Staff Publications

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.

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A histological description of the salivary gland system of some aphid species of the Adelgidae and Aphididae (Homoptera, Aphidoidea)
Ponsen, M.B. - \ 2015
Wageningen : Wageningen University, University for Life Sciences (Wageningen Agricultural University papers 06.1) - 64
adelgidae - aphididae - speekselklieren - dieranatomie - histologie - salivary glands - animal anatomy - histology
A histological description of the alimentary tract, salivary glands, and related organs of Mindarinae (Homoptera, Aphidoidea)
Ponsen, M.B. - \ 2012
Wageningen etc. : Wageningen University [etc.] (Wageningen Agricultural University papers 06.1) - 60
mindarus - mindaridae - aphididae - dieranatomie - spijsverteringsstelsel - speekselklieren - proboscis - histologie - animal anatomy - digestive system - salivary glands - histology
Postnatal development of articular cartilage
Turnhout, M.C. van - \ 2010
Wageningen University. Promotor(en): Johan van Leeuwen, co-promotor(en): Sander Kranenbarg. - [S.l. : S.n. - ISBN 9789085857839 - 189
kraakbeen - bindweefsel - beenderen - collageen - biologische ontwikkeling - histologie - zoogdieren - cartilage - connective tissue - bones - collagen - biological development - histology - mammals
Articular cartilage (AC) is the thin layer of tissue that covers the ends of the bones in the synovial joints in mammals. Functional adult AC has depth-dependent mechanical properties that are not yet present at birth. These depth-dependent mechanical properties in adult life are the result of a depth-dependent composition and structure that develops in postnatal life. Our knowledge on how postnatal AC remodelling proceeds, and how the functional depth-dependent mechanical properties develop in postnatal life is still limited.

In this thesis, we investigated the properties of the postnatal collagen network in AC, and the contribution of postnatal collagen network remodelling to the adult depth-dependent mechanical properties of AC. We used horses and (mostly) sheep as experimental animals to obtain measurements on three parameters of the postnatal collagen network (predominant collagen fibril orientation, collagen densities and collagen network anisotropy). We used a composition-based finite element model for computational analysis of the role of this collagen network in the postnatal development of depth-dependent mechanical properties.

We first investigated how collagen structure in AC affects the parameters that are measured by quantitative polarised light microscopy (qPLM), because qPLM is the most popular technique to investigate properties of the collagen network in AC. We quantified the contributions of the three collagen network parameters (orientation, density and anisotropy) to the measured predominant fibril orientation and the measured total tissue birefringence (retardance), and showed that collagen network anisotropy can be quantified when the retardance from polarised light microscopy is corrected for collagen densities.

In the study on horses, we investigated differences in predominant collagen orientation for equine articular cartilage in stillborn and adult animals with scanning electron microscopy and quantitative polarised light microscopy (qPLM). In the study on sheep, we first investigated the predominant collagen orientation in animals divided over ten sample points between birth and maturity (72 weeks) with qPLM.
Both studies confirmed the remark by Archer et al. [Archer2003] that the collagen fibrils in perinatal animals lie predominantly parallel to the articular surface, and we confirmed and quantified the adult 'Benninghoff' structure in the mature animals in both studies. We further observed a transitional layer with weak fibril anisotropy in the perinatal animals that is not correlated to changes in predominant collagen fibril orientation as in the adult Benninghoff structure.

To investigate the contribution of collagen reorientation to the development of depth-dependent mechanical properties, we implemented the results on postnatal predominant collagen fibril orientation in the sheep in a composition-based finite element model. We described the interactions between collagen orientation, free swelling strains, osmotic pressures and effective AC stiffness in confined compression.
Based on the results, we hypothesised that collagen densities increase most in the deep tissue due to increased collagen fibril strains that result from postnatal collagen fibril reorientation.

In sheep, we measured collagen densities with Fourier transform infrared micro-spectroscopy. Collagen density increased in postnatal life, and they increased most in the deep tissue (near the bone), which supported our earlier hypothesis. Perinatal animals showed a valley in collagen densities near the articular surface, i.e. at the position of the transitional layer. We showed that this valley disappears in early postnatal life. We corrected the qPLM retardance from our sheep data with the collagen densities from the sheep data to assess collagen network anisotropy. The results showed that anisotropy is relatively constant in the deep tissue, and that anisotropy is stronger in the transitional layer of perinatal animals compared with the transitional layer of adult animals.


To investigate interactions in postnatal collagen network remodelling, we implemented the three collagen network parameters (orientation, density and anisotropy) that we obtained from the sheep in the finite element model. Based on the results, we suggested different functional roles for the three collagen network parameters: collagen fibril reorientation contributes most to the development of depth-dependent mechanical properties, collagen density increases appear to equalise collagen fibril strains, and the weak anisotropy in the transitional layer appears to smooth gradients in the mechanical state of the tissue in adult animals.


Variation in commercial sources of soybean meal influences the severity of enteritis in Atlantic salmon (Salmo salar L.)
Urán, P. ; Schrama, J.W. ; Jaafari, S. ; Baardsen, G. ; Rombout, J.H.W.M. ; Koppe, W. ; Verreth, J.A.J. - \ 2009
Aquaculture Nutrition 15 (2009)5. - ISSN 1353-5773 - p. 492 - 499.
trout oncorhynchus-mykiss - rainbow-trout - distal intestine - antinutritional factors - fish-meal - products - diets - histology - growth - soy
Soybean meal (SBM) is a potential alternative for the replacement of fishmeal in aquafeeds. In Atlantic salmon, however, dietary SBM causes an inflammation of the distal intestine, known as SBM-induced enteritis. The objective of the present study is to verify whether different (geographically spread) commercial sources of SBM yield contrasting inflammatory responses. To do so, six SBM batches from different origins were included in the Atlantic salmon diets at the level of 20%. After 4 weeks of feeding, the distal intestine of the salmon was sampled and scored by a semi-quantitative scoring system, which assessed six separated parameters, characterizing the extent of enteritis. The overall mean score as well as the score of the separate parameters varied between the different commercial sources of SBM included in the diet. The variation in SBM caused different degrees of disparity in the score of the separate parameters. The parameter that was most affected by the variation in the source of SBM was the disappearance of supranuclear vacuoles in enterocytes. In contrast, the increase in goblet cells showed the smallest variation between the different SBM sources. This study shows that different commercial sources of SBM can result in differences in the severity of SBM-induced enteritis in Atlantic salmon
Soybean meal induces intestinal inflammation in common carp (Cyprinus carpio L.)
Urán, P. ; Gonçalves, A. ; Taverne-Thiele, J.J. ; Schrama, J.W. ; Verreth, J.A.J. ; Rombout, J.H.W.M. - \ 2008
Fish and Shellfish Immunology 25 (2008)6. - ISSN 1050-4648 - p. 751 - 760.
salmon salmo-salar - trout oncorhynchus-mykiss - atlantic salmon - distal intestine - rainbow-trout - immunocytochemical localization - induced enteritis - expression - histology - immune
The development of soybean meal (SBM) induced enteritis in the hindgut of the omnivorous common carp (Cyprinus carpio L.). The developed condition was assessed when carp, continuously fed on animal protein, were transferred to a diet in which 20% of the protein was replaced by SBM. After week 1, most of the inflammation parameters were already present, but at week 3, a strong aggravation of the condition was observed which included a shortening of the mucosal folds, the disappearance of the supranuclear vacuoles, an increased number of goblet cells, a thickened lamina propria and sub-epithelial mucosa with increased numbers of basophilic granulocytes as well as a decreased uptake capacity of enterocytes (impaired endocytosis and microvilli). Contrary to previous observations made with respect to Atlantic salmon, common carp start to recover from the fourth to the fifth week after switching to SBM feeding. At this stage, the supranuclear vacuoles refill and most of the parameters revert to basal levels. During the enteritis process, a real-time quantitative PCR analysis was conducted to measure the expression of inflammatory and anti-inflammatory cytokine genes in the isolated intraepithelial lymphocytes (IEL). The pro-inflammatory interleukin 1ß (IL-1ß) and tumour necrosis factor ¿1 (TNF-¿1) genes were up-regulated during the inflammation process while the anti-inflammatory interleukin 10 (IL-10) was down-regulated after an initial up-regulation at week 1. Transforming growth factor ß (TGF-ß) expression showed an up-regulation from week 3 onwards despite the high Ct value and the low primer efficiency shown. This study confirms the contribution of IEL (mainly T-like cells) and basophils in the enteritis process. In addition, the results show a clear involvement of up- and down-regulated cytokine genes in both the onset and recovery of the SBM-induced enteritis in the hindgut of carp.
A histological description of the salivary gland system of Phylloxeridae (Homoptera, Aphidoidea)
Ponsen, M.B. - \ 2006
Wageningen etc. : Wageningen University [etc.] (Wageningen Agricultural University papers 06.1 supplement) - 30
phylloxeridae - speekselklieren - histologie - salivary glands - histology
A histological description of the alimentary tract and related organs of Adelgidae (Homoptera, Aphidoidea)
Ponsen, M.B. - \ 2006
Wageningen : Wageningen University (Wageningen Agricultural University papers 06-1 (2006)) - ISBN 9789057821745 - 103
adelgidae - spijsverteringskanaal - histologie - digestive tract - histology
Effecten van combinaties van androgenen bij kalveren: histologie en detectie van residuen van hormoonesters in haren
Groot, M.J. ; Nielen, M.W.F. - \ 2005
Wageningen : RIKILT (Rapport / RIKILT 2005.008)
androgenen - kalveren - histologie - residuen - haaranalyse - nadelige gevolgen - kennis - androgens - calves - histology - residues - hair analysis - adverse effects - knowledge
The ABC transporter MgAtr4 is a virulence factor of Mycosphaerella graminicola that affects colonization of substomatal cavities in wheat leaves
Stergiopoulos, I. ; Zwiers, L.H. ; Waard, M.A. de - \ 2003
Molecular Plant-Microbe Interactions 16 (2003)8. - ISSN 0894-0282 - p. 689 - 698.
atp-binding cassette - septoria-tritici - blotch pathogen - resistance - infection - gene - sensitivity - histology
The role in virulence of the ATP-binding cassette (ABC) transporters MgAtr1, MgAtr2, MgAtr3, MgAtr4, and MgAtr5 from Mycosphaerella graminicola was analyzed by gene disruption or replacement on seedlings of the susceptible wheat cultivar Obelisk. Disruption strains of MgAtr1 and MgAtr2 and replacement strains of MgAtr3 and MgAtr5 displayed the same phenotype as control strains, while virulence of the MgAtr4 disruption strains was significantly reduced. This reduction in virulence was independent of the wheat cultivar used. Histopathological analysis of the infection process revealed that MgAtr4 disruption strains colonize substomatal cavities less efficiently and display reduced intercellular growth in the apoplast of wheat leaves. In vitro growth experiments in different media showed no fitness penalty associated with the disruption of MgAtr4. Expression analysis demonstrated that transcripts of the constitutively expressed gene CYP51 encoding the fungal-specific cytochrome P450 sterol 14alpha-demethylase from M. graminicola were not detectable in interaction RNA from wheat infected with MgAtr4 disruption strains, thus confirming the reduced intercellular growth of these strains. The results indicate that MgAtr4 is a virulence factor of M. graminicola during pathogenesis on wheat and may function in protection against fungitoxic compounds present around the substomatal cavities of wheat leaves. MgAtr4 is the first virulence factor cloned from this important plant pathogen.
A Gene-for-Gene Relationship Between Wheat and Mycosphaerella graminicola, the Septoria Tritici Blotch Pathogen
Brading, P.A. ; Kema, G.H.J. ; Verstappen, E.C.P. ; Brown, J.K.M. - \ 2002
Phytopathology 92 (2002)4. - ISSN 0031-949X - p. 439 - 445.
disease resistance genes - cladosporium-fulvum - host cultivars - pathosystem - avirulence - virulence - histology - infection - cf-9
Specific resistances to isolates of the ascomycete fungus Mycosphaerella graminicola, which causes Septoria tritici blotch of wheat, have been detected in many cultivars. Cvs, Flame and Hereward, which have specific resistance to the isolate IPO323, were crossed with the susceptible cv. Longbow. The results of tests on F1 and F2 progeny indicated that a single semidominant gene controls resistance to IPO323 in each of the resistant cultivars. This was confirmed in F3 families of Flame x Longbow, which were either homozygous resistant, homozygous susceptible, or segregating in tests with IPO323 but were uniformly susceptible to another isolate, IPO94269. None of 100 F2 progeny of Flame x Hereward were susceptible to IPO323, indicating that the resistance genes in these two cultivars are the same, closely linked, or allelic. The resistance gene in cv. Flame was mapped to the short arm of chromosome 3A using microsatellite markers and was named Stb6. Fifty-nine progeny of a cross between IPO323 and IPO94269 were used in complementary genetic analysis of the pathogen to test a gene-for-gene relationship between Stb6 and the avirulence gene in IPO323. Avirulence to cvs. Flame, Hereward, Shafir, Bezostaya 1, and Vivant and the breeding line NSL92-5719 cosegregated, and the ratio of virulent to avirulent was close to 1: 1, suggesting that these wheat lines may all recognize the same avirulence gene and may all have Stb6. Together, these data provide the first demonstration that isolate-specific resistance of wheat to Septoria tritici blotch follows a gene-for-gene relationship.
Non-homologous chromosome synapsis during mouse meiosis : consequences for male fertility and survival of progeny
Peters, A.H.F.M. - \ 1997
Agricultural University. Promotor(en): C. Heyting; P. de Boer. - S.l. : Peters - ISBN 9789054857761 - 182
muridae - muizen - meiose - geslachtelijke voortplanting - parthenogenese - polyembryologie - vruchtbaarheid - overleving - levensvatbaarheid - interacties - milieu - uitsterven - stofverplaatsing - chromosoomtranslocatie - chromosomen - cytologie - histologie - muridae - mice - meiosis - sexual reproduction - parthenogenesis - polyembryony - fertility - survival - viability - interactions - environment - extinction - translocation - chromosome translocation - chromosomes - cytology - histology
In the mouse, heterozygosity for several reciprocal and Robertsonian translocations is associated with impairment of chromosome synapsis and suppression of crossover formation in segments near the points of exchange during prophase of meiosis. This thesis describes the analysis of the consequences of the occurrence of non-homologous synapsis and/or suppression of meiotic crossover formation over many successive generations for male fertility and viability of the progeny.

For studying chromosome synapsis, we modified a drying down technique which results in high yields of nuclei of all first meiotic prophase stages in both male and female from only small amounts of tissue (chapter 2). Preparations are suitable for synaptonemal complex (SC) analysis by normal light and electron microscopy (chapters 2, 3 and 7), for fluorescence immunocytochemistry and in situ hybridization (chapters 2, 8).

In the study presented in chapter 3, we analysed the variation in male fertility of mice double heterozygous for two near identical reciprocal translocations T(1;13)70H and T(1;13)1Wa in relation to the synaptic behaviour of two differently sized heteromorphic bivalents during meiotic prophase. Male fertility rises when non-homologous synapsis in the small 1 13heteromorphic bivalent, leading to a "symmetrical" SC, is more frequent at the initial prophase stages. Based on the data presented, we favour the "unsaturated pairing site" model as the primary cause for male sterility.

In T70H/T1Wa females not all heterologous synapsis within the small heteromorphic bivalent is effectuated during the early stages of meiosis; some is achieved lateron by the mechanism of "synaptic adjustment" (chapter 3). Each heteromorphic bivalent contains a copy of the chromosome 1 region between the T70H and T1Wa breakpoints which is about 10 cM in size (Δ1 segment). Although axial elements representing these Δ1 segments are seen to approach each other during early meiotic prophase stages, they never successfully constitute a synaptonemal complex in either sex (chapter 3). This agrees with the fact that in earlier cytogenetic studies quadrivalents were never seen at both male and female diakinesismetaphase 1.

In chapter 7, we demonstrate that male fertility of the T70H/T1Wa mice is not only determined by the chromosomal constitution of the carrier but is additionally influenced by the pairing or synaptic history in previous meioses of especially the T70H and T1Wa short translocation chromosomes. Fertility of T70H/T1Wa males is more impaired after one or more successive transmissions of the T1Wa translocation chromosomes through a heteromorphic bivalent configuration, irrespective of the sex of the transmitting parent.

Furthermore, we show that the introduction of the Robertsonian translocation Rb(l1.13)4Bnr into the T70H/T1Wa karyotype restores fertility of double heterozygous males by stimulating non-homologous synapsis of the small heteromorphic bivalent. We speculate that this Rb4Bnr effect is mediated by a prolongation of the early stages of meiotic prophase I.

Successive female transmissions of the T1Wa translocation chromosomes in the presence of Rb4Bnr inititially resulted in an increase of the capacity for early meiotic nonhomologous synapsis within the small heteromorphic bivalent, leading to a restoration of fertility for the majority of carriers. Subsequently, a decrease of the capacity of the small heteromorphic bivalent to fully synapse was noticed, although a higher than original (F1) background level of male fertility remained.

These variations in male fertility are most likely based on epigenetic variance, reflected as the capacity to engage into non-homologous synapsis early in male meiosis leading to a "symmetrical" SC, despite the different amounts of chromatin to accommodate.

In chapter 4, the localization of several microsatellite markers and single copy genes relative to the T70H and T1Wa breakpoints, using quantitative PCR, quantitative Southern blotting and in situ hybridization, is described.

In chapter 5, we investigated the level of suppression of meiotic recombination and impairment of chromosome synapsis in T70H heterozygotes in relation to the viability of the progeny. For T70H/+ females, the introgression of the D1Mit4, D1Mit20 and D1Mit122 microsatellite marker alleles positioned distal of the T70H breakpoint on the normal chromosome 1 into the 13 1T70H long translocation chromosome was suppressed in a distance dependent manner. This effect was more pronounced in T70H/+ females, additionally homozygous for Rb4Bnr. The delay in introgression was paralleled by a reduction of the frequency and extent of non-homologous synapsis in segments near the T70H breakpoints of the pachytene translocation multivalents in T70H/+ and Rb4BnT70H/Rb4Bnr+ males. The extend of non-homologous synapsis around the centre of the synaptic cross configuration in these males correlated with fluctuations in prenatal viability of segregating translocation homozygotes in crosses between (Rb4Bnr)T70H homozygous males and heterozygous females when meiotic drive at the female second meiotic division is excluded. The reduction in viability is explained by the gain of mutations resulting from incorrect processing of recombination intermediates which is due to non-homologous synapsis around the translocation breakpoints.

In chapter 6, we analysed the consequences of the absence of crossing over for regions between the T70H and T1Wa breakpoints (Δ1 and Δ13 segments) of the Rb4BnrT1Wa translocation chromosomes, which have been transmitted for over 20 generations via heteromorphic bivalents in Rb4BnrT70H/Rb4BnrT1Wa females. Survival of heterozygous and homozygous carriers for these segments was taken as the phenotypic endpoint. The viability of progeny of crosses between Rb4BnrT70H homozygous males and Rb4BnrT70H/Rb4BnrT1Wa females, of which the latter principally produce 4 types of gametes, was estimated using a haplotype analysis of microsatellites in the Δ1 segment for genotyping (see chapter 4). We observed no differences in the pre- and postnatal survival rates of the double heterozygous and 13 1H, 13 1H, 1 13Wa 1 13H "duplication" progeny in which the Δ1 and Δ13 segments of the T1Wa translocation chromosomes had either no, an onegeneration or a multi-generation history of non-homologous synapsis in heteromorphic bivalents during previous female meioses. In addition, intercrossing of Rb4BnrT70H/Rb4BnrT1Wa double heterozygotes after genetic isolation of these Δ1 and Δ13 segments for 20 to 22 generations, showed that the viability of the Rb4BnrT1Wa homozygotes was not different from the Rb4BnrT70H homozygous and Rb4BnrT70H/Rb4BnrT1Wa karyotypes generated by this cross. Thus, exclusion of the Δ1 and Δ13 segments from meiotic crossing over within non-homologous synapsed heteromorphic bivalents during 20 to 25 successive generations does not result in an accumulation of recessive lethal mutations or an increased susceptibility for gaining dominant lethal mutations.

For the D1Mit122 microsatellite used in offspring haplotyping a higher mutation frequency was observed after transmission through a double heterozygous than after transmission through a T70H homozygous karyotype (chapter 6). On the basis of the identity of the mutations, the ectopic pairing of the St2 gene copies (containing D1Mit122) during meiosis of T70H/T1Wa males (chapter 8) and the observation of ectopic homologous contacts of the Δ1 segments during the zygotene stage without SC formation (chapter 3), we speculate that these mutations are the result of ectopic homologous gene conversion events most likely occurring in the absence of a synaptonemal complex.

The crossover suppressive influence of the Rb translocation on the Δ1 segment (chapter 5) enabled us to analyze the effects of introgression of genetic material from the Swiss +/+ stock into the translocation karyotypes. Introgression of "new" genetic material correlated with an increase in littersize of Rb4BnrT70H homozygotes (chapter 5), an improvement of the life expectancy of Δ1 duplication offspring from double heterozygous mothers (chapter 6) and a clear improvement of male fertility in double heterozygous and T70H homozygous males also carrying Rb4Bnr (chapter 7). These pleiotrophic findings are discussed in chapter 8 in terms of genetic versus epigenetic mechanisms of inheritance.

Finally, when T1Wa was backcrossed for many generations to the Rb4BnrT70H/Rb4BnrT70H karyotype, essentially precluding genetic recombination in the Δ1 and Δ13 segments, or when T1Wa was combined with Rb4Bnr after many successive transmissions via alternating T1Wa heterozygotes and homozygotes, stable Rb4BnrT1Wa homozygous lines could not be bred (chapter 8). Especially female reproductive performance decreases after repeated male and female homologous meiosis. As non-homologous synapsis in the centre of the synaptic cross configuration in T1Wa/+ males is common too (unpublished results), more work into the genetic stability of chromosome segments, that have a history of hindered homologous interaction, is indicated (chapter 8).

Imaging of polarity during zygotic and somatic embryogenesis of carrot (Daucus carota L.)
Timmers, A.C.J. - \ 1993
Agricultural University. Promotor(en): M.T.M. Willemse; J.H.N. Schel. - S.l. : Timmers - ISBN 9789054851004 - 123
somatische embryogenese - daucus carota - penen - apiaceae - planten - embryologie - cytologie - histologie - immunocytochemie - somatic embryogenesis - daucus carota - carrots - apiaceae - plants - embryology - cytology - histology - immunocytochemistry

In this thesis a study of the regulation of coordinated growth and the development of polarity during embryogenesis of carrot, Daucus carota L., is described. To this end, several microscopical techniques were used, such as light microscopy, fluorescence microscopy, confocal scanning laser microscopy and electron microscopy. Next to this, immunocytochemical methods were used frequently to localize proteins in plant tissue sections.

Plants are composed of several types of organs and tissues, each of them having a characteristic structure and function. For the development of a full-grown germling from one cell, the zygote, a tight regulation of growth and differentiation is required. During this process of embryogenesis, growth proceeds through a number of developmental stages which are described subsequently as globular, oblong, heartshaped and torpedo-shaped.

Despite the large number of observations on embryogenesis, made in various plants, the molecular and cellular basis of this developmental pathway is still poorly understood. The divalent cation Ca 2+participates in the initiation and maintenance of a great variety of physiological processes, including the regulation of cell polarity, cell division, cell growth, cell volume, hormone action and distribution, and enzyme synthesis and activation. Considering the diversity of processes in which Ca 2+is involved, it is to be expected that an investigation of the distribution of Ca 2+, and Ca 2+binding proteins, during plant embryogenesis, will lead to a deeper understanding of the regulation of this process.

Studies on zygotic embryogenesis are hampered by the presence of surrounding maternal tissue. Therefore, somatic embryos of carrot are used often as experimental substitutes for zygotic embryos, since the discovery of in vitro embryogenesis in carrot cultures in 1958. Carrot somatic embryos can be obtained, relatively easily, in great amounts, essentially free of surrounding tissue, just by transferring cell clusters, designated as proembryogenic masses, from medium supplemented with the growth regulator 2,4-D to medium without 2,4-D. This feature makes carrot an ideal model system for the study of plant embryogenesis.

In Chapter 1, the general introduction, the zygotic and somatic embryogenesis of carrot is described structurally. Similarities and differences between both processes are mentioned. Many external factors, which are described extensively in the literature, influence the development of somatic embryos. For normal growth and development to occur, the presence of Ca 2+in the medium is absolutely required, and embryogenesis is enhanced specifically by a rise of [Ca 2+]. In this chapter, the role and distribution of Ca 2+in plants is briefly described.

The principal targets of calcium signals in eukaryotes are calciumbinding proteins of which calmodulin, a protein present in all plant cells, has been studied most extensively. The structure, activity and localization of this acidic, small and heatresistant protein is described from the literature. Chapter 1 ends with a survey of techniques which are nowadays available for the localization of Ca 2+and calmodulin in plants.

In Chapter 2, chlorotetracycline and fluphenazine, two fluorescent indicators, are being used to localize Ca 2+and activated calmodulin respectively, during carrot somatic embryogenesis. Embryogenesis appears to coincide with a rise in [Ca 2+] and activated calmodulin is mainly found in the future root side of the embryo. It is concluded, that the polarity in the distribution of calmodulin is already present before polarity is visible morphologically.

Fluphenazine visualizes only activated calmodulin. In Chapter 3, the distribution of both activated and non- activated calmodulin has been studied with the aid of antibodies. Besides the various developmental stages of somatic embryogenesis, also zygotic embryos and a number of stages of zygotic embryo germination have been studied in this chapter. The most striking observation is that the distribution of calmodulin in somatic embryos differs strongly from the distribution in zygotic embryos, but resembles the distribution during zygotic embryo germination. Both in somatic embryos and in germinated zygotic embryos, calmodulin appears to be present mainly in amyloplasts, while in zygotic embryos calmodulin predominantly was found to be localized in the cytoplasm.

For a detailed analysis of the distribution of Ca 2+in living, intact plant cells in tissues with fluorescent indicators, confocal laser scanning microscopy is the method of choice. A suitable indicator is fluo-3. Unfortunately, the plasma membrane is not permeable for this compound. Therefore, a method had to be developed with which fluo-3 could easily be loaded into plant cells. In Chapter 4, it is described that with the aid of digitonin fluo-3 can be used successfully for the localization of Ca 2+in embryogenic plant cells, in combination with confocal scanning laser microscopy.

As has already been noticed in Chapter 2, carrot somatic embryogenesis coincides with a rise in [Ca 2+]. In Chapter 5, a detailed analysis has been made of the distribution of free cytosolic Ca 2+during carrot somatic embryogenesis with the aid of the method described in Chapter 4. It appeared that [Ca 2+] is especially high in the protoderm of the embryos, and gradients in [Ca 2+] along the longitudinal axis of torpedo-shaped embryos were frequently observed. Very obvious was the high [Ca 2+] in the nuclei of protoderm cells, This nuclear localization was confirmed by antimonate precipitation, by which Ca 2+is visualized as electron dense precipitates in the electron microscope.

The concentration of Ca 2+in the cytosol is not only linked with the concentration in the vacuole, but also with the pH of the cytosol and the vacuole. Since both pH and [Ca 2+] are important factors during embryo genesis, in Chapter 6 a study is described of the distribution of pH in vacuoles during somatic and zygotic embryogenesis and during zygotic embryo germination of carrot. Neutral red and acridine orange were used as indicators of vacuolar pH and their distribution has been compared with the distribution of fluphenazine. Strikingly, major similarities were found between the distribution of the three probes used, and all three reacted similarly on treatments with A231 87, EGTA or propionic acid. Confocal microscopy revealed a network of vesicles and tubules, predominantly present in the protoderm of somatic embryos and germinated zygotic embryos after incubation in acridine orange. Proposed is, that calmodulin is possibly involved in the digestion of cell material in autophagic vacuoles or is involved in the regulation of the movements of vacuolar tubules. However, additional research is necessary to explain the observed distribution patterns satisfactory.

From the previous chapters it appeared that noticeable differences exist between somatic and zygotic embryos in the distribution of anti calmodulin, fluphenazine fluorescence, neutral red and acridine orange. In Chapter 7 a possible structural basis of these differences has been searched for. The distribution of anti-calmodulin, and perhaps also fluphenazine fluorescence, could be linked with the presence of amyloplasts, which were abundant in somatic and germinated zygotic embryos, but which were not found in zygotic embryos. Differences in the localization of neutral red and acridine orange are related to differences in vacuolation between somatic and zygotic embryos.

Chapter 8 is the general discussion. Here, somatic embryo initiation, its early development and the formation of polarity during somatic and zygotic embryogenesis of carrot are the main topics. It is concluded, that important differences exist between somatic and zygotic embryogenesis and that the process of somatic embryogenesis shows similarities with zygotic embryo germination. The chapter ends with some concluding remarks about the methods used for the localization of Ca 2+and calmodulin.

Immunofluorescence microscopy and dilution-plating for the detection of Xanthomonas campestris pv. campestris in crucifer seeds : methods to determine seed health and seed infection
Franken, A.A.J.M. - \ 1992
Agricultural University. Promotor(en): J. Dekker. - S.l. : Franken - 165
plantenziekten - plantenziekteverwekkende bacteriën - koolsoorten - kleuring - fluorescerende kleurstoffen - immunologische technieken - elisa - xanthomonas - cytologie - histologie - immunologie - antigenen - plant diseases - plant pathogenic bacteria - cabbages - staining - fluorescent dyes - immunological techniques - elisa - xanthomonas - cytology - histology - immunology - antigens
Black rot is one of the most threatening diseases of crucifers. The causal agent of this disease is the bacterium Xanthomonas campestris pv. campestris . The bacterium attacks all cultivated brassicas, radishes and numerous weeds, and is able to survive on plant debris in the soil. The primary source of inoculum is often infected seed. The most important ways to control black rot are the use of resistant cultivars and the use of 'healthy' seed. To obtain healthy seed, chemical or physical seed treatments may be used. These seed treatments may, however, seriously damage the seed quality (germination), may cause phytotoxicity or may not sufficiently eliminate the black rot pathogen from the seed (chapter 2). To check the seed health, sensitive and specific methods are needed to detect X. c. pv. campestris in the seed. Several methods for detecting X. c. pv. campestris are summarized in chapter 2. The most commonly used assays are plating assays, in which seed washings are plated onto isolation media, such as BSCAA (basal starch cycloheximide agar with nitrofurantoin and vancomycin), CS20ABN (a starch medium with bacitracin, neomycin and cycloheximide), FS (a medium with starch trimethoprim, cephalexin, cycloheximide, methyl green), NSCA (nutrient starch cycloheximide agar), NSCAA (NSCA with nitrofurantoin and vancomycin), and SMA-medium (starch-methionine agar with cephalexin and nitrofurantoin). Serological assays such as enzyme immuno-assays, Ouchterlony double diffusion, agglutination and immunofluorescence microscopy (IF) can be used for identification of pure cultures of X. c. pv. campestris . Cross-reactions with other pathovars of X. campestpis have, however, been reported. So far, IF using polyclonal antisera was the only serological technique employed for detecting X. c. pv. campestris in seed washings.

The aim of this study was to analyse important characteristics of IF and plating assays, and to improve their use for identification and detection of X. c. pv. campestris in crucifer seeds. Methods for the detection and identification of X. c . pv. campestris are reviewed in chapter 2. In chapter 3 some aspects of plating assays for isolation of Xanthomonas campestris pv. campestris from crucifer seeds are discussed. Little differences were found between results obtained with NSCA, NSCAA and FS medium. It was, however, noted that the performance of the media often depends on the seed lot and extraction method used. Therefore, probably other methods such as immunofluorescence microscopy (IF) are needed to confirm presence of X. c. pv. campestris with higher certainty. With the extraction methods 2.5 h shaking and 1.5 h soaking, more colonyforming units were recovered from some seed lots than with the standard 5 min shaking of seed lots. However, prolonged extraction did not result in finding more seed lots infected. However, the use of two methods for extracting X. c. pv. campestris from crucifer seed, will enhance the chance of isolating the pathogen from the seed (chapter 3 and 6).

In chapter 4 the specificity of polyclonal antisera and monoclonal antibodies for identification of X. c. pv. campestris is discussed. Polyclonal antisera reacted in IF with all strains of X. c. pv. campestris and other xanthomonads (e.g. X. c. pv. vesicatoria and amoraciae ) at low dilution (1:100). Non-xanthomonads also reacted with 2 out of 3 polyclonal antisera at this dilution. At higher dilutions (1:900), however, most crossreactions with non-xanthomonads disappeared as well as reactions with some strains of X. c. pv. campestris and other pathovars. Six monoclonal antibodies (MCA 17C12, MCA 16B5, MCA 20H6, MCA 2F4, MCA 18G12, MCA 10C5), produced against X. c. pv. campestris were tested in immunoblotting (IB), an enzyme immunoassay (EIA), dot-blot immunoassay (DBI) and IF. The monoclonal antibodies reacted with the lipopolysaccharide (MCA 20H6, 2F4, 18G12, and IOC5) or membrane proteins (MCA 17C12 and 16B5) of X. c. pv. campestris in IB Two monoclonal antibodies (MCA 17C12 and 16B5) reacted with all xanthomonads tested in DBI, but not in IF and EIA. The other monoclonal antibodies (MCA 20H6, 2F4, 18G12, and 1OC5) did not react with all strains of X. c. pv. campestris and did react with some other xanthomonads, such as X. c. pv. vesicatoria and amoraciae, in IF, EIA and DBI. It was concluded that some polyclonal antisera and monoclonal antibodies may be used for identification of (a group of) strains of X. c. pv. campestris. The question, whether monoclonal antibodies, as compared to a polyclonal antiserum, may give rise to false- negative or false-positive results when testing seed lots, is dealt with in chapter 5. IF with one polyclonal antiserum
and monoclonal antibodies was used for direct detection of X. c. pv. campestris in seed washings and identification of colonies. For direct detection of X. c. pv. campestris in seed washings, it was found that IF-results (cell counts) depend in part on the seed lot tested, the extraction method and antibodies used. This confirmed the interactions found in plating assays. The polyclonal antiserum tested (PCA 94) did not always result in detection of more fluorescent cells in IF. On the contrary, for some seed lots and extraction methods the opposite was found; monoclonal antibodies sometimes detected more fluorescent cells than polyclonal antisera. When using two extraction methods (5 min and 2.5 h shaking at room temperature) for one seed lot the risk of false-negative results in IF could be minimized (chapter 5 and 6). When monoclonal antibodies were used for identification of colonies, it was found that some X. c. pv. campestris colonies did not react with antibodies MCA 16B5, MCA 17C12, and to a lesser extent with MCA 1OC5, MCA 2F4, and MCA 18G12. Saprophytic isolates of one seed lot cross-reacted with MCA 17C12 and to a lesser extent with MCA 2F4, 18G12 and PCA 94. On the basis of this study MCA 20H6 was considered to be the most suited antibody for detection and identification of X. c. pv. campestris .

Chapter 6 shows that the correlation between IF and dilution-plating was similar for one monoclonal antibody (MCA 20H6) and one polyclonal antiserum tested (PCA 94). With increasing cell numbers in IF the chance of isolating X. c. pv. campestris also increased. With IF generally much more seed lots were found positive than with dilution-plating. It was shown that the correlation between IF and dilution-plating depended in part on the volume of seed extract examined. When examining relatively large volumes (e.g. 50 μl), the sensitivity of IF will be enhanced and the risk of false-negative reactions in IF as compared to dilution-plating will be decreased. On the other hand the correlation with dilution-plating decreased. It was concluded that IF generally gives a good prediction of 'health' of a seed lot and that dilution-plating generally gives a good prediction of 'disease'. Chapter 7 summarizes the problems with serological techniques for seed-borne bacteria and gives possible solutions to be worked out in future.

To minimize the risk of false-negative results, it is advisable to use at least two plating media (e.g. NSCAA and CS20ABN) in dilution-plating and two extraction methods (5 min and 2.5 h shaking at room temperature) in both IF and dilution-plating. For IF, it is advisable to screen either with different monoclonal antibodies separately, with a mixture of monoclonal antibodies (e.g. MCA 20H6, MCA 2F4 and MCA 18G12), or both a monoclonal antibody (MCA 20H6 or 2F4) and a high quality polyclonal antiserurn (PCA 94).

More research is needed, however, to assess the significance of interference by saprophytes and antibiotic substances, released by the seed or saprophytes, with the detection assay and the survival of the pathogen in the seed.

Isolation and characterization of Spinacia oleracea L. sperm cells
Theunis, C.H. - \ 1992
Agricultural University. Promotor(en): J.L. van Went; H.J. Wilms. - S.l. : Theunis - 89
spinacia oleracea - spinazie - chenopodiaceae - gameten - spermatozoön - eicellen - cytologie - histologie - spinacia oleracea - spinach - chenopodiaceae - gametes - spermatozoa - ova - cytology - histology

Gametes are specialized cells with the natural capacity to fuse in a well determined way. The fusion products are intended to develop into new individuals. Basic knowledge of gametes is of great importance for both traditional plant breeding as well as for modem biotechnology and gene manipulation. For applications in these fields, more knowledge is necessary of the characteristics of gametes and the mechanisms involved in the process of gamete recognition and fusion. Isolated gametes form ideal material to investigate this. The present study was focused on the isolation and characterization of the male gametes, the sperm cells.

In chapter 1 an introduction is given. The current information we have on in situ sperm cells, and on the subject of sperm cell isolation, is summarized in this chapter.

In chapter 2 the ultrastructure is described of the pollen grains of Spinacia oleracea and the sperm cell pair therein. The pollen grain is trinucleate and consists of a vegetative cell and two sperm cells. The pollen grain wall is tectate, with many germination pores, which have a hexagonal distribution. The vegetative nucleus together with the sperm cells are located in the periphery of the pollen grain and are organized in a "male germ unit". The cytoplasm of the vegetative cell contains vacuoles and electron dense vesicles. The mitochondria have a size of 0.3 μm to 0.5 μm. The ER is often organized in single elements, and bears ribosomes. The plastids are filled with starch and only the outer membrane is visible. The high amount of starch may be used in an autotrophic way of germination, or for osmotic stabilization during germination. Microtubules are not found in the vegetative cytoplasm.

The sperm cell inside the pollen grain contains a heterochromatic nucleus, mitochondria, dictyosomes, and ER. The two sperm cells are attached to each other. They form a pair which is surrounded by a vegetative plasma membrane. Only a few microtubules have been shown in the sperm cell cytoplasm. In previous studies, microtubules have been clearly demonstrated inside the sperm cells. Therefore, it was concluded that the used method of freeze substitution does not completely stop the breakdown of microtubules.

In order to release the sperm cells, the vegetative cells has to be opened. The osmotic shock method, which has been used in some species, does not work for pollen grains of Spinacia oleracea. Even in pure water only a small percentage of the pollen grains bursts. For this reason, a mechanical method has been developed, using a glass roller to squash large quantities of pollen grains. This method is described in chapter 3.

Because of the squashing of the grain, the sperm cells are released from the pollen grain together with most of the vegetative cytoplasm. Since physical breaking is applied, any medium can be chosen in which the breaking is performed. After the squashing, the mixture of pollen grains, free sperm cells, vegetative nuclei, vegetative organelles, and pollen grain fragments is filtered over a 25 μm nylon filter. Subsequently, the filtrate is centrifuged on a 20% percoll layer for further elimination of small debris. With this method, a fraction is obtained which contains numerous sperm cells, but which is still contaminated with small vegetative organelles, and small pollen fragments. The yield is approximately 5-10% with a final concentration of 4x10 6sperm cells/ml.

The free sperm cells are elongated just after squashing, but become spherical after a short time. The originally paired sperm cells separate. The close association of the sperm cell pair with the vegetative nucleus is not maintained during squashing and is therefore, not a firm binding. The sucrose concentration of the medium does not influence the change in shape of the free sperm cells. The volume, however, is influenced by the osmotic value of the medium. The 25 % sucrose concentration was chosen for the rest of the experiments in order to avoid damage caused by osmotic swelling. The diameter of the isolated sperm cells can vary from 4 μm to 9 μm, depending on the sucrose concentration.

Immediately after isolation, more than 90% of the sperm cells is viable (tested with the fluorescein diacetate test). Soon after isolation, however, some of the cells loose their viability. After 18 h, only 50% of the isolated cells is still viable. Storage of the isolated sperm cells at low temperatures (0°C) doubles the lifespan. Addition of 1 % vitamin C also enlarges the lifespan. It is concluded that depletion of energy is not the cause of the loss of viability, since addition of 0.1 M ATP makes no difference for the lifespan.

With histochemical tests, using calcofluor white MR2 for cellulose, analine blue for callose, and the PAS reaction for carbohydrates, no cell wall material was observed around the isolated sperm cells. This indicates that the cells are true protoplasts.

In chapter 4 the results are presented of the analysis of the numbers of mitochondria in isolated sperm cells. To visualize the mitochondria, two staining methods have been used. The DiOC 6 (3) staining (in a concentration of 0.1 μg/ml) gives better results with less background staining, than the Rhodamine 123 staining.

The analysis was carried out on individual sperm cells, as well as on sperm cell pairs. If individual sperm cells were used, two populations of sperm cells seemed to be present, with an average of respectively 10.3 and 17.8 mitochondria per sperm cell. However, by counting the mitochondria in sperm cell pairs, it is found that there is only one population of sperm cells. The average is 12.4 ± 4.6 mitochondria per sperm cell. The number of mitochondria per sperm cell varies from 2 to 25, which is a high variation. This high variation can be explained in two ways. It is possible that already after the division of the microspore, a high variation exists in number of mitochondria per generative cell. The second explanation can be, that during the development of the generative cell and/or sperm cells, mitochondria are produced or lost.

With the technique of freeze-fracturing, the plasma membranes of the isolated sperm cells were examined, including the intra-membrane particles (IMP's). In chapter 5 the results are presented. Also with this method, no remnants of the vegetative plasma membrane were found around the sperm cells. Only incidentally, transverse fracture planes through whole sperm cells are found. Most of the fracture planes of sperm cells follow the sperm cell plasma membrane exposing either the PF or the EF face. Neither the ES or the PS faces are found. Both the PF as well as the EF face show IMP's. These IMP's are randomly distributed, and no pattern can be recognized. The PF face has a density of 719 IMP's/μm 2. The EF face has a density of 2088 of IMP's/μm 2. Evidently, the EF half of the sperm cell membrane contains approximately 3 times more IMP's than the PF half. For sporophytic protoplast it has been reported that the PF half contains more IMP's than the EF half. The specific IMP distribution in the sperm cell plasma membrane may be related to the process of gamete recognition and subsequent fusion. With respect to IMP's density, only one type of sperm cells was observed and therefore for this character, no dimorphism could be established.

Morphometrical and ultrastructural characterization of the isolated sperm cells has been reported in chapter 6. Experiments with various fixations demonstrate that sperm cells are fragile, and difficult to fixate. The osmotic value of the fixation media appear to be of great importance. Also with this method it is clear that the isolated sperm cells are separate and completely spherical. The surrounding vegetative plasma membrane has disappeared. No cell wall material is observed. The sperm cell contains a large nucleus which can be either heterochromatic or euchromatic. The mitochondria are spherical and frequently appear to be clustered in groups of 5 to 10 mitochondria, but also individual mitochondria have been observed. The dictyosomes have 4 to 5 cisterns with associated small vesicles. Small vacuoles are present. The endoplasmatic reticulum is sparse, and often dilated. Ribosomes are sometimes grouped in polysomes. No microtubules have been observed. From these observations it is clear that isolated cells contain a similar set of organelles as the in situ sperm cells. After measuring the section diameters, the average diameter of the complete cell is calculated to be 3.66 μm with the used fixation conditions. From surface area's in the sections is calculated that 50 % of the sperm cell is occupied by its nucleus, 2.5 % of the cell is mitochondria, and 0. 6 % of the cell is dictyosome. The ultrastructural analysis did not give any indication that in Spinacia oleracea sperm cell dimorphism in regard of sperm cell size, exists.

In chapter 7 the results of the present thesis are discussed in general sense and in a broader context. Results which have already been discussed in previous chapters are not further discussed in this general discussion.

The isolation technique of "physical breaking", developed for Spinacia oleracea is compared with the "osmotic shock" technique, used for other species. The advantages and disadvantages of both techniques are presented and discussed. The usefulness of the two techniques in further research of isolated gametes are explained. Preferences for one of the two technique clearly depends on the aims of the further research.

The general discussion highlights the two major phenomena sperm cells show when isolated: the changing of a spindle shaped cell to a spherical cell, and the loss of viability. The change in shape occurs in all species observed thus far. Likely, the change in shape is a natural process which also occurs during fertilization. The loss of viability can be slowed down with low temperature, and anti-oxidantia. During the natural fertilization process, extended viability is not necessary, since the free sperm cells fuse rapidly with the female partners. So also for this character it may be a natural phenomenon.

Sperm cell dimorphism, which has been reported in some species, is not found in Spinacia oleracea. The isolated sperm cells form good material to study dimorphism because of the large ~ties of cells which can be analyzed.

Histologische veranderingen bij koeien en schapen die in de praktijk en experimenteel blootgesteld zijn aan polygechloreerde dibenzo-p-dioxinen en -furanen
Groot, M.J. ; Berende, P.L.M. ; Ossenkoppele, J.S. ; Tuinstra, L.G.M.Th. - \ 1991
Wageningen : DLO-Rijks-Kwaliteitsinstituut voor Land- en Tuinbouwprodukten (Rapport / RIKILT-DLO 91.25) - 31
koeien - dioxinen - polychloordibenzofuranen - microscopie - biopsie - schapen - histologie - cows - dioxins - polychlorinated dibenzofurans - microscopy - biopsy - sheep - histology
Histologisch onderzoek is verricht op organen van koeien en schapen, welke chronisch en experimenteel bloot hebben gestaan aan dioxinen. Alle dieren waren op het moment van slachten klinisch gezond. Bij de meeste dieren werden histologische afwijkingen waargenomen. De waargenomen veranderingen kunnen niet zonder meer aan dioxinen worden toegeschreven aangezien de bodem van het gebied van herkomst van de dieren vervuild was met andere toxische stoffen (o.a. lood , cadmium, fluor, molybdeen), welke ook een bijdrage aan de waargenomen weefselveranderingen zullen hebben geleverd. Daarnaast betrof het oudere dieren waarvan de ziektegeschiedenis weinig bekend was.
A histochemical study of root nodule development
Wiel, C. van de - \ 1991
Agricultural University. Promotor(en): A. van Kammen. - S.l. : Van de Wiel - 189
wortelknolletjes - knobbelvorming - histologie - cytologie - root nodules - nodulation - histology - cytology

In cooperation with soil bacteria of the genera Rhizobium , Bradyrhizobium or Azorhizobium , many members of the legume family are able to form specialized organs on their roots, called root nodules. The bacteria, wrapped up inside a plant membrane, are accomodated in large parenchymatic cells located centrally in these root nodules. For this, they reward their host by converting atmospheric nitrogen into a form usable for the plant. The central infected tissue of the nodule is surrounded by a peripheral tissue provided with vascular bundles through which metabolites are exchanged with the other parts of the plant.

In the interaction with the bacteria, the host plant expresses specific genes that are not transcribed at a detectable level in other parts of the plant. The products of several of these genes are made during the formation of the nodule and are named early nodulins.

The present study aims at elucidating the role of these early nodulins in the formation and infection of the root nodules. For that purpose, we set out to combine the molecular approach of studying gene expression with the microscopical approach of studying the structural development of the nodule.

To provide a background to these studies, chapter 11 summarizes existing knowledge about nodule development from an anatomical/cytological point of view, supplemented with data on already described nodulins and with brief excursions into physiological phenomena relevant to the rest of our study.

In chapter III and IV, nodulin gene expression is analysed in common vetch ( Vicia sativa ) nodules elicited by a panel of bacterial strains with various defined genetic changes. Such nodules were blocked at different stages in the development of the central tissue depending on the bacterium involved; the precise stage at which the blockade occurred was determined by light- and electron-microscopical observations. In that way, insight could be gained in the diverse genetic information supplied by the bacterium for nodule development to proceed through the successive developmental stages and the induction of the appropriate nodulin genes going with it. Furthermore, the start of the expression of individual nodulin genes, for instance the early nodulin Nps-40', could be related to certain stages of central tissue development. In the case of the leghemoglobin genes, such a correlation between nodulin gene expression and specific developmental stages could be confirmed by the direct localization of the leghemoglobin proteins in pea ( Pisum sativum ) nodule sections comprising different consecutive developmental stages, by immunolabeling.

Such direct approach of studying nodulin gene expression in nodule sections was further pursued in the chapters V, VI, VII and VIII. In chapters V, VI and VII early nodulin gene transcripts for which sequenced cDNA clones had become available were localized by in situ hybridization: in chapter V, ENOD2 in soybean ( Glycine max ) and pea nodules, respectively; in chapter VI, ENOD2 in alfalfa ( Medicago sativa ) nodules; and in chapter VII, PsENOD12 in pea. In chapter VIII an attempt to localize the Nps-40' protein by immunolabeling in pea nodules is described. By these in situ localization methods, different temporal and spatial patterns of gene expression for each early nodulin were determined. Speculations about the functions of the individual nodulins are made based upon the gene expression patterns and the amino acid sequences of the nodulins as deduced from the nucleotide sequence of the corresponding cDNA clones.

In addition, in chapter VI, in situ localization of MsENOD2 transcripts was performed on alfalfa nodules induced by certain engineered bacterial strains or by auxin transport inhibitors. Such nodules do not have bacteria in their central tissue and also differ in other structural details from effective nodules, but nevertheless were shown to exhibit a tissue- specific expression pattern of the MsENOD2 gene similar to effective nodules. In chapter VII the results of further experiments are reported pertaining to the influence of the bacterium on nodulin gene expression, particularly the involvement of bacterial factors and the bacterial nod genes in the induction of the expression of the PsENOD12 genes.

Finally, chapter IX summarizes the results of the in situ localization of early nodulin gene products. In the light of these results, the significance of our histochemical approach to elucidating the role of nodulins in root nodule development is discussed.

Elongation and contraction of the plant axis and development of spongy tissues in the radish tuber (Raphanus sativus L. cv. Saxa Nova).
Magendans, J.F.C. - \ 1991
Unknown Publisher (Wageningen Agricultural University papers 91-1) - ISBN 9789067541848 - 57
raphanus sativus - radijsjes - histologie - planten - plantenanatomie - plantenmorfologie - onderzoek - methodologie - radishes - histology - plants - plant anatomy - plant morphology - research - methodology
Histological, genetical and epidemiological studies on partial resistance in wheat to wheat leaf rust
Broers, L.H.M. ; Jacobs, T. - \ 1989
Agricultural University. Promotor(en): J.E. Parlevliet; J.C. Zadoks. - S.l. : Broers [etc.] - 201
plantenziekteverwekkende schimmels - triticum aestivum - tarwe - hexaploïdie - pucciniales - puccinia - planten - plaagresistentie - ziekteresistentie - plantenveredeling - plantenplagen - plantenziekten - epidemiologie - distributie - histologie - puccinia recondita - partiële resistentie - plant pathogenic fungi - triticum aestivum - wheat - hexaploidy - pucciniales - puccinia - plants - pest resistance - disease resistance - plant breeding - plant pests - plant diseases - epidemiology - distribution - histology - puccinia recondita - partial resistance

Partial resistance (PR) of wheat to wheat leaf rust acted in two steps. The first step led to a reduced infection frequency (IF), the second step to a longer latency period (LP).

The genes for a prolonged LP inherited in a recessive way and showed additive gene action. The PR-genotype Akabozu carried two and Westphal 12A three LP-prolonging genes. BH 1146 contained one HR gene and two or three LP genes. Transgression was observed in the progenies.

PR, measured as LP, was best expressed in the young flag leaf stage, at low temperatures. LP is a good estimator for PR.

Three epidemiological parameters were used to assess PR in the field., 1) disease severity at the time that susceptible controls are severely rusted, 2) AUDPC, 3) the logistic growth rate. 1) and 2) are reliable estimators for PR, 3) appeared unsuitable. Long LP and low IF were correlated.

Conclusive evidence for or against race specificity of PR has not been obtained. PR was expressed in a wide range of environments, indicating that PR is stable. The ranking orders were similar at all locations and years. The PR studied might be durable. The PR in BH 1146 is effective since 1955.

Onderzoek van de chemische en histologische samenstelling van snijworst
Cazemier, G. ; Herstel, H. - \ 1988
Wageningen : RIKILT (Rapport / RIKILT 88.06) - 6
vleeswaren - chemische samenstelling - histologie - meat products - chemical composition - histology
Localization of viral antigens in leaf protoplasts and plants by immunogold labelling
Lent, J.W.M. van - \ 1988
Agricultural University. Promotor(en): J.P.H. van der Want; B.J.M. Verduin. - S.l. : van Lent - 125
bromovirus - cellen - vignabonen - cytologie - histologie - methodologie - plantenziekten - plantenvirussen - relaties - technieken - vigna - virologie - virussen - bromovirus - cells - cowpeas - cytology - histology - methodology - plant diseases - plant viruses - relationships - techniques - vigna - virology - viruses

This thesis describes the application of an immunocytochemical technique, immunogold labelling, new in the light and electron microscopic study of the plant viral infection. In Chapter 1 the present state of knowledge of the plant viral infection process, as revealed by insitu studies of infected cells, is briefly reviewed. Until now, light and electron microscopic studies have merely described morphological changes in cells and tissue as a result of viral infection, but have failed to provide information on the functional role of these structures in the viral infection process and their association with viral components. A common cytopathological feature of many different plant viruses seems to be the induction of membranous vesicles or membranous bodies, which have been implicated in viral replication. However, only in a few cases some evidence was obtained with regard to the Intracellular location of viral replication and the association of replication and membranes. Available cytochemical techniques have apparently failed to provide a tool for the identification of virus particles and virus-encoded proteins within cellular structures. The Impact of a suitable detection techniques to elucidate the molecular processes of viral replication and transport insitu is obvious, as it would link findings obtained by invitro experiments to the events observed in the cell.

Immunogold labelling seems to provide such a tool for the tracing of antigens in light and electron microscopic preparations of biological specimens. Gold particles are excellent markers for electron microscopy, because of their high electron density which makes them appear as black dots In EM preparations. Furthermore, by a simple silver staining following gold labelling, viral antigens can be dete cted in semi-thin sections with the light microscope. The application of immunogold labelling for the light and electron microscopic localization of antigens is described in Chapters 2, 3, 4, 5, 6 and 7.

In Chapter 2 the preparation of homodisperse suspensions of colloidal gold particles is described. By adsorption of protein A to the surface of the gold particles, a marker (protein A-gold, pAg) is obtained which can be used for labelling antigen-antibody complexes. The specificity of the technique was demonstrated by gold labelling of antibodies bound to plant viruses in mixed suspensions of two viruses. Each virus was labelled using its homologous antiserum and pAg, and no significant cross-reaction with the other virus occurred. Simultaneous identification of two different viruses (CCMV and SBMV) with similar morphological appearance was achieved by double labelling with pAg-complexes containing gold particles of 7 and 16 nm, respectively. Immunogold labelling of viral antigens in suspension has been applied to distinguish between different serologically related viruses like strains of TMV (Pares and Whitecross, 1982), and the potyvirus sugarcane mosaic virus and maize dwarf mosaic virus (Alexander and Toler, 1986; 1985). A clear advantage of the immunogold labelling over conventional decoration of antigens is that the discrete gold particles allow quantification of the results.

The immunogold labelling of viral antigen in ultrathin sections of infected protoplasts is described in Chapter 3. Best results were obtained when the protoplasts were only mildly fixed with aldehydes, dehydrated and finally embedded in Lowicryl K4M at -30°C. The antigenicity of viral coat protein was well preserved. A disadvantage of the method is the limited preservation of cell structures, especially membranes due to extraction of lipids. Weibull etal. (1983) reported that approximately 50% of the lipid content of cells may be extracted, despite the low temperatures used in the Lowicryl K4M embedding procedure. Ashford etal. (1986) questioned the low temperature character of Lowicryl embedding, and found that during polymerization of the resin, temperature rises due to the exothermic nature of the reaction. With plant tissue (not protoplasts), low temperature dehydration and infiltration of the embedding resin must be prolonged, to allow sufficient penetration of the chemicals through the thick walls surrounding the plant cells, and this may result in even more extraction than reported by Weibull and colleagues. Rapid dehydration in ethanol and infiltration of plant tissue with a polar resin like LR White at ambient temperatures, therefore, seems to be a good alternative (Newman etal. , 1983; Causton, 1984; Newman and Jasani, 1984).

Light microscopic localization of viral antigen in semi-thin sections of LR White embedded plant tissue is described in Chapter 6. CCMV was successfully localized in petiolules of systemically inoculated cowpea plants by immunogold labelling and subsequent silver staining (immunogold/silver staining: IGSS). The silver stain could be observed in the light microscope by brightfield, darkfield and phase-contrast illumination. Most sensitive detection, however, was obtained with epi- illumination using polarized light (epipolarization microscopy). Combining epipolarization illumination with brightfield illumination allowed the simultaneous observation of silver stain and cell morphology.

Immunogold labelling and IGSS in combination with appropriate fixation and embedding of biological specimens, appear to be efficient and simple techniques for the insitu identification and localization of antigens, with many advantages over other immunochemical and cytochemical techniques, like ferritin- labelling, peroxidase-anti-peroxidase, immunofluorescence and autoradiography, which have only incidentally been used in plant virus research. Recently, Patterson and Verduin (1987) have reviewed the literature on the use of immunogold labelling in animal and plant virology, showing numerous fields of applications and discussing progress made in virus research. With respect to the technique the authors rightly concluded that immunogold labelling is a flexible technique with little limitation for the improvement of existing assays and the development of new ones.

Using immunogold labelling to identify and localize virus particles and coat protein, CCMV- infection in cowpea protoplasts was studied as function of the infection time. Observations with regard to virus entry into protoplasts are reported in Chapter 3. Upon inoculation aggregates of virus particles were observed attached to the plasmamembrane, or sometimes penetrating the plasmamembrane at places where the membrane appeared to be damaged. Virus was also found inside vesicles formed by invagination of the plasmamembrane. These vesicles with inoculum-virus particles were stable over long periods of time. Large vesicles (vacuoles) containing viral antigen were also detected at 24 h post-inoculation in protoplasts which were not infected by CCMV.

The mechanism by which plant viruses enter their host cells is still disputed (Shaw, 1986). Passage of the plasmalemma by endocytosis was suggested by Takebe (1975), and through pores or lesions by Burgess etal. (1973) and Watts etal. (1981). Our observations do not favour endocytosis to be the mechanism of virus entry leading to infection of the protoplasts as virus containing vesicles are stable. Recently, Roenhorst etal. (1988) presented data supporting a mechanism of virus entry by initial physical association of virus particles with the protoplast membrane and subsequent invasion of virus particles through membrane lesions. Such a mechanism may be also applicable to the cytoplasmic extrusions observed by Laidlaw (1987) after puncturing plant epidermal cells. The author suggested that virus particles may adsorb to the plasmalemma covering the extrusions, which are then withdrawn into the cell. Invasion of whole particles through membrane lesions may then be followed by a uncoating and initial translation (cotranslational disassembly) at the cytoplasmic ribosomes as suggested by Wilson (1985).

Ultrastructure of RNA-inoculated protoplasts was studied in sections of aldehyde- and osmium-fixed protoplasts (Chapter 4). Cytological alterations attributed to virus infection consisted of dilation of the endoplasmic reticulum (ER) and the formation of vesicles early in infection. Distended ER and vesicles seemed to form a kind of membranous area in the cytoplasm. In protoplasts fixed and embedded in Lowicryl K4M newly synthesized virus particles or coat protein were first localized in restricted areas of the cytoplasm at 6-9 h post-inoculation. The rough appearance of the cytoplasm in these areas suggested the presence of membranous structures like observed in osmium-fixed protoplasts. However, due to poor membrane preservation in Lowicryl embedded material this could not be proven. Within one protoplast several of these labelled areas were identified. At later stages of infection viral antigen was located throughout the cytoplasm, but also in the nucleus and in particular the nucleolus. No viral antigen was detected in or specifically associated with chloroplasts, mitochondria, microbodies and vacuoles. The specificity of gold labelling was demonstrated by quantification of the labelling density on sections of infected and non-infected protoplasts. These results indicate that CCMV coat protein synthesis and virus assembly take place in the cytoplasm of plant cells, but the involvement of cellular structures, in particular membranes, remains to be established. Protein synthesis and virus assembly may occur in certain restricted sites (compartments) in the cytoplasm possibly formed by the membranous bodies. Compartmentalization of the cytoplasm, creating different environments in the cell, may explain the occurrence of both disassembly and assembly in the same cell, and furthermore account for the phenomenon of specific assembly of viral RUA and homologous coat protein in cells infected with two related viruses like CCMV and BMV (Sakai etal. , 1983 ; Zaitlin and Hull, 1987). Whether RNA-replication also occurs in the same location as coat protein synthesis and virus assembly could be established by localization of non-structural virus encoded proteins involved in viral replication. However, antisera against these products of the CCMV-genome were not available. The function of CCMV coat protein or virus in the nucleus and especially the nucleolus is not known. Coat protein may have an affinity for ribosomal proteins and/or fulfill some functional role in the viral replication. Kim 1977 described the occurrence of filamentous inclusions (FI) in the nucleus often associated with the nucleolus. These FI were not found in the nuclei of cowpea protoplasts (this study) or tobacco protoplasts (Burgess etal. , 1974), but may be formed later in the infection by excess coat protein. Bancroft etal. (1969) showed the ability of CCMV-coat protein to form narrow tubules under specific conditions. The (FI) described by Kim (1977) may represent this type of coat protein aggregation, although the chemical composition of the (FI) is not yet known.

In Chapter 5 preliminary observations are reported on the localization of sites of CPMV replication in cowpea protoplasts, by in situ detection of coat proteins and non-structural proteins involved in viral replication and proteolytic processing. With regard to virus entry and subsequent locations of inoculum virus inside vesicles, similar phenomena were observed as in infection with CCMV. Infection of CPMV generates large inclusion bodies in the cytoplasm, consisting of membranous vesicles with fibrillary material and adjoining amorphous electron-dense material which have been observed as early as 12 h post- inoculation. Virus particles and/or coat protein were first detected 24 h after inoculation throughout the entire cytoplasm and in between the membranous vesicles and electron dense material. The 24K, 170K and their precursor proteins were exclusively localized in the electron dense material and not in association with the membranous vesicles or any other location in the cell. These results show that the electron-dense material consists at least in part of CPMV-encoded non-structural proteins and may represent a site for accumulation of non-functional proteins. The membranous vesicles have been implicated in viral RNA synthesis (Goldbach and Van Kammen, 1985). The failure to detect non- structural proteins in association with these membranes may be explained by either a low concentration of these proteins at the site of replication or by extraction of these proteins during the fixation and embedding procedure, despite the low temperature.

With IGSS the distribution of CCMV in cowpea plants was monitored at different times after systemic inoculation according to Dawson and Sehlegel (1976) (Chapters 6 and 7). No virus was detected at the time of temperature shift (t=0) in petiolule and leaves of plants subjected to 3 days of differential temperature treatment. Virus was first localized in phloem parenchyma cells of petiolule and veins at t=3 h and from there it spread to neighbouring tissues. Twenty four hours after systemic inoculation virus was located in the phloem, bundle sheath, cortex, but also in the cambium and some xylem cells. These results show that CCMV is transported from the inoculated primary leaves to the secondary leaves through the phloem, apparently following the route of metabolites. This finding is in agreement and further supports the generally accepted concept of plant virus long-distance transport through phloem. tissue (Matthews, 1982; Atabekov and Dorokhov, 1984). The failure to detect CCMV in differentiated sieve elements may indicate that the form in which the infectious entity is transported is another than virus particles (Atabekov and Dorokhov, 1984), or that the amount of virus transported through the sieve elements is below detectable levels. The true character of the synchrony of infection of leaf mesophyll cells obtained by differential temperature treatment is disputed. Infection of mesophyll tells may have been accomplished after shifting the plants to higher temperature by fast transport of infectious particles from the vascular tissue, as was also suggested by Dorokhov etal. (1981).

For the first time a suitable method for localization of antigens is available, which can be routinely applied for both light and electron microscopic study of the plant viral infection process. The application of the gold labelling technique in the localization of viral structural and non-structural proteins has been demonstrated, using CCMV- and CPMV-infections of plant cells as model system.

With regard to the technique, future work must be done on the improvement of the preservation of cellular structures, especially membranes, as these appear only poorly in Lowicryl embedded plant tissue even with dehydration, infiltration and polymerization at low temperatures. Alternatives, may be found in cryofixation and cryosectioning or freeze-substitution techniques.

With regard to the study of the plant viral infection process, the localization of virus-encoded proteins involved in replication and transport, but also the localization of plant viral nucleic acids by insitu hybridization, will contribute to the understanding of the mechanisms underlying these events. New biochemical techniques like the production of infectious transcripts from cloned viral cDNA (Ahlquist etal. 1984) enabling genetic manipulation of the viral genome, and integration of plant viral genes into the plant genome (Gardner etal. , 1984; Abel etal. , 1986) will supply future model systems for the study of virus-host interactions.

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