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.

    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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    Silky gels for cells : Self-assembling protein-based polymers for use in tissue engineering
    Wlodarczyk, M.K. - \ 2016
    Wageningen University. Promotor(en): Martien Cohen Stuart; Marleen Kamperman; S.C.G. Leeuwenburgh. - Wageningen : Wageningen University - ISBN 9789462576230 - 194
    polymers - proteins - biomedical engineering - biomaterials - recombinant dna - transplantation - compatibility - encapsulation - heparin - biodegradation - physical properties - polymeren - eiwitten - biomedische techniek - biomaterialen - recombinant dna - transplantatie - compatibiliteit - inkapselen - heparine - biodegradatie - fysische eigenschappen

    Tissue engineering is a relatively new, but actively developing field of biomedical science. It aims at organ or tissue regeneration by use of scaffolds, which are usually seeded with cells prior to implantation, and stimulated by bioactive cues or growth factors. It is a promising and valuable alternative to the use of transplants, for which the demand is greater than the supply, and for which application is connected with high risk of rejection and infection due to immunosuppressant medication. One of the main challenges of tissue engineering, that we tried to address in this thesis, is the design of biocompatible and functional biomaterials that could serve as cell scaffold. We investigated, if protein-based polymers, more specifically, if the de novo designed, C2SH48C2 copolymer, which self-assembles into fibers upon a pH-trigger, is a suitable material for cell scaffolds.

    In Chapter 2 we described the design and production, by means of recombinant DNA technology, of C2SH48C2. The protein was efficiently secreted by Pichia pastoris at high yields of g/l levels and we proposed an effective purification method. We showed that fibers and gels form by self-assembly upon pH adjustment, and that rheological properties of the obtained hydrogels depend on the total protein concentration. In view of potential biomedical applications, erosion studies were performed, which indicated that the gels exhibited long term stability in conditions mimicking those in body fluid. The biocompatibility of the gel scaffolds was demonstrated in a 2D cell culture study. However, despite the cell viability, a low proliferation rate was observed.

    To improve cell performance in contact with C2SH48C2 hydrogels (Chapter 3) we incorporated active domains in the C2SH48C2 protein by recombinant functionalization. We described the synthesis of two protein variants: (1) BRGDC2SH48C2, N-terminally enriched in integrin-binding domains (RGD) and (2) BKRSRC2SH48C2, N-terminally enriched in heparin binding domains (KRSR). We showed precise control over the amount of active domains in the final gels, by simply mixing the variants of the proteins in the desired molar ratio before inducing gelation. A 23-day cell culture study, performed using MG-63 cells, revealed that the presence of RGD and KRSR domains positively influenced cell attachment, spreading and activity. A synergistic effect was observed, i.e. scaffolds containing both bioactive domains yielded fully confluent layers of cells at an earlier stage during cell culture than the other gels. We concluded that cell behavior can be controlled by tuning the content of functional domains.

    In Chapter 4, we tested the suitability of the C2SH48C2 protein, enriched in RGD domains, for cell encapsulation, as the conditions of 3D cell culturing are more similar to the environment of cells in the body. We independently varied gel stiffness (by means of protein concentration) and functional motif (RGD) density, and analyzed the influence of these parameters on the cellular response. The viability and proliferation of MG-63 cells, encapsulated in the gels at different protein concentrations and RGD densities, was investigated with a cell activity assay, and by quantitative analysis of confocal pictures of nuclei (DAPI stain) and F-actin (phalloidin). We showed that optimal cell behavior is obtained in the presence of RGD domains and at low protein concentrations. The results indicated that RGD functionality is not the sole requirement; the gel matrix needs to exhibit the right mechanical properties and architecture to allow for cell growth, cytoplasmic extension and migration.

    Finally, in Chapter 5, we showed that active domains (here KRSR) can serve multiple functions in the material. We demonstrated the cross-linking ability of KRSR domains in the presence of heparin, leading to structural and mechanical changes in the scaffolds. In dilute systems (0.1 % (w/v)), heparin increases the rate of fiber growth, and induces fiber bundling. At higher protein concentrations, leading to the hydrogel formation (2 % (w/v)), the gelation rate and final storage modulus can be tuned by the amount of heparin and KRSR domain density. We concluded that with this approach, the material properties of C2SH48C2 protein gels can be effectively and simply controlled in a straightforward and biocompatible way.

    In Chapter 6 we described the main requirements for biomaterials and discussed to what extent they are fulfilled by protein-based polymers, and in particular, by the presented C2SH48C2 protein. The main advantages over alternative materials, and the challenges that need to be addressed before application in tissue engineering becomes a reality, were discussed. We ended with suggestions to improve the properties of C2SH48C2 protein for use as a biomaterial, especially its biodegradability, and its structural and mechanical properties.

    Bird-flu (avian influenza): application of recombinant DNA techniques in research and vaccine development
    Peeters, B.P.H. - \ 2008
    Tijdschrift voor Diergeneeskunde 133 (2008)22. - ISSN 0040-7453 - p. 956 - 958.
    aviaire influenzavirussen - genetische modificatie - recombinant dna - onderzoek - avian influenza viruses - genetic engineering - research
    Informatie omtrent de stand van zaken betreffende het influenzavirusonderzoek
    Faag-display als bron voor diagnostische antistoffen
    Speksnijder, A.G.C.L. ; Beekwilder, M.J. ; Doorn, J. van - \ 2008
    detectie - schimmelsporen - bacteriofagen - antilichamen - recombinant dna - moleculaire detectie - detection - fungal spores - bacteriophages - antibodies - recombinant dna - molecular detection
    In deze poster informatie over de ontwikkeling van procedures voor de selectie van specifieke fagen voor de herkenning van schimmelsporen en de ontwikkeling van een recombinant antilichaam voor de detectie van Tulp virus X
    GMO is dood, lang leve de gentechniek
    Arendonk, J.A.M. van - \ 2004
    Food Management 22 (2004)6. - ISSN 0168-325X - p. 26 - 29.
    genetische modificatie - recombinant dna - plantenveredeling - dierveredeling - biotechnologie - genetic engineering - plant breeding - animal breeding - biotechnology
    Nu de consument met geen stok aan de gmo-voeding is te krijgen, bloeit de aandacht voor klassieke veredeling op. Dit traditionele proces plukt echter ook de vruchten van gentech. Er kan gerichter veredeld worden, met als resultaat tropisch fruit dat niet bederft tijdens de verscheping, of wellicht melk met extra veel onverzadigde vetzuren. Het bedrijf Genetwister (een spin-off van Wageningen Universiteit) is gespecialiseerd in het ontrafelen van de genetische achtergrond van vruchtenrijping en verricht onderzoek naar rijpingsgenen
    Levensvatbaarheid van Ralstonia en Clavibacter meetbaar met behulp van RNA-detectiemethoden
    Wolf, J.M. van der; Beckhoven, J.R.C.M. van - \ 2003
    Gewasbescherming 34 (2003)5. - ISSN 0166-6495 - p. 157 - 160.
    aardappelen - plantenziekteverwekkende bacteriën - clavibacter michiganensis subsp. sepedonicus - epidemiologie - diagnostische technieken - tracer technieken - recombinant dna - ralstonia solanacearum - potatoes - plant pathogenic bacteria - clavibacter michiganensis subsp. sepedonicus - epidemiology - diagnostic techniques - tracer techniques - recombinant dna - ralstonia solanacearum
    Studie naar de epidemiologie van de bacterieziekten bruinrot (Ralstonia solacearum) en ringrot (Clavibacter michiganensis subsp. sepedonicus). De nieuwe methode is gebaseerd op amplificatie van 16S rRNA sequenties met behulp van NASBA (nucleic acid sequence based amplification). NASBA is een methode waarmee nucleïnezuren, maar in het bijzonder RNA-sequenties, efficiënt geamplificeerd kunnen worden
    Recombinant gelatin and collagen from methylotrophic yeasts
    Bruin, E.C. de - \ 2002
    Wageningen University. Promotor(en): N.C.M. Laane; F.A. de Wolf. - S.l. : S.n. - ISBN 9789058085832 - 110
    collageen - gelatine - recombinant dna - hansenula - Hansenula polymorpha - Pichia pastoris - collagen - gelatin - recombinant dna - hansenula - Methylotrophic yeast - Hansenula polymorpha - Pichia pastoris - Fermentation - Secretion - Recombinant expression

    Based on its structural role and compatibility within the human body, collagen is a commonly used biomaterial in medical applications, such as cosmetic surgery, wound treatment and tissue engineering. Gelatin is in essence denatured and partly degraded collagen and is, as a result of its unique functional and chemical properties, also used in many medical and pharmaceutical products. Collagen and gelatin are traditionally extracted from animal tissues. The quality and the characteristics of the proteins are not very reproducible in today's batch-to-batch production processes and recently, potential contamination of collagen and gelatin with viruses and prions (causing BSE) became a matter of concern. BSE is thought to cause a new variety of the brain- wasting Creutzfeldt-Jacob disease in humans.

    Recombinant DNA technology may provide safe collagen and gelatins from which the quality and characteristics can precisely be controlled and reproduced and, in addition, opens up possibilities for novel functional "tailor-made" proteins.For the heterologous production of animal proteins yeasts are frequently used. Since yeasts are eukaryotes, most translational modification, needed for functionality and stability of recombinant animal proteins, normally occur. However prolyl 4-hydroxylation, essential for gelling properties of recombinant gelatin and thermal stability of recombinant collagen, is generally considered to be absent in yeast systems.

    In this study we explored the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris for their use as recombinant production systems of natural and "tailor-made" gelatins and human collagen.We found that both yeasts are well able to cope with the repetitive gene sequences encoding animal gelatin and human collagen and showed that P. pastoris can produce synthetic gelatins with highly hydrophilic properties at high levels. Furthermore, it was discovered that H. polymorpha unexpectedly produced endogenous collagen-like proteins with 4-hydroxyproline amino acid residues. This finding indicated that the yeast H. polymorpha , in contract to what was generally believed, must contain intrinsic proly 4-hydroxyalse activity. Indeed, expression of murine gelatin in H. polymorpha yielded a secreted and hydroxylated product. We also investigated if H. polymorpha could be used for the production of recombinant human collagen. Intract human collagen trimers were obtained but they were not stable at temperatures higher than 15 °C, indicating that hydroxylation in the product was poor.

    In the course of this study we found putative prolyl 4-hydroxylase genes in different eukaryotic microbial systems. In the future these genes may be used to further develop yeasts into cell factories for the production of animal gelatins and thermally stable human collagen.

    Microarraytechnologie en veiligheidsbeoordeling: verandering in genexpressie in of door GGO's opsporen
    Kok, E. ; Peijnenburg, A. ; Keijer, J. ; Kuiper, H. - \ 2001
    Voeding Nu 3 (2001)11. - ISSN 1389-7608 - p. 21 - 23.
    genexpressie - genomic imprinting - gewassen - voedselsamenstelling - voedselveiligheid - voedselvergiftiging - toxiciteit - interdisciplinair onderzoek - landen van de europese unie - onderzoeksprojecten - genetische modificatie - recombinant dna - gene expression - crops - food composition - food safety - food poisoning - toxicity - interdisciplinary research - european union countries - research projects - genetic engineering
    De toepassing van de DNA-microarraytechnologie voor de veiligheidsbeoordeling van genetisch gemodificeerde gewassen en daarvan afgeleide producten
    A calf is born : a reconstruction of the public debate on animal biotechnology
    Theune, E. - \ 2001
    Wageningen University. Promotor(en): M.J.J.A.A. Korthals; F.W.J. Keulartz. - S.l. : S.n. - ISBN 9789058085122 - 184
    biotechnologie - genetische modificatie - recombinant dna - ethiek - filosofie - nederland - dierenwelzijn - openbare mening - kranten - stieren (bulls) - dierenbescherming - dierverzorging - moraal - biotechnology - genetic engineering - recombinant dna - bulls - animal welfare - ethics - philosophy - public opinion - newspapers - netherlands - animal protection - care of animals - moral
    'How should public debates be understood?' is the central question of this study. And it is answered by reconstructing a single public debate, namely the debate about the transgenic bull Herman. Herman the bull was created by Gene Pharming in 1989. An extra gene has been inserted in his genome as to induce the production of lactoferrin in the milk of his daughters. A debate started in the media about whether this should be allowed or not. This media debate has lasted for nine years. In this study the debate is analysed and interpreted in order to make visible for others what has happened during this particular debate. It thus provides handles for disclosing other public debates and for better understanding public debates in general.

    Insight is provided in what has kept this debate going. This media debate, in which the public is involved as an audience, has needed a concrete cause (the case) to develop. This seems to be obvious, but in the Netherlands many debates are being organised without such a case. And one may witness that the media, and so the public, are hardly involved in these organised debates. But not only will a case be needed to induce a public debate. The urge of the debate has to be articulated as well. Virtually everybody can articulate problematic aspects of a case, but for a debate to have impact actors are needed that can provide continuity to the debate. In this particular debate this continuity has been provided by the Dierenbescherming (the Dutch Society for the Protection of Animals). But, this still is not enough. If the media are not interested, the public will not know what is going on. So, for a vivid public debate, a case is needed to make the debate urgent, an organisation has to provide continuity, and the media have to publish about it.

    With respect to the content of the debate two discussions can be distinguished: a) the general controversy that has evoked the debate and b) the discussion about several specific applications of this technology.

    ad a) The general controversy. Four general principles are usually mentioned as integrating of the many norms that regulate interpersonal conduct. Two of these are generally reckoned to be applicable to animals as well, to wit 'do not harm' and 'do good'. With respect to the other two principles ('respect for autonomy' and 'be just') people do not agree about whether they apply to animals as well. It is obvious that these last principles cannot be applied directly to animals, as animals are not capable of being autonomous in the sense humans are. This implies for many people that the principle of justice does not apply to animals as well. At least a transformation of these principles is required, but this will alter their content substantially. Respect for autonomy can be transformed into respect for intrinsic value and bodily integrity, and justice can be transformed into equal and fair treatment. The debaters can now be divided into two groups: people that do and people that do not recognise these altered principles as applicable to animals. People that do not recognise these principles will argue in terms of 'do the benefits outweigh the harms' and they will be in favour of a 'yes, if the relevant conditions are met' policy (e.g. Gene Pharming and the biomedical researchers). People that do recognise these principles will either be opponents of animal transgenesis (the Dierenbescherming and some other societal organisations) or will be in favour of a 'no, unless there are good reasons to do so' policy (e.g. the Minister of Agriculture, most animal experts and animal ethicists). They will stress the search for alternatives and will ask for sufficient reasons before they enter a discussion about harms and benefits.

    ad b) Applications and contexts. With respect to the specific case at hand, it turned out that people have been arguing from different values and standards. These values and standards were connected with different practices of animal keeping and using. This has resulted, dependent on the specific applications of the lactoferrin obtained, in a discussion about what kind of animal Herman the bull is. Will his lactating daughters produce milk in the context of dairy cattle farming? Will they be better protected against mastitis, a cattle disease, which would mean that Herman the bull is functioning in a veterinary context? Will they produce medicines like many laboratory animals do, which would refer to a biomedical context? Or will his daughters produce food for patients in a specialised food production context? It turned out that these contexts, which are called practices, imply different standards for the treatment of animals.

    Neither the standards of veterinary practice, nor the standards of cattle farming practice do allow for the production of transgenic cattle. As the production of specialised food was mainly considered from the point of view of ordinary milk production practice, the standards of this practice were applied and so the production of transgenic cattle was rejected. In the case of the production of medicine by way of transgenic cattle, the standards for dealing with this case have not become clear at the end of the debate. People did not considered the standards of the biomedical practice adequate nor the standards of the dairy farming practice. Both perspectives remain open. It even may be the case that animal transgenesis develops as a practice of its own or that animal transgenesis will only be judged from the point of view of general standards and values instead of from contextual standards.

    The process of debating has been subject of the debate as well. The debaters have criticised each others inputs and have reacted to critique with respect to their conduct. These critiques provide insight in the implicit rules of debating in public.

    Public debates are debates in front of the public as an audience. If people are debating in public, they will not in it for understanding and convincing each other. Their main objective will be to influence public opinion and to gain public support for their views. They will reason as lucid as possible. And this has consequences for the evaluation of the conduct of the debaters. This does not mean that there are no rules, but it does mean that there will be specific rules for these kind of discussions. So, people will be criticised if they go too far, that is to say if they violate the rules of decent reasoning.

    Debating in public is more than reasoning only. There are all kinds of theatrical elements as well, like suggestive photo's or posters, or telling stories about other debaters, or suggestive reasoning. Here too, rules of decent conduct can be reconstructed from the critiques on several of these theatrical elements that were uttered during the debate at hand.

    This study ends with a reply to the people that have criticised this debate in general. From their critiques it can be derived that these critics have raised too high (too idealistic) expectations of public debates. The fruitfulness and sense of a public debate should not be assessed on the basis of convergence of opinions or on the orientation at consent of the debaters, but on the quality and rationality of the reasoning of the debaters. One may witness that all debaters have developed their views and reasoning; they have listened to others and have reacted to these inputs by better articulating their own reasoning or by adapting their actions. It is evident that the participants have not always conducted according to the rules of decent debating. But, they have taken critique on their conduct seriously and have adapted their behaviour.

    New developments in crop plant biotechnology and their possible implications for food product safety : literature study under commission of the foundation 'Consument and biotechnologie'
    Kleter, G.A. - \ 2000
    Wageningen : RIKILT (Report / RIKILT 2000.004) - 66
    biotechnologie - genetische modificatie - recombinant dna - voedselveiligheid - voedsel - plantaardige producten - literatuuroverzichten - biotechnology - genetic engineering - food safety - food - plant products - literature reviews
    This study reports recent developments in the application of biotechnology in agriculture in order to assess whether current food safety evaluations strategies are adequate in view of these new and presumably more far reaching developments. Trends are observed that may require additional regulatory measures by the government. Finally, specific new developments in gene technology may be the subject of a public debate in order to decide whether these developments are socially acceptable or not.
    Toekomstige genetisch gemodificeerde voedselgewassen
    Kleter, G. ; Noordam, M. ; Kok, E. ; Kuiper, H. - \ 2000
    Voeding Nu 2 (2000)12. - ISSN 1389-7608 - p. 24 - 25.
    genetische modificatie - biotechnologie - voedselgewassen - voedingsmiddelen - recombinant dna - chromosomen - technologie - voedselveiligheid - toekomst - genetic engineering - biotechnology - food crops - foods - chromosomes - technology - food safety - future
    Een korte samenvatting van het rapport over recente ontwikkelingen en de gevolgen voor de voedselveiligheid, dat door het Rikilt werd samengesteld in opdracht van de Stichting Consument en Biotechnologie
    Crops of uncertain nature? Controversies and knowledge gaps concerning genetically modified crops: an inventory
    Visser, A.J.C. de; Nijhuis, E.H. ; Elsas, J.D. van; Dueck, T.A. - \ 2000
    Wageningen : Plant Research International - 70
    genetica - recombinant dna - genetische modificatie - biotechnologie - gewassen - voedselveiligheid - volksgezondheid - risicoschatting - kennis - voedsel- en voedingsgeschilpunten - genetics - recombinant dna - genetic engineering - biotechnology - crops - food safety - public health - risk assessment - knowledge - food and nutrition controversies
    Agro-ecologische risico's van transgene gewassen
    Lotz, L.A.P. ; Kempenaar, C. - \ 2000
    Gewasbescherming 31 (2000)4. - ISSN 0166-6495 - p. 100 - 102.
    genetische modificatie - agronomie - plantenveredeling - recombinant dna - landbouwproducten - duurzaamheid (sustainability) - milieueffect - ecologie - agro-ecologie - genetic engineering - agronomy - plant breeding - recombinant dna - agricultural products - sustainability - environmental impact - ecology - agroecology
    Agro-ecologische effecten van het telen van transgene gewassen (GGO's = genetisch gemodificeerde organismen) zijn afhankelijk van een complex van interacties tussen de betreffende genetische modificatie, het overige deel van het erfelijk materiaal, de invloed van het milieu op het organisme en tenslotte de gevoeligheid van het agro-systeem voor de betreffende effecten. Deze interacties maken het noodzakelijk dat de analyse en beoordeling van deze agro-ecologische risico's "case by case" gebeurt
    Regeneration and transformation by particle bombardment in leek (Allium ampeloprasum L.)
    Schavemaker, C.M. - \ 2000
    Agricultural University. Promotor(en): E. Jacobsen; R.G.F. Visser. - S.l. : S.n. - ISBN 9789058082343 - 100
    preien - allium ampeloprasum - verjonging - genetische transformatie - transformatie - genetische modificatie - somatische embryogenese - recombinant dna - transgene planten - allium porrum - biolistiek - leeks - allium ampeloprasum - regeneration - biolistics - genetic transformation - transformation - genetic engineering - somatic embryogenesis - recombinant dna - transgenic plants - allium porrum

    In this thesis the results are presented of experiments aiming at the genetic modification of leek ( Allium ampeloprasum L.). Leek is a vegetable grown for its edible (false) stem and belongs to the Alliaceae, together with onion ( Allium cepa ) and garlic ( Allium sativum ). The production of leek is mainly confined to Europe. In the last few years production has increased along with consumer demands. It is propagated through seeds and gives rise to heterogeneous progeny. Problems in cultivation of leek are rust ( Puccinea allii , P.mixtu ), yellow stripe virus and the lack of uniformity. The most suitable system able to cope with these problems seems to be hybrid breeding. However, hybrid breeding is hampered by the lack of a suitable emasculation system or male sterility system and the severe inbreeding depression. Therefore, emphasis has been put on the application of genetic modification in order to solve some of these problems. This relatively new technique opens the possibility to add or alter traits which cannot easily be achieved with conventional breeding methods. The most important prerequisites for genetic modification were investigated during this thesis research. The first prerequisite is an efficient regeneration system. Starting from seed, a cyclic somatic embryogenesis regeneration system was developed with long term regeneration potential, providing a regeneration system where it should be possible to obtain true transformants from chimeric structures (Chapter 2).

    A comparison was made between the first cycle, starting from zygotic embryos, and latter cycli, starting with somatic embryos. All genotypes tested were able to produce somatic embryos although genotypic differences in somatic embryo production occurred. The first cycle, using zygotic embryos, was the most effective in somatic embryo production compared to the later cycli using somatic embryos. On average the first cycle produced 23.8 somatic embryos per zygotic embryo and the later cycli ranged from 11.1 to 16.0 somatic embryos per initial somatic embryo. Shoot regeneration from somatic embryos was satisfactory, obtaining normal looking greenhouse plantlets.

    In Chapter 3 this cyclic somatic embryogenesis system was analyzed for its suitability in a genetic transformation system, like particle bombardment. The relatively new reporter gene luciferase was used in the transformation experiments. Leek, like most monocotyledons, seemed to be very persistent to selective media. Neither the selective agent kanamycin nor hygromycin could prevent leek cells from growing. The selective agent phosphinothricin had a better inhibitory effect on cell growth. A histological and morphological study showed that regeneration occurred from the deeper cell layers inside the somatic embryo, in the same way that leaves are produced on a mature leek plant. These cell layers are hardly reached by particles, explaining the poor results of the transformation experiments using somatic embryos.

    In Chapter 4 a newly developed regeneration system from flower stalk strip explants was used to determine the optimal conditions for particle bombardment with the luciferase reporter gene. After a vernalisation period leek starts to develop a flower stalk. This flower stalk, still inside the plant, is harvested and stripped. Regeneration from these strips occurs just beneath the epidermis, which is easy to reach for the particles of the particle gun. The regeneration frequency was neither influenced by the stripping nor by the bombardments. Over 300 plantlets could be obtained from 1 flower stalk. Important factors for transformation experiments like pre-culture time, pressure, distance and coating of the particles were analyzed and optimized for these highly regenerative explants. Flower stalk strips of leek as explants, cut from the basal part of the flower stalk, pre-cultured for 2 days, should be bombarded at a distance of 5 cm, with a pressure of 1800 psi to obtain a high transient expression. Gold particles should be used and coated following the procedure of Christou (1991). After bombardment the explants should be transferred to selection medium.

    In Chapter 5 these optimal conditions were applied to the flower stalk strip explants. A comparison of the use of the reporter genes GUS and luciferase was made. Both genes, combined in one plasmid, showed similar expression patterns. Expression of both genes was still present 7 weeks after bombardment, but local increases in gene activity were not observed. The reporter gene luciferase facilitates the investigations in the genetic modification research as the chemical reaction with this reporter gene is non-lethal to the plant tissue whereas the reaction with GUS-reporter gene is. The non-detrimental effect of the luciferin treatment made it possible to investigate gene expression in time. Still, using a novel reporter gene means also unexpected results like the long transient expression time of the luciferase gene product even after application of the substrate luciferin. Eventually, 16 chimeric plantlets were obtained. Probably, the regeneration from flower stalk strip explants is a multicellular event. Seeds harvested from potentially chimeric plants did not show any GUS or luciferase activity after germination.

    In the near future leek transformation research has to focus on the development of embryogenic cell suspension or protoplast cultures, to obtain true transformants of the chimeric plants and an efficient selection system to select and favor the transgenic cells. To facilitate success in transformation research, efforts could also be directed at plastid transformation, in order to come to true breeding transgenic lines

    Estimation of the ecological risks involved with genetically modified plants based on model experiments: Quantification of ecological key traits
    Dueck, T.A. ; Jordi, W. ; Werf, A. van der - \ 1999
    Wageningen : AB (Note / AB, Research Institute for Agrobiology and Soil Fertility 207) - 22
    genetische modificatie - recombinant dna - milieueffect - schatting - modellen - planten - nadelige gevolgen - genetic engineering - environmental impact - estimation - models - plants - adverse effects
    Biosafety of site-specific recombination-mediated and homing endonuclease-mediated chromosome modifications in plants
    Gilissen, L.J.W.J. ; Nap, J.P. - \ 1999
    Wageningen : CPRO - 45
    genetische modificatie - planten - bioveiligheid - recombinatie - recombinant dna - genetic engineering - plants - biosafety - recombination
    Environmental risks of transgenic multiple herbicide resistance
    Kempenaar, C. ; Lotz, L.A.P. - \ 1999
    Wageningen : AB - 30
    genetische modificatie - recombinant dna - milieueffect - milieubescherming - agronomie - gewassen - plantenveredeling - transgene planten - geïnduceerde resistentie - resistentie tegen herbiciden - risicoschatting - gewasbescherming - genetic engineering - environmental impact - environmental protection - agronomy - crops - plant breeding - transgenic plants - induced resistance - herbicide resistance - risk assessment - plant protection
    Geen machtsmiddel in discussie over gemodificeerd voedsel: gentech en de consument
    Dagevos, H. - \ 1999
    Spil 159-160 (1999). - ISSN 0165-6252
    voedselindustrie - genetische modificatie - recombinant dna - biotechnologie - voedingsmiddelen - voedseltechnologie - consumentenaangelegenheden - food industry - genetic engineering - biotechnology - foods - food technology - consumer affairs
    Pathogenen beheersen de kunst van het veranderen
    Aantrekker, E.D. den; Janssen, A.G.W. - \ 1999
    Voedingsmiddelentechnologie 32 (1999). - ISSN 0042-7934 - p. 44 - 46.
    voedselmicrobiologie - micro-organismen - pathogenen - kiemgetal - antibiotica - immunologie - antigenen - antilichamen - antiseptica - reticulo-endotheliaal systeem - antimicrobe-eigenschappen - biotechnologie - genetica - recombinant dna - voedselbewaring - conserveermiddelen - programmaeffectiviteit - detectie - technieken - monitoring - voedselveiligheid - conferenties - food microbiology - microorganisms - pathogens - bacterial count - antibiotics - immunology - antigens - antibodies - antiseptics - reticuloendothelial system - antimicrobial properties - biotechnology - genetics - food preservation - preservatives - program effectiveness - detection - techniques - food safety - conferences
    De proceedings van dit congres zijn verkrijgbaar bij TNO Voeding te Zeist. Info: 030-6944144
    The influence of the agricultural use of genetically modified plants on biodiversity, with emphasis on agrobiodiversity
    Gilissen, L.J.W.J. ; Nap, J.P.H. - \ 1998
    Wageningen : CPRO-DLO - 35
    genetische modificatie - recombinant dna - soortendiversiteit - transgene planten - transgenic plants - genetic engineering - species diversity
    Regeneration and transformation of Alstroemeria
    Schaik, C.E. van - \ 1998
    Agricultural University. Promotor(en): E. Jacobsen; M.J. de Jeu. - S.l. : Van Schaik - ISBN 9789054858904 - 111
    sierplanten - amaryllidaceae - genetische modificatie - recombinant dna - alstroemeria - ornamental plants - amaryllidaceae - genetic engineering - recombinant dna - alstroemeria

    Alstroemeria or Inca Lily is an economically important cut flower in the Netherlands. The monocotyledonous ornamental Alstroemeria is mainly cultivated for the production of cut flowers, but there are also Alstroemeria pot plants and garden plants on the market. The increasing popularity for Alstroemeria can be attributed to its wide range of flower colours, long vase life and low energy requirement in the greenhouse. Since 1960 many efforts have been made to create elite cultivars by conventional breeding in the Netherlands. Nowadays a novel technique, called genetic modification opens the possibility to add or alter traits which cannot easily be achieved by conventional breeding.

    The aim of this thesis was to develop a regeneration and transformation procedure for genetic modification of Alstroemeria, which could be used routinely. The most important prerequisites for genetic modification were investigated during this thesis. The first prerequisite for complete stable transformation is an efficient regeneration system. When we started this research only a very inefficient plant regeneration system was described by Gonzalez-Benito. This plant regeneration system had a regeneration frequency of 4% and used mature zygotic embryos as explants. We developed a more efficient and embryogenic regeneration system with immature zygotic embryos as explants with a regeneration frequency of 40-50% (see Chapter 2). In Chapter 3 we tried to optimise our somatic embryogenesis system and turned it into a cyclic system, thus becoming less dependent on flowering plants.

    For genetic modification a regeneration system not only needs to be embryogenic and efficient, but the cells capable of regeneration need to be accessible to transformation. Histological observations revealed that the cells capable of regeneration and the transient gene expression after particle bombardment were located in cells at the surface of the callus. So regeneration and transformation took place in the same cell layers of the Alstroemeria callus. Another prerequisite, namely selection for transformed cells was investigated in Chapter 4. Transformed cells need to be selected between non-transformed cells, to prevent overgrow by the non-transformed cells. The herbicide PPT was found to be a good selective agent in the somatic embryogenesis system.

    The two main transformation techniques nowadays are transformation by the vector Agrobacterium tumefaciens or direct gene transfer by the particle gun. The plant pathogenic soil bacterium Agrobacterium has developed a sophisticated mechanism for stably integrating part of its extrachromosomal DNA (T-DNA) into the nuclear genome of receptive plants. The particle gun is a direct gene transfer technique, small particles coated with DNA accelerate by helium pressure to penetrate the target tissue. Both transformation techniques were investigated in Alstroemeria (see Chapter 5).

    The marker genesβ - glucuronidase (GUS) and luciferase (LUC) were used. The GUS assay, which is a destructive test, shows expression in the target tissue by a blue product. Later during our research we could use the non destructive LUC assay. This LUC gene from the firefly is capable of emitting light which can be detected by a very sensitive camera. By using both marker genes we optimised the two different transformation protocols. We were unable to find stably transformed callus by the Agrobacterium -mediated transformations. The combined transformation of the somatic embryogenesis system with particle gun bombardment revealed only chimeric transformed plants. On the other hand, the particle gun-mediated transformation of Alstroemeria cell suspension cultures resulted in a complete stably transformed cell suspension culture. This cell suspension had a clear positive LUC activity and PCR positive result, indicating that the culture was indeed transformed. Unfortunately, despite many attempts, we were not able to regenerate plants from this transgenic culture. Most likely, one of the reasons for failing to do so was the long time period, which this culture had to be maintained in vitro .

    We concluded from our transformation experiment, that it is very important to know where the origin of regeneration exactly takes place. In our Alstroemeria somatic embryogenesis system the origin of the regenerants was from the surface of the callus, but not from single epidermal cells. Probably this is the reason why only chimeric plants were found after combined Alstroemeria somatic embryogenesis and particle gun bombardment transformations. The prospects for the use of embryogenic cell suspension cultures for Alstroemeria transformation are good. So in the near future, Alstroemeria transformation research has to focus on the development of embryogenic cell suspensions or a de novo regeneration system from single epidermal or subepidermal cells. The advantage of a cell suspension is that in a cell suspension there are many active cells (good explants for transformation) and that first a stable transformed cell suspension can be formed. The regeneration efficiency of the cell suspension is then of less importance.

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