the basis of individual compounds.
    The Salmonella /microsome assay has been a major assay used for monitoring the mutagenic potential of complex environmental mixtures such as airborne particulate matter. Results obtained with this test system may also be useful as a general air pollution parameter, representing particle bound extractable organics.
    The Salmonella /microsome assay involves subsequently sample collection, extraction, exposure of the test strain and the quantitative assessment of the revertants. Extraction is carried out routinely with organic solvents, while generally liver homogenates are used to identify mutagens which require metabolic activation. In the first part of this study methanol and liver homogenates from Aroclor 1254-pretreated Wistar rats were used to study the occurrence of particle bound mutagens collected indoors and outdoors. In the second part of this study physiological fluids and lung homogenates, which are more representative for the environment particles encounter in lungs, are used as solvent and metabolizing system in the Salmonella /microsome assay. Also cytotoxicity of airborne particulate matter to rat alveolar macrophages was determined in order to study some toxicological implications of inhalation of particle bound organic compounds.

    Mutagenic activity of extracts of airborne particulate matter from outdoor and indoor environments

    Chapter 1 provides a summary of literature data on physical and chemical characteristics of airborne particulate matter.
    The occurrence of mutagens in the environment and their sources are discussed.
    Chapter 2 presents data on in vitro testing of airborne particles, collected outdoors. Much evidence exists on the mutagenicity of airborne particles at urban and industrial locations. This study shows that particulate matter at background (Terschelling) and rural locations (Wageningen) may also bear mutagenic compounds. In Wageningen the level of mutagenicity follows a yearly cycle, the highest activity being found in winter. Over the years considerable differences in mutagenicity were found. The correlation of mutagenicity of samples simultaneously collected on the same day at both locations and the relation between mutagenicity of air samples and wind direction and with air trajectories suggest that the mutagenic potential of suspended matter originates not from local sources but rather depends on large scale processes. This is supported by literature data which show that the mutagenic potential mainly occurs on the smallest particles which, as a result of their long residence time, may be transported over distances of thousands of kilometers.
    The air, sampled at rural and background locations contains direct as well as indirect acting mutagens. The addition of liver microsomes gives variable effects on the level of mutagenic activity. Increases or decreases, if found are mostly relatively small. Furthermore, mutagenic activity was correlated with commomly measured air pollution parameters; multiple regression showed that SO 2 , NO 2 , NO, CO and 0 3 together account for 70% of the variation' in direct mutagenicity and 80% of the variation of indirect activity. SO 2 and NO 2 , and SO 2 , NO 2 and CO were significantly associated with the variation in direct and indirect activity respectively.
    As variations in mutagenic activity can be explained to a large extent by commonly registered air pollutants, monitoring the mutagenic burden of aerosols does not contribute to our knowledge already obtained by monitoring SO 2 , NO x and CO.
    Chapter x 3 and chapter 4 deal with the comparison of particle bound mutagens collected indoors and outdoors. In chapter 3 the contribution of smoking and cooking and in chapter 4 the contribution of wood combustion to mutagenicity of indoor aerosols is established. The results show that not only extracts of outdoor particles, but also indoor particulate matter may contain mutagenic compounds. Comparison of the mutagenic activity indoors and outdoors indicates that outdoor extracts show a direct mutagenic activity which is clearly detectable in nearly all samples, whereas indoor samples mostly show a low or undetectable direct activity. It is found that the indirect mutagenic activity is generally larger in indoor samples than in outdoor samples. Morever in indoor extracts cytotoxic effects are more pronounced.
    One of the sources of indoor mutagens may be penetration of outdoor particles. In the winter 82/83 no contribution of outdoor sources to mutagenic activity indoors was observed, while in the winter 84/85 a correlation between the extremely high levels of outdoor mutagenicity and indoor mutagenic activity was found. However, the important differences in composition of simultaneously collected indoor and outdoor samples which manifest themselves by the ratio - S9/+S9 justifies the conclusion that penetration of outdoor particles may be one but certainly not the only source of mutagenic activity indoors. Mostly the contribution of outdoor sources to indoor mutagenicity is only small.
    From our studies it is obvious that cigarette smoking is the predominant source of airborne genotoxicity in homes. wood combustion appeared to be a second important factor producing genotoxic compounds as sometimes a 2-3 fold increase of mutagenic activity is found. Volatilization of cooking products represents a less important source of mutagens.
    From these experiments it may be concluded that the removal of mutagens to the outdoor environment is not complete.

    Exposure is a function of concentration and time. In The Netherlands people spend most of the time at home. As all indoor samples show a strong indirect mutagenic activity, it may be concluded that exposure to genotoxins will be determined to a large extent by the level of pollution inside homes, which implies that exposure to indirect acting mutagens is quantitatively of far greater concern than exposure to direct acting mutagens.

    Biological availability of particle bound organic compounds

    Chapter 5 reviews a number of studies which deal with some aspects of the biological fate of mutagenic compounds and their 'carrier' particles. Respirable airborne particles to which most potential mutagenic compounds, detected in the Ames assay, are adsorbed may deposit in various parts of the respiratory tract. one of the defence mechanisms with regard to possible harmful action of these particles is clearance, as it reduces residence time on potentially sensitive epithelial surfaces. Alveolar macrophages provide the initial defence of the lower respiratory tract towards particulate matter.
    In chapter 6 results show a significant reduction of phagocytic activity in rat alveolar macrophages in vitro after exposure to extracts of airborne particulate matter, and the effect was greatest with indoor air. Metabolizing enzymes, together with transformed or not transformed particles may be released in the alveolar spaces as a result of damage to alveolar macrophages.
    In chapter 7 dissolution of particle bound organics into newborn calf serum and lung lavage fluid from pigs is described. Although from our results it is clear that physiological fluids, especially when obtained from lung lavage are less efficient in removing mutagens than organic solvents, the suggestion seems to be justified that a certain elution of environmental chemicals into body fluids takes place.
    Chapter 8 deals with metabolic activation of extracts of indoor and outdoor particulate matter. Drug metabolizing enzyme systems may also be seen as a defence mechanism towards chemicals invading the body. These enzyme systems enable the organism not only to convert lipid soluble harmful drugs into harmless water soluble metabolites, but also more toxic or mutagenic metabolites may be formed. Our results show that in addition to liver, lung homogenates of rat (Wistar) and mouse (Swiss) are also able to activate extracts of airborne particulate matter in a comparable way. Uninduced liver and lung homogenates showed only minor differences in activation capacity in the metabolism of airborne particles. In contrast to liver homogenates, Aroclor 1254- pretreatment of test animals did not give a strong induction in metabolic activation capacity of lung homogenates.
    In liver, almost all cells contribute to metabolic capacity, whereas in the lung metabolic capacity is almost exclusively located in the Clara cells, which is only one of the 40 different lung cell types. In certain parts of the lungs, especially in the terminal bronchioles, in which Clara cells are located, a rather high metabolic activation may take place. Therefore these results suggest that the respiratory system may be an important site for in vivo bioactivation of respirable particles.

    Verslag van een dienstreis naar Belgie en Frankrijk, van 23 - 26 april 1985
    Lange, W. - \ 1985
    Wageningen : SVP - 14
    genetica - mutaties - mutagenese - mutagenen - onderzoek - frankrijk - genetics - mutations - mutagenesis - mutagens - research - france
    Mutagene activiteit van het binnenluchtaerosol
    Boleij, J.S.M. ; Houdt, J.J. van; Alink, G.M. ; Jongen, W.M.F. - \ 1984
    Lucht en Omgeving 1 (1984)2. - ISSN 0168-8138 - p. 49 - 52.
    atmosfeer - aërosolen - samenstelling - stof - luchtverontreiniging - luchtkwaliteit - mutagenen - chemicaliën - schade - verwarming - ventilatie - binnenklimaat - atmosphere - aerosols - composition - dust - air pollution - air quality - mutagens - chemicals - damage - heating - ventilation - indoor climate
    Organic mutagens and drinking water in The Netherlands : a study on mutagenicity of organic constituents in drinking water in The Netherlands and their possible carcinogenic effects
    Kool, H.J. - \ 1983
    Landbouwhogeschool Wageningen. Promotor(en): J.H. Koeman. - Wageningen : Kool - 116
    carcinoom - chemicaliën - schade - drinkwater - koolwaterstoffen - mutagenese - mutagenen - mutaties - neoplasma's - verontreinigingsbeheersing - waterverontreiniging - waterzuivering - organische scheikunde - carcinoma - chemicals - damage - drinking water - hydrocarbons - mutagenesis - mutagens - mutations - neoplasms - pollution control - water pollution - water treatment - organic chemistry
    Several mutagenic and carcinogenic organic compounds have been detected in Dutch surface waters and in drinking water prepared from these surface waters. Although the levels of these compounds in drinking- and surface water are relatively low, in general below μg per litre, it appeared that organic concentrates tested in the Ames/microsome assay, showed mutagenic activity in 50 ml surface- and 500 ml drinking water.

    Such a result however was not expected based on the concentration of organic mutagens identified in these waters. Therefore the conclusion had been drawn that a number of unknown organic mutagens in combination with the identified mutagens were responsable for the level of mutagenic activity. With this in mind, an extensive investigation was carried out in an attempt to answer the following questions :
    - do drinking waters prepared from surface water, groundwater or a mixture of both show mutagenic activity;
    - do different water treatment processes during the preparation of drinking water influence the mutagenic activity;
    - what kind of physical-chemical properties have the organic compounds in drinking water concentrates showing mutagenic activity in the Ames test;
    - do mutagenic organic concentrates prepared from drinking water show carcinogenic properties?

    Against this background an inventory study was made on the presence of mutagenic activity in drinking water of 18 cities in The Netherlands.

    Besides this study, the influence of different water treatment processes on the mutagenic activity was examined in a number of water works. Furthermore, an attempt was made to characterize the organic compounds which are responsable for the mutagenic activity. Finally, a carcinogenicity study was carried out to see whether mutagenic drinking water concentrates induce carcinogenic effects in rats.

    Chapter 1, describes the aim of this investigation and the factors which have led to the present concern regarding the possible toxic effects of organic (micro)pollutants in drinking water in The Netherlands.

    Furthermore a summary is given of the literature on the identification of individual compounds and some data are given of the mutagenic and (suspect) carcinogenic organic compounds which are detected in surface- and drinking water in The Netherlands.

    In Chapter 2, materials and methods are described which were used for the inventory study on the presence of mutagenic activity in drinking water of 18 cities (Chapter 3), the characterization of organic mutagens (Chapter 4) and for a carcinogenicity study with mutagenic drinking water concentrates (Chapter 5).

    This chapter describes the XAD resins which have been used for concentrating organic mutagens from water, the concentration procedures with the aid of XAD resins and freeze drying, a mutagenicity assay viz. the Ames Salmonella/microsome assay (Ames test). The methods for chemical analysis which are used in the 18 city survey, fractionating techniques like thinlayer chromatography (TLC), high performance liquid chromatography (HPLC) and gelfiltration are described. The treatment, dose levels and experimental design and conduct which are used in a carcinogenicity study are explained. Finally, statistical procedures which have been used for analyzing tumour incidence in the carcinogenicity experiment and relating chemical parameters in drinking water to mutagenic activity in organic concentrates prepared from these drinking waters are described.

    In Chapter 3, the need for concentrating organic mutagens is explained. Mutagenicity results (Ames test) of five surface waters in The Netherlands are presented, in which the organic mutagens are concentrated with the XAD procedures. Depending on the concentration factor applied (103-4.103) it appeared that in all five surface waters mutagenic activity could be detected. The river Rhine showed the highest mutagenic activity viz. a doubling of revertants in 50 ml water.

    To be certain that the XAD procedure is a selective concentration method for organic mutagens, this procedure was compared with a freeze drying technique. The results of this comparative investigation showed that the XAD procedure is a reliable method for concentrating organic mutagens from surface water. on the basis of the surface water results, the XAD procedure was also applied for concentrating organic mutagens from drinking water. It was found that drinking water of four out of six cities showed mutagenic activity in volumes varying from 0.5 to 3 litre.

    To see whether the mutagenic results obtained in the six cities are representive for drinking water in The Netherlands, an extensive inventory study on the presence of mutagenic activity in several types of drinking water was carried out. In this study eighteen cities (twenty drinking waters) were investigated three times for the presence of mutagenic activity and the following chemical parameters : AOCL, EOCl, THM, VOCl, TOC, Tot. N, over a period of 2 years. The chemical parameters were measured to see whether a relationship could be found between one or more of these parameters and mutagenic activity.

    The selection of the drinking water was based on the water source (ground water, surface water) the storage facility (dune filtration, bankfiltration, storage reservoir) and the application of a chlorine treatment during the preparation of drinking water. The results of this study showed that in fourteen of the twenty drinking waters, mutagenic activity could be detected in volumes varying from 0.5 to 3 litre. When the cities were classified according to their water source, storage facility and type of treatment, it appeared that only three of the fifteen cities which prepare their drinking water from surface water or a mixture of surface- and groundwater, did not show mutagenic activity. Two of the five cities which use groundwater as a drinking water source showed mutagenic activity although in one city the activity was marginal.

    Correlating the chemical parameters with the mutagenic activity, it was demonstrated that AOCL showed the highest correlation with the direct mutagenic activity in strain TA 98 and TA 100. It was also shown that a chlorine treatment applied during drinking water preparation correlated well with the direct mutagenic activity in strain TA 98 and TA 100. In addition, chemical parameters which showed a significant difference (p< 0.01) in concentration between mutagenic and non mutagenic samples (Mann-Whitney) and a significant correlation (p< 0.01, Kendall tau) with mutagenic activity were used for straight curve fitting. From these fitted regressions, concentration levels were calculated above which a doubling of revertants may be expected.

    In the second part of Chapter 3 (3.4) the influence of different treatment processes on the mutagenic activity and some chemical parameters were investigated in three waterworks. Application of a chlorine treatment, generally increased the direct and promutagenic activity, but the amount of increase proved to be dependent on the type of water chlorinated. The use of ozone in the preparation of drinking water decreased the mutagenic activity in the water. The amount of reduction was dependent on the type of water ozonated. Dune filtration greatly reduced the mutagenic activity.

    Slow sand filtration could not be evaluated, because of the toxicity of the organic concentrates for the bacterial strains. Filtration over active carbon filters which operated for about 1 year, reduced the mutagenic activity below the detection level. Carbon filters which operated more than 1.5 years in a pilot plant, showed a break-through of mutagenic activity. This result suggests that carbon filters are able to remove organic mutagens only for a short time.

    From the results of the chemical parameters before and after the different treatment processes it appeared that the level of AOCL, behaved very similar with the mutagenic activity in the neutral fraction. These results confirm those obtained in the eighteen city survey and support the idea that A0C1 might be a useful indicator for mutagenic activity in drinking water.

    The physical-chemical characterization of organic mutagens in drinking water concentrates is described in Chapter 4. The first approach was to examine the influence of the pH on the adsorption behaviour of organic mutagens on the XAD resins. Lowering the original pH of drinking water with HCl to pH = 2- 3, revealed the presence of another class of organic mutagens the so-called acid fraction which hardly adsorb on the XAD resins at pH = 7.5.

    Further it was investigated whether different organic solvents are able to eluate the organic mutagens from the XAD column. it was found that diethylether only eluted a minor part of the mutagenic activity from the XAD resins and subsequent elution with acetone eluted the major part of the activity.

    This result showed that it is not likely that the organic mutagens in the acetone fraction are identical with the already identified organics in the ether fraction in this drinking water. Furthermore it appeared that the organic mutagens arenot only less volatile but also resistent to boiling. Another approach to characterize the organic mutagens was to apply fractionation techniques. Using TLC and HPLC, it was found with HPLC analysis that the mutagenic activity was present predominantly in two fractions. Finally, gelfiltration on Sephadex LH20, showed that organic mutagens which demonstrated mutagenic activity with strain TA 98, had a molecular weight in the order of 200.

    Chapter 5, presents the results of a carcinogenicity study, in which Wistar SSP TOX rats were exposed to mutagenic drinking water concentrates of one city in The Netherlands. Drinking water concentrates were prepared every week using the XAD concentration procedure and the organic concentrates (DMSO) were mixed with the non mutagenic drinking water of the Wistar SSP TOX rats.

    Dosage levels were based on multiples of expected human exposure levels. In the calculation the average human exposure was assumed to be approximately 29 ml/kg bw./day. Furthermore it was assumed that a rat of 250 gram would consume about 30 ml water per day. In the experiment rats were divided into four groups (50 males and 50 females per group), a control group and groups which received respectively 10, 30 and 90 times the human exposure level in their drinking water.

    During the experiment (106 weeks) the water consumption of the rats was measured weekly. Body weights were recorded weekly in the first two months of the experiment and once a month thereafter. The mutagenic activity of concentrates which were mixed with non mutagenic drinking water were measured after a week. It was found that the mutagenic activity with strain TA 98 could be recovered for 60-90%, while the TA 100 activity hardly could be recovered. The latter was explained by the observed partial decrease of TA 100 activity after mixing the mutagenic drinking water concentrate with drinking water.

    During the experiment it appeared that the assumed water consumption of 30 ml per day per rat, on which the dose level was based, not was reached.

    Moreover, it appeared that Wistar SSP TOX rats became much heavier than was expected and therefore the actual dose levels in ml/kg bw. of the three exposed groups were not 10, 30 and 90 times the expected human exposure levels, but 4.5, 14 and 40 for male rats respectively and 7, 22 and 68 for female rats. Exposing Wistar SSP TOX rats to these dose levels for 106 weeks, did not result in a significant increase (p <0.05) in tumour induction.

    Furthermore it was shown, that the development and types of tumours were similar in the treated and control groups. The number of animals with tumours and the animals which died of tumours in the exposed groups was not significantly different (p < 0.05) from the control group. The negative results in this carcinogenicity study indicate that mutagenic drinking water concentrates did not contain very potent carcinogens in effective concentrations like for instance N-Nitrosodiethylamine because when these kind of compounds are present, the carcinogenicity should be positive at the dose levels tested.

    On the other hand one cannot exclude the presence of weak carcinogens in the mutagenic concentrates, because one cannot detect a carcinogenic effect at the dosis tested. Based on the carcinogenicity results, an estimation of a risk factor was made for the people who consumed this mutagenic drinking water.

    For the estimation the following three assumptions had been made:
    - when organic carcinogens had been present in drinking water, they were present solely in the mutagenic drinking water concentrates
    - results obtained in the present carcinogenicity study with rats may be extrapolated to man
    - linear extrapolation of high dosis to low dosis give correct results.

    When all these assumption are correct it appeared after statistical analysis that less than 356 of 110.000 people might be at risk. On this basis the contribution of drinking water, if there is a contribution at all, seems relatively small viz. less than 1. 1 % in comparison to the expected tumour incidence of 33.000. The latter value is based on an average tumour incidence of 30 per 100, due to background processes.

    To improve, however the reliability of an estimation of the risk factor for people who consume polluted drinking water, organic concentratres of drinking water which have not been included in the present carcinogenicity study should be examined for complete carcinogenicity in combination with higher doses of concentrates of organic mutagens as used in this study.

    Besides this investigation it is recommended that both organic concentrates will be tested for tumour promotion and initiation activity as well as identification of the organic responsable for these effects should receive priority. Finally Chapter 6 describes a number of conclusions emerging from this investigation. The data presented show that mutagenic activity was detectable in drinking water of many cities in The Netherlands. The number of cities which showed mutagenic activity in their drinking water was the least where groundwater was used as a drinking water source. Therefore it is recommended that groundwater is preferable over other sources of drinking water and the quality of the groundwater should be protected very carefully so that oxidation and disinfection with a chlorine treatment will not be necessary. This recommendation is not only based on the eighteen city survey but also from data obtained in waterworks which prepare their drinking water mainly from surface water, because mutagenic activity is significantly increased by a chlorine treatment which may often result in mutagenic activity in the end product. It is recommended further that the organic mutagens should be identified in order to evalute them with respect to their possible toxic properties. Also higher dosis of mutagenic drinking water concentrates in combination with concentrates prepared from the rest of the organics in drinking water, should be tested for carcinogenicity and chronic toxicity for a more reliable risk estimation. Besides, this investigation it is recommended that both organic concentrates should be investigated for tumour promotion and initiation activity.

    The genetics of some planthormones and photoreceptors in Arabidopsis thaliana (L.) Heynh.
    Koornneef, M. - \ 1982
    Landbouwhogeschool Wageningen. Promotor(en): J.H. van der Veen. - Wageningen : Koornneef - 157
    brassicaceae - genetische variatie - genetica - heritability - overerving - mutagenese - mutagenen - mutaties - fytochroom - plantengroeiregulatoren - plantenpigmenten - brassicaceae - genetic variation - genetics - heritability - inheritance - mutagenesis - mutagens - mutations - phytochrome - plant growth regulators - plant pigments - cum laude
    This thesis describes the isolation and characterization in Arabidopsis thaliana (L.) Heynh. of induced mutants, deficient for gibberellins (GA's), abscisic acid (ABA) and photoreceptors.

    These compounds are known to regulate various facets of plant growth and differentiation, so mutants lacking one of these substances are expected to be affected in several aspects of their physiology. It is shown in this thesis that the earliest expression of these mutants occurs during seed development and seed germination. Therefore these processes form an excellent phase to screen for these mutants.

    Planthormone and photoreceptor mutants in relation to seed physiology.

    In general three major periods may be distinguished in the history of a seed: 1) Seed development and maturation, 2) developmental arrest of the mature seed, characterized either by a dormant state in which seeds even do not germinate under favourable environmental conditions, or by a quiescent state in which seeds only require rehydration, and 3) germination, starting with water uptake and often requiring breaking of dormancy, which is triggered by specific environmental factors such as light and temperature. Planthormones may play a regulatory role in all three phases.

    Non-germinating GA-responsive mutants as described in Chapter 1 have a strongly reduced gibberellin biosynthesis (Barendse, pers.comm.) which may lead to an increased level of dormancy and/or to the inability of the seeds to
    break dormancy after imbibition of mature seeds. Clearly the presence of GA's, either by de novo GA synthesis, or by hydrolysis of bound forms, is not always a prerequisite for seed germination: genotypes that combine GA- and ABA deficiency like the revertants of non-germinating ga-1 mutants described in Chapter 2 do readily germinate.

    Apart from the absence of endogenous factors such as gibberellins, also the lack of receptors for environmental factors that normally break dormancy might prevent germination. An example are the hy-1 and hy-2 mutants (Chapter 4), which are characterized by an increased hypocotyl length in white light and the absence of detectable phytochrome in dark grown hypocotyls. It was shown by Spruit et al. (1980), that these mutants hardly show any germination and correspondingly, have strongly reduced levels of phytochrome in their seeds. Their reduced germination capacity is restored by (relatively high) concentrations of exogeneously applied GA 4+7 (Koornneef et al., 1981). Consequently one might expect such phytochrome deficient mutants to occur among the GA responsive non-germination mutants in Arabidopsis, like van der Veen and Bosma actually found for a tomato mutant (see Koornneef et al., 1981). Remarkably this was not the case in Arabidopsis. The reason for this seems to be the absence of a light requirement in the hy mutants from the M 2 populations screened for non-germinating mutants of Arabidopsis. It happened that these M 2 seeds in all cases were harvested from M 1 plants grown in winter, in contrast to the seeds studied by Spruit et al. (1980) which were harvested in summer. We have observed during a number of years that seeds (including wild-type seeds), which developed in winter (natural daylight with additional continuous light by Philips TL 57) were less dormant than seeds from summer grown mother plants (long days, high light intensity, no additional light). Relevant environmental factors in this respect may be light intensity, light quality (McCullough and Shropshire, 1970) and daylength (Karssen, 1970; Luiten, 1982). The effect of light quality (McCullough and Shropshire, 1970) indicates that phytochrome may be involved in the determination of the level of dormancy.

    To select mutants with a reduced or absent seed dormancy, one may choose those conditions, where the wild-type is clearly dormant. However, the high and probably complex environmental variability of this character and the relatively rapid change in the level of dormancy during dry storage of the seeds makes this selection system less attractive.

    Selection for revertants in the progeny of mutagen treated non-germinating ga-1 mutants proved to be an effective procedure to isolate mutants with a reduced dormancy (Chapter 2). As the reverting effect (restored germination) was caused by a mutation at a different locus, the ga-1 allele could be replaced by its wild-type allele by crossing the revertant with the wild-type parent followed by selection in F 2 . These newly selected monogenic recessive mutants had a reduced level of ABA in the leaves and in both the developing and ripe seeds. Correspondingly the mutant allele was called aba (ABA-types are aba/aba plants).

    The germination of seeds collected at different stages of their development on both ABA- and wild-type plants showed that dormancy developed during the last part of seed maturation in wild-type, but not in the aba -mutant. This shows that the function of ABA is dormancy induction. ABA determinations in unripe siliquae showed a peak level of ABA at about 10-12 days after anthesis, followed subsequently by a decrease, a short period at a constant level and a further decrease (Chapter 3). In addition to ABA-type mothers with ABA-type embryo's and wild-type mothers with wild-type embryo's, one can also obtain by means of the appropriate reciprocal crosses ABA-type mothers with wild-type embryo's and wild-type mothers with (50%) ABA type embryo's. So the effects of maternal and embryonic genotype can be separated. It was found (Chapter 3) that the genotype of the mother plant regulated the sharp rise in ABA content halfway seed development (maternal ABA). The genotype of the embryo and endosperm was responsible for a second ABA fraction (embryonic ABA), which reached lower levels; but persisted for some time after the maximum in maternal ABA. The onset of dormancy showed a good correlation with the presence of the embryonic ABA fraction and not with the maternal ABA.

    Another category of mutants which also may give some understanding of the role of ABA in seed germination are the ABA tolerant mutants recently isolated by us in Arabidopsis. Compared to wild-type these mutants require an upto 20 fold higher concentration of exogeneously applied ABA to inhibit seed germination. These mutants too are characterized by a reduced seed dormancy.

    Other genetically determined factors than those mentioned above are certainly also involved in seed development and seed germination. Thus in Arabidopsis mutations leading to the absence of seed coat pigments (transparent testa) and simultaneously to the absence of a mucilage layer around the seed have a reduced dormancy (Koornneef, 1981). The latter seedcoat characters are determined purely by the maternal genotype.

    Planthormone and photoreceptor mutants in relation to other physiological effects.

    Non-germinating mutants at the loci ga-1, ga-2 and ga-3, when made to germinate by adding gibberellin, initially develop into normal looking seedlings. Later on they become dark green bushy dwarfs with reduced petals and stamens. Regular GA-spraying from the seedling stage onwards maintains the wild-type phenotype completely or nearly so (Chapter 1). The strong and quick response of the dwarfs to GA sprays (the elongation of the petals of older dwarfs becomes visible within two days) clearly demonstrates the essential role of gibberellin in elongation growth.

    Recently the non-germinating ga alleles were shown to have a strongly re duced kaurene synthetase activity in young siliquae compared to wild type. These analyses were performed by Dr. G.W.M. Barendse (pers.comm.). This indicates that these genes control some early step(s) in GA biosynthesis.

    Apart from mutants that do not germinate without GA, also more or less normally germinating GA responsive dwarfs were isolated. Half of these were found to be allelic to the non-germinating ga-1 , ga-2 and ga-3 mutants. These mutant alleles behave like so called "leaky alleles", i.e. the alleles are only partly defective and produce sufficient GA for seed germination, but not enough to give normal elongation growth.

    GA sensitive dwarfs were also found at two other loci ( ga-4 , ga-5 )of which no non-germinating alleles have been isolated so far (Chapter 1). These mutants have normal or slightly reduced kaurene synthetase activity (Barendse, pers.comm.), which indicates that these genes regulate steps beyond kaurene, or affect GA metabolism in another way. It is also possible that in the mutants cell elonga tion factors are blocked for which the relatively high concentration of exogeneously applied GA may substitute. Locus ga-4 seems to control interconversions between GA's, which is suggested by the insensitivity of ga-4 dwarfs to GA 9 . which gibberellin is effective with mutants at the other 4 loci.

    Abscisic acid (ABA) deficient mutants are characterized not only by reduced seed dormancy but also by disturbed water relations (wiltiness, withering), probably as a result of failure to close the stomata upon conditions of water stress (Chapter 2). This is characteristic for ABA deficient mutants in tomato (Tal and Nevo, 1973) and potato (Quarrie, 1982). ABA deficient mutants in maize are in addition to a reduced seed dormancy (viviparous mutants, gene symbol op) characterized by the failure to synthesize carotenoids and they accumulate precursors of these pigments (Robichaud et al., 1980). As ABA deficient mutants in Arabidopsis, tomato and potato have normal pigments, it is suggested that in the latter species the ABA biosynthesis may be blocked in the last part of the pathway, whilst in the maize mutants it is blocked at an earlier stage, i.e. where ABA and carotenoids still have a common pathway.

    Some of the photoreceptor mutants are affected in their germination behaviour as discussed above. However, the most conspicuous effect observed is the partial lack of light induced inhibition of hypocotyl elongation (Chapter 4). Mutants in Arabidopsis, and also in tomato and cucumber (Koornneef et al., 1981; Koornneef et al., unpublished), that have an elongated hypocotyl when grown in white light, were shown to have locus-specific alterations in the spectra of light inhibition when grown in light of restricted spectra] regions. In these "colour blind" mutants at two loci (hy-1 and hy-2) little or no spectrophotometrically detectable phytochrome was present in dark grown hypocotyls, nor was it in the seeds. In these mutants the inhibitory effect of red and farred was almost absent. Mutants of other genes were characterized by the absence only of red inhibition (hy-3) or by a decreased sensitivity to the shorter wavelengths of the spectrum (hy-4, hy-5). Hy-5 also showed a reduced inhibitory effect of far-red light. The differential sensitivity of the genotypes to specific spectral regions strongly suggests the involvement of more than one pigment in the inhibition by light of hypocotyl elongation and probably also in other photomorphogenetic processes. Some authors ascribed this role solely to phytochrome (Schäfer, 1976).

    Since under specific conditions phytochrome could nevertheless be detected in so called phytochrome deficient mutants (Koornneef and Spruit, unpublished) the genes hy-1 and hy-2 probably do not represent the structural genes of the phytochrome protein or the phytochrome chromophore, but instead may play a role in the regulation of phytochrome metabolism.

    Further genetic aspects of plant hormone and light receptor mutants.

    Mutation frequencies for the different groups of loci were estimated for ethylmethanesulphonate (EMS), fast neutrons and X-rays (Chapter 5). Average mutation frequencies calculated per diploid cell, per locus and per MM EMS during 1 hr at 24 °C, were for ga-1, ga-2, ga-3 8.0 ± 1.8 x 10 -6, for hy-1, hy-2, hy-3 4.2 ± 1.4 x 10 -6and for the aba locus about 27 x 10 -6. These mutation frequencies are relatively high compared to other loci studied by us and others. It is not excluded that in these categories loci escaped detection simply because of a low mutation frequency.

    It is a good custum to locate newly induced mutations on the organisms gene map, especially when they are the basis of extensive research like our ga, aba and hy mutants. Unfortunately, the gene map of Arabidopsis was rather fragmentary, and contradictory or wrong conclusions about linkage relations could be found in literature. Since we had gradually built up the complete set of 5 primary trisomics supplemented with a number of telotrisomics (one chromosome arm extra) and also made a collection of mutations at many loci, induced in the course of various experiments at our department and supplemented with mutants described in literature, we had a good starting point to construct a more representative gene map for Arabidopsis. The required scale of operations was only feasable thanks to the accurate assistance of many students who performed trisomic analysis and gene mapping as part of their university training program. Important further additional data were obtained from the department of Genetics of Groningen University and from literature.

    The trisomic analysis aimed at assigning linkage groups (via representative markers) to the different chromosomes is described in Chapter 6. The gene maps in centimorgans for the five Arabidopsis chromosomes is presented in Chapter 7. On the basis of 76 loci mapped the genetic length of the Arabidopsis chromosomes now compares well with that of individual chromosomes in e.g. tomato and maize. This notwithstanding the small size of the Arabidopsis chromosomes.

    Genes with a similar mutant phenotype (and probably comparable functions) seem to be distributed at random over the Arabidopsis genome.

    Our set of mutants at the ga-1 locus of Arabidopsis provides an excellent opportunity for fine structure analysis of the gene. The system has a very high resolving power, for the intragenic recombinants are found as the rare wild-type seedlings among thousands of non-germinating seeds per petri dish. The results show (Chapter 8) that 8 different alleles could be arranged into an internally consistent map on the basis of the frequencies of intragenic recombinants. One fast neutron induced allele behaved as an intragenic deletion. The order of the sites with respect to other genes on chromosome 4 could be established.

    Symposium of the Mutation Breeding Contact Group : Wageningen, 6 March 1963
    Broertjes, C. - \ 1963
    Wageningen : ITAL
    plant breeding - mutations - mutagens - radiation - plantenveredeling - mutaties - mutagenen - straling
    Mutatieproblemen
    Prakken, R. - \ 1961
    Wageningen : Veenman - 16
    colleges (hoorcolleges) - mutagenese - mutagenen - mutaties - lectures - mutagenesis - mutagens - mutations
    Rede Wageningen 9 maart 1961
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