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Staff Publications

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

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

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

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

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    From harmful to useful algae
    Blaas, Harry - \ 2017
    Wageningen University. Promotor(en): Carolien Kroeze. - Wageningen : Wageningen University - ISBN 9789463430357 - 117
    algae - algae culture - adverse effects - nitrogen - phosphorus - rivers - eutrophication - waste water treatment - europe - algen - algenteelt - nadelige gevolgen - stikstof - fosfor - rivieren - eutrofiëring - afvalwaterbehandeling - europa

    Eutrophication of coastal waters is a worldwide phenomenon. This study focuses on eutrophication in the coastal waters of Europe. Eutrophication is mainly a result of the increased transport of nutrients from watersheds by rivers to the coastal waters. Nutrient losses from watersheds are generally from agriculture, sewage, atmospheric deposition and from natural sources. In case of an overload of nutrients in the coastal waters, algal blooms may develop which increase the risk of hypoxia, fish mortality, and loss of biodiversity.

    Algae can also be useful. They are increasingly considered an interesting product. For instance, micro-algae can be grow on land to produce proteins, lipids and fatty acids. Some studies indicate that micro-algae can be an important feedstock in the future for, for instance, the production of biodiesel. Moreover, macro-algae can be produced in seawater in sea farms. Macro-algae can be edible, or be used as a feedstock. By yielding macro-algae, nutrients are removed from the water, reducing coastal eutrophication.

    The objective of this study is to analyse past and future trends in nutrient export by rivers to European seas with a focus on the role of algae. Three types of algae will be distinguished: (1) harmful algal blooms in coastal seas, (2) cultivation of micro-algae on land for the production of proteins, lipids and fatty acids, and (3) cultivation of multi cellular algae in seaweed farms for human consumption or other products.

    To meet the objective the following research questions are addressed:

    RQ1 To what extent do N and P loads exceed levels that minimize the risk of harmful algal blooms, and what are the relative shares of sources of N and P in rivers of the European Union?

    RQ2 What are the potential consequences of large-scale land-based production of biodiesel from cultivated micro-algae in Europe for coastal eutrophication?

    RQ3 Would it possible to cultivate and process micro-algae in a factory, and what is the environmental performance?

    RQ4 To what extent can seaweed farming in combination with nutrient management in agriculture and waste water treatment reduce the potential for coastal eutrophication?

    These questions are answered through model analyses. The Global NEWS (Nutrient Export from WaterSheds) model simulates river export of nutrients as function of human activities on land. It includes more than 6000 rivers worldwide. It can be used to quantify nutrient flows from land to sea for the years 1970, 2000, 2030 and 2050. For future years four scenarios have been implemented. One of these scenarios is named Global Orchestration and mostly used as a reference in this thesis. This scenario assumes a globalised world, with a reactive approach towards environmental problems. The model was released in 2010, has been validated for the years 1970 and 2000. The nutrients considered in the model are nitrogen (N) and phosphorus (P). In this thesis Global NEWS is used to calculate transport of nutrients to the coastal waters of Europe. The model uses ICEP (Indicator for Coastal Eutrophication Potential) values at the river mouths as an indicator for potentially harmful effects of nutrient enrichment. These ICEP values reflect the ratio of nitrogen and phosphorus to silica in coastal seas. A positive ICEP value indicates that nitrogen or phosphorus levels are too high, favouring conditions for potentially harmful algae to bloom.

    In chapter 2 Global NEWS is used to calculate the transport of nutrients and ICEP values for 48 European rivers for the years 2000 and 2050. The model calculates a positive ICEP for 38 rivers in the year 2000, and for 34 rivers in the year 2050. This indicates that current policies are not so effective in reducing the river transport of nutrients. For polluted rivers the anthropogenic sources of the nutrients are investigated. For most rivers the dominant polluting sources are agriculture or sewage. The results indicate that a basin-specific policy is needed to reduce the risks of coastal eutrophication.

    In chapter 3 the focus is on useful algae: micro-algae cultivation on land for, for instance, biodiesel production. The consequences of large-scale production of biodiesel on nutrient export by rivers to the European coastal waters are investigated. A scenario is developed assuming that a production of 0.4 billion m3 diesel from cultivated micro-algae. The cultivation is assumed to be in the open air, for instance in ponds or in closed tube systems. Such production levels would need a land surface area as large as Portugal. The Global NEWS model is used to calculate the amount of waste water from micro-algae production that will be transported to the coastal waters in this scenario. The results indicate that large-scale cultivation of micro-algae on land can become a source of nutrient pollution in rivers. In the scenario with large-scale micro-algae cultivation the future transport of nitrogen and phosphorus is considerably higher than in the reference scenario. To ensure sustainable production of biodiesel from micro-algae it is important to develop cultivation systems with low nutrient losses to the environment.

    Chapter 4 presents a design of a factory for the cultivation and processing of micro-algae in an environmentally sound way. The factory does not use fossil fuels and applies maximum recycling of water and nutrients. In this factory it is possible to produce lipids, carbohydrates, proteins and minerals. The factory can be built on any piece of land, so there is no need to use arable land. The factory is independent of weather and climate. Energy can be delivered by wind mills. In this chapter an example of producing diesel in the factory is shown. In the 12 stories factory with a cultivation area of 1 hectare, 810 ton micro-algae can be cultivated per year. This is enough for the production of 386 ton diesel per year.

    Chapter 5 focuses on mitigation of eutrophication in European coastal waters. A scenario is presented assuming different types of measures. The scenario first assumes that nutrient use efficiencies in agriculture are higher than today, and that waste water treatment in sewage systems is improved. In addition, it assumes that all excess N and P in coastal waters is harvested in seaweed farms producing edible macro-algae. In our scenario for 2050 there is seaweed farming in the coastal waters of 34 rivers mouths in Europe .NEWS The areas needed to ensure that ICEP values remain below 0 (low potential for coastal eutrophication) range between 0 and 952 km2 per river mouth.

    This thesis shows that algae can be both harmful and useful. River export of nutrients can lead to coastal eutrophication increasing the risks of harmful algal blooms. On the other hand, micro-algae can be produced without environmental harm on land, and macro-algae can be useful in reducing pollution levels in coastal seas. This thesis could serve as a basis for environmental policies to stimulate the production of these useful algae. The methods to mitigate algal blooms and to use algae in a sustainable way in this thesis are also useful for other parts of the world.

    Excessive nitrogen and phosphorus in European rivers : 2000-2050
    Blaas, Harry ; Kroeze, Carolien - \ 2016
    Ecological Indicators 67 (2016). - ISSN 1470-160X - p. 328 - 337.
    Algal blooms - Coastal waters - Eutrophication - Indicator - Nutrients - Rivers

    Rivers export nutrients to coastal waters. Excess nutrient export may result in harmful algal blooms and hypoxia, affecting biodiversity, fisheries, and recreation. The purpose of this study is to quantify for European rivers (1) the extent to which N and P loads exceed levels that minimize the risk of harmful algal blooms and (2) the relative shares of sources of N and P in rivers. This may help to identify effective management strategies to reduce coastal eutrophication. We focus on 48 rivers in 27 countries of the European Union (EU27). We used the Global Nutrient Export from Watersheds (NEWS) model to analyze nutrient export by rivers and the associated potentials for coastal eutrophication as reflected by Indicator for Coastal Eutrophication Potential (ICEP). In 2000, 38 of the 48 EU rivers indicated in our study had an ICEP > 0, indicating a relatively high potential for harmful algal blooms. These 38 rivers cover 60% of EU27 land area. Between 2000 and 2050 nutrient export by European rivers is projected to decrease. However, by 2050 still 34 EU rivers, covering 48% of the land area, have an ICEP > 0. This indicates that in these scenarios little progress is made in terms of environmental improvement. About one-third of the rivers with ICEP > 0 are N limited, and about two-thirds P limited. In N-limited rivers reducing N loads is a more effective way to reduce the risk for coastal eutrophication than reducing P, and vice versa. For N-limited rivers agriculture or sewage are the dominant sources of nutrients in river water. In P-limited rivers, sewage is found to be the dominant source of P, except for rivers draining into the Atlantic Ocean, where agriculture can also be dominant. A basin-specific approach is needed to effectively reduce N and P loads.

    Possible future effects of large-scale algae cultivation for biofuels on coastal eutrophication in Europe
    Blaas, H. ; Kroeze, C. - \ 2014
    Science of the Total Environment 496 (2014). - ISSN 0048-9697 - p. 45 - 53.
    life-cycle assessment - biodiesel - microalgal
    Biodiesel is increasingly considered as an alternative for fossil diesel. Biodiesel can be produced from rapeseed, palm, sunflower, soybean and algae. In this study, the consequences of large-scale production of biodiesel from micro-algae for eutrophication in four large European seas are analysed. To this end, scenarios for the year 2050 are analysed, assuming that in the 27 countries of the European Union fossil diesel will be replaced by biodiesel from algae. Estimates are made for the required fertiliser inputs to algae parks, and how this may increase concentrations of nitrogen and phosphorus in coastal waters, potentially leading to eutrophication. The Global NEWS (Nutrient Export from WaterSheds) model has been used to estimate the transport of nitrogen and phosphorus to the European coastal waters. The results indicate that the amount of nitrogen and phosphorus in the coastal waters may increase considerably in the future as a result of large-scale production of algae for the production of biodiesel, even in scenarios assuming effective waste water treatment and recycling of waste water in algae production. To ensure sustainable production of biodiesel from micro-algae, it is important to develop cultivation systems with low nutrient losses to the environment.
    The effects of large-scale algae cultivation for biofuels on coastal eutrophication in Europe
    Blaas, H. ; Kroeze, C. - \ 2013
    dentification of the volatile component(s) causing the characterisc foxy odor in various cultivars of Fritillaria imperialis L. (Liliaceae)
    Helsper, J.P.F.G. ; Bucking, M.W. ; Muresan, S. ; Blaas, J. ; Wietsma, W.A. - \ 2006
    Journal of Agricultural and Food Chemistry 54 (2006)14. - ISSN 0021-8561 - p. 5087 - 5091.
    defensive spray - tenax ta - skunk - mephitis - release - coffee - beer
    To identify the component(s) causing the foxy odor, characteristic for some Fritillaria imperialis cultivars, the headspace of flower bulbs was analyzed using gas chromatography-olfactometry (GC-O) and GC-mass spectrometry (GC-MS). Six Fritillaria species and cultivars were selected as follows: F. imperialis cv. Premier (very strong foxy odor), F. imperialis cv. Lutea (strong foxy odor), F. imperialis ssp. Inodora (no odor), Fritillaria eduardii (weak mousy odor), Fritillaria raddeana (no odor), and an F1 of F. imperialis Lutea × Inodora (weak foxy odor). Volatiles from these flower bulbs were accumulated on Tenax and injected into the GC by thermodesorption. The majority of the volatiles consisted of low molecular weight aliphatic compounds. GC-O revealed that the foxy odor was caused by a single component, identified as 3-methyl-2-butene-1-thiol on the basis of smell in GC-O analyses (two GC columns), mass spectra, and retention times. Chemical identification was substantiated by GC-O and GC-MS of an authentic standard of 3-methyl-2-butene-1-thiol, prepared by organic synthesis
    Market linkages in the Slovak agri-food sector
    Bozik, M. ; Blaas., G. ; Berkum, S. van - \ 2005
    Den Haag : LEI (LEI rapport ) - 47 p.
    Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon
    Lücker, J. ; Schwab, W. ; Hautum, B. van; Blaas, J. ; Plas, L.H.W. van der; Bouwmeester, H.J. ; Verhoeven, H.A. - \ 2004
    Plant Physiology 134 (2004)1. - ISSN 0032-0889 - p. 510 - 519.
    s-linalool synthase - biosynthetic-pathway - volatile compounds - nicotiana-tabacum - gene-expression - floral scents - flowers - transformation - emission - clarkia
    Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one plant by crossings, we show that it is possible to increase the amount and alter the composition of the blend of monoterpenoids produced in tobacco plants. The transgenic tobacco plant line with the three introduced monoterpene synthases is emitting -pinene, limonene, and -terpinene and a number of side products of the introduced monoterpene synthases, from its leaves and flowers, in addition to the terpenoids emitted by wild-type plants. The results show that there is a sufficiently high level of substrate accessible for the introduced enzymes
    Studies on the pro-oxidant chemistry of flavonoids
    Awad, H.M. - \ 2002
    Wageningen University. Promotor(en): I.M.C.M. Rietjens; P.J. van Bladeren; J. Vervoort. - S.l. : S.n. - ISBN 9789058085887 - 133
    flavonoïden - oxidatiemiddelen - antioxidanten - quercetine - structuuractiviteitsrelaties - hplc - kernmagnetische resonantiespectroscopie - massaspectrometrie - flavonoids - oxidants - antioxidants - quercetin - structure activity relationships - hplc - nuclear magnetic resonance spectroscopy - mass spectrometry

    There is currently much interest in the development of functional foods aiming at the prevention of the development of some diseases, for example cancer, by the introduction of selected natural substances at elevated levels into the diet. The rationale for this approach is based especially on epidemiological data that indicate that food items containing such chemicals may reduce the risk of these diseases in humans. Epidemiological studies indicate, for example, that diets rich in fruit and vegetables protect against a variety of diseases, including heart diseases and certain forms of cancer. However, identification of the actual ingredient in a specific diet responsible for the beneficial health effects remains an important bottleneck for translating observational epidemiology to development of a functional food ingredient. The protection against cancer afforded by fruit and vegetables has been attributed to antioxidant micronutrients such as vitamin C, beta-carotene and vitamin E, which may act at many sites, including the stomach, intestine, lung and bladder. However, present scientific attention is focusing as well on the significance of other minor dietary components, notably the flavonoids as protectants against disease. Flavonoids are widespread in nature and are found in considerable quantities in fruits, vegetables, seeds, peel and tubers. The average Western diet may provide up to 1 g of flavonoids per day. Numerous in vitro studies show that flavonoids are potent antioxidants and metal chelators. Their potential as anti-inflammatory, antiallergic and antiviral compounds has also attracted attention. These studies provide the basis for the present rapidly increasing interest for the use of flavonoids as functional food ingredients. As a result increased human exposure to flavonoids can be expected in the near future. In shops and at the internet, food and food supplements based on (iso)flavonoids as functional ingredients are marketed. This, although hard scientific data supporting the health claims as well as data allowing a balanced risk-benefit evaluation are lacking. For flavonoids increased future human exposure regimens induce the question on their pro-oxidant chemistry. There is considerable evidence that some flavonoids are mutagenic in both bacterial and mammalian experimental systems. A high incidence of gastric cancer in some human populations has been linked to consumption of wine containing potentially mutagenic flavonoids (Tamura et al. , Proc. Natl. Acad. Sci. USA. 77, 4961-4965, 1980, Hoey et al. , Am. J. Epidemiol., 113, 669-974, 1981). Relatively little is understood about either the toxicity or protection afforded by flavonoids in humans.

    Since flavonoid quinone/quinone methides have been suggested as the major metabolites responsible for the possible pro-oxidant toxicity and mutagenicity of flavonoids, characterisation of flavonoid quinone chemistry is of importance. However, little information is available on the structure and reactivity of these flavonoid oxidation products. Therefore, the objective of this thesis was to investigate the pro-oxidant chemistry of flavonoids and to perform structure activity studies on the chemical behaviour of 3',4'-dihydroxyflavonoids with special emphasis on the nature and reactivity of the quinone/quinone methide type metabolites formed. Using the GSH trapping method, HPLC, LC/MS, MALDI-TOF, 1H NMR, 13C NMR and quantum mechanical computer calculations the quinone/quinone methide chemistry of a series of 3',4'-dihydroxyflavonoids could be characterised.

    The results provide insight in structure-activity-relationships for the pro-oxidant chemistry of these electrophilic quinone/quinone methide flavonoid metabolites. The results obtained also reveal an unexpected pH-dependent electrophilic behaviour of B ring catechol flavonoids. Furthermore the results of this thesis also reveal, for the first time, evidence for the pro-oxidative chemistry of quercetin in a cellular in vitro model. The formation of these glutathionyl-flavonoid adducts provides evidence for the actual pro-oxidative formation of reactive quinone type metabolites from B ring catechol flavonoids in the selected cellular in vitro model using melanoma cells. Oxidation of the catechols to quinones and their isomeric quinone methides generates potent electrophiles that could alkylate DNA. Interestingly, the structural requirements essential for good antioxidant activity match the requirements essential for pro-oxidant action and quinone methide formation. Altogether, the pro-oxidant behaviour of flavonoids and their quinone/quinone methides are far from straight forward and need to be re-evaluated especially in the framework of the risk-benefit evaluation of the use of these flavonoids as functional food ingredients and/or food supplements.


    Er is momenteel veel interesse voor de ontwikkeling van functionele voedingsmiddelen (functional foods), met als doel het voorkomen van het ontstaan van ziekten zoals bijvoorbeeld kanker, via het in verhoogde mate introduceren van geselecteerde natuurlijke bestanddelen in het dieet. De basis voor deze aanpak wordt momenteel met name gevonden in epidemiologische studies die laten zien dat diëten rijk aan specifieke voedselcomponenten of ingrediënten de kans op bepaalde ziekten bij de mens verlagen. Zo geven epidemiologische studies bijvoorbeeld aan dat diëten die rijk zijn aan fruit en groenten beschermen tegen een aantal ziekten zoals hartziekten en bepaalde vormen van kanker. Echter, het identificeren van de belangrijke ingrediënten in het betreffende dieet die het gezondheidsbevorderende effect tot stand brengen is een knelpunt voor het vertalen van de resultaten uit de epidemiologie naar de ontwikkeling van een functioneel voedingsingrediënt.

    De bescherming tegen kanker door groenten en fruit is toegeschreven aan antioxidanten zoals vitamine C, beta-caroteen en vitamine E, die op vele plaatsen in het lichaam, zoals de maag, darmen, long en de blaas actief zijn. Wetenschappelijk wordt momenteel veel aandacht besteed aan het mogelijke belang van andere belangrijke dieet componenten, zoals flavonoïden, als beschermende ingrediënten tegen ziekte. Flavonoïden komen in de natuur veel voor, en worden met name in hoge concentraties gevonden in fruit, groenten, knollen en zaden. Het gemiddelde Westerse dieet bevat ongeveer 1 gram aan flavonoïden per dag.

    Vele in vitro studies tonen aan dat flavonoïden goede antioxidanten en metaal chelatoren zijn. Daarnaast hebben ze anti-inflammatoire, anti-allergische en anti-virale eigenschappen die van belang worden geacht. Deze bevindingen verschaffen de basis voor de momenteel snel groeiende interesse om flavonoïden te gebruiken als functionele voedingsingrediënten. Als gevolg hiervan zou er in de nabije toekomst een toename in de opname van flavonoïden via het dieet verwacht kunnen worden. In winkels en via het internet worden voedingsmiddelen en voedingssupplementen gebaseerd op (iso)flavonoïden als functionele voedingsingrediënten verkocht. Dit, terwijl zowel de wetenschappelijke onderbouwing voor de gezondheidsclaims als gegevens die een gebalanceerde "risk-benefit" analyse mogelijk maken, nog ontbreken. In het geval van verhoogde toekomstige blootstelling van mensen aan flavonoïden worden voor de risk-benefit evaluatie vragen van belang rond hun mogelijk pro-oxidatieve chemisch gedrag. Er zijn aanwijzingen dat sommige flavonoïden mutageen zijn in zowel bacteriële als zoogdier in vitro test systemen. Een verhoogde mate aan maagkanker in bepaalde humane populaties is in verband gebracht met de consumptie van wijn met daarin mogelijk mutagene flavonoïden (Tamura et al. , Proc. Natl. Acad. Sci. USA. 77, 4961-4965, 1980, Hoey et al. , Am. J. Epidem., 113, 669-974, 1981). Alles samenvattend is er eigenlijk weinig bekend van de schadelijke maar ook van de gezondheidsbevorderende effecten van flavonoïden.

    Omdat flavonoid chinon/chinon methides genoemd zijn als de belangrijkste metabolieten die verantwoordelijk zouden zijn voor de mogelijke pro-oxidatieve toxiciteit en mutageniteit van flavonoïden, is karakterisering van deze pro-oxidant chemie van flavonoïden van belang. Echter er is weinig bekend over de structuur en de reactiviteit van deze flavonoid oxidatie producten. Daarom was het doel van deze studie de pro-oxidant chemie van flavonoïden te onderzoeken en een structuur-activiteits studie uit te voeren naar het chemische gedrag van 3',4'-dihydroxyflavonoïden. Daarbij werd speciale aandacht besteed aan de aard en reactiviteit van de gevormde chinon/chinon methide metabolieten. Met behulp van de GSH-trapping methode, HPLC, LC/MS, MALDI-TOF, 1H-NMR, 13C-NMR en kwantum-chemische computerberekeningen kon de chinon/chinon methide chemie van een serie 3',4'-dihydroxyflavonoiden gekarakteriseerd worden.

    De verkregen resultaten geven inzicht in de structuur-activteits relaties voor de pro-oxidatieve chemie van de electrofiele chinon /chinon methides metabolieten van de flavonoïden. De resultaten laten ook een onverwacht effect zien van de pH op het electrofiele gedrag van de B-ring catechol flavonoïden. Bovendien laten de resultaten van het proefschrift zien dat zelfs onder reducerende omstandigheden in een cellulair in vitro model (melanoma cellen) de pro-oxidatieve chemie van quercetine van belang kan zijn. Met name de vorming van glutathion-flavonoid conjugaten is een bewijs dat in het gekozen cellulaire model de pro-oxidatieve vorming van reactieve flavonoid chinon/ chinon methide metabolieten is opgetreden. Oxidatie van de catecholen naar chinonen en hun isomere chinon methides genereert electrofielen die DNA kunnen alkyleren. Van belang is dat de structurele randvoorwaarden die een flavonoid een goede antioxidant maken gelijk blijken te zijn aan de structurele kenmerken die essentieel zijn voor pro-oxidant gedrag en chinon methide vorming.

    Al met al is de pro-oxidant chemie van flavonoïden en van hun chinon /chinon methides verre van recht toe recht aan gebleken en zou de pro-oxidatieve chemie en de toxiciteit van de flavonoïden in het kader van hun gebruik als functional food ingredienten beter onderzocht en afgewogen moeten worden, rekening houdend met hun mogelijk gezondheidsbevorderende effecten.

    Expression of Clarkia S-linalool synthase in transgenic petunia plant results in the accumulation of S-linalyl-b-D-glucopyranoside
    Lücker, J. ; Bouwmeester, H.J. ; Schwab, W. ; Blaas, J. ; Plas, L.H.W. van der; Verhoeven, H.A. - \ 2001
    The Plant Journal 27 (2001). - ISSN 0960-7412 - p. 315 - 324.
    Petunia hybrida W115 was transformed with a Clarkia breweri S-linalool synthase cDNA (lis). Lis was expressed in all tissues analysed, and linalool was detected in leaves, sepals, corolla, stem and ovary, but not in nectaries, roots, pollen and style. However, the S-linalool produced by the plant in the various tissues is not present as free linalool, but was efficiently converted to non-volatile S-linalyl--d-glucopyranoside by the action of endogenous glucosyltransferase. The results presented demonstrate that monoterpene production can be altered by genetic modification, and that the compounds produced can be converted by endogenous enzymatic activity.
    Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA micro-arrays
    Aharoni, A. ; Keizer, L.C.P. ; Bouwmeester, H.J. ; Sun, Z. ; Alvarez-Huerta, M. ; Verhoeven, H.A. ; Blaas, J. ; Houwelingen, A.M.M.L. van; Vos, R.C.H. de; Voet, H. van der; Jansen, R.C. ; Guis, M. ; Davis, R.W. ; Schena, M. ; Tunen, A.J. van; O'Connel, A.P. - \ 2000
    The Plant Cell 12 (2000). - ISSN 1040-4651 - p. 647 - 661.
    Genetic modification of flavour and fragrance production in plants
    Verhoeven, H.A. ; Aharoni, A. ; Wein, M. ; Blaas, J. ; Bouwer, R. ; Tunen, A.J. van - \ 1998
    In: Natural product analysis / Schreier, P., Herderich, M., Humpf, H.U., Schwab, W., - p. 311 - 312.
    Identification and molecular analysis of transgenic potato chromosomes transferred to tomato through microprotoplast fusion
    Rutgers, E. ; Ramulu, K.S. ; Dijkhuis, P. ; Blaas, J. ; Krens, F.A. ; Verhoeven, H.A. - \ 1997
    Theoretical and Applied Genetics 94 (1997)8. - ISSN 0040-5752 - p. 1053 - 1059.
    Results are reported on the integration sites and copy number of alien marker genes neomycin phosphotransferase II (nptII) and β-glucuronidase (uidA), introduced into diploid potato Solanum tuberosum through transformation by Agrobacterium tumefaciens. Also, the transgenic potato chromosomes 3 and 5 harbouring the nptII and uidA genes, which were transferred to tomato (wild species Lycopersicon peruvianum) by microprotoplast fusion, as revealed by genomic in situ hybridization (GISH), were identified by RFLP analysis using chromosome-specific markers. The data revealed three integration sites in the donor potato genome, each containing the uidA gene, and two also harbouring the nptII gene. Analysis of monosomic-addition hybrid plants obtained after microprotoplast fusion showed that each of these three integration sites is located on a different potato chromosome. The microprotoplast hybrid plants contained only the chromosomes that carried the selectable gene nptII. The data on sexual transmission of the donor potato chromosome carrying the uidA and nptII genes were obtained by analyzing the first backcross progeny (BC1) derived from crossing a monosomic-addition hybrid plant to tomato (L. peruvianum). The glucuronidase (GUS) assay and PCR analysis using primers for the uidA gent indicated the presence of the potato chromosome in GUS-positive and its absence in GUS-negative BC1 plants. RFLP analysis confirmed sexual transmission of the potato chromosome carrying the nptII and uidA genes to the BC1 plants. A few BC1 plants contained the nptII and uidA genes in the absence of the potato additional chromosome, indicating that the marker genes were integrated into the tomato genome. The potential applications of the transfer of alien chromosomes and genes by microprotoplast fusion technique are discussed.
    Intergeneric transfer of a partial genome and direct production of monosomic addition plants by microprotoplast fusion
    Ramulu, K.S. ; Dijkhuis, R. ; Rutgers, E. ; Blaas, J. ; Krens, F.A. ; Verbeek, W.H.J. ; Colijn-Hooymans, C.M. ; Verhoeven, H.A. - \ 1996
    Theoretical and Applied Genetics 92 (1996). - ISSN 0040-5752 - p. 316 - 325.
    Microprotoplast-mediated transfer of single, specific chromosomes between sexually incompatible plants
    Ramulu, K.S. ; Dijkhuis, P. ; Rutgers, E. ; Blaas, J. ; Krens, F.A. ; Dons, J.J.M. ; Colijn-Hooymans, C.M. ; Verhoeven, H.A. - \ 1996
    Genome 39 (1996). - ISSN 0831-2796 - p. 921 - 933.
    A monoclonal antibody against the alkaline extracellular beta-glucosidase from Trichoderma reesei.
    Hofer, F. ; Weissinger, E. ; Mischak, H. ; Messner, R. ; Meixner-Monori, B. ; Blaas, D. ; Visser, J. ; Kubicek, C.P. - \ 1989
    Biochimica et biophysica acta-protein structure and molecular enzymology 992 (1989). - ISSN 0167-4838 - p. 298 - 306.
    Induction and characterization of micronuclei in plant cells : perspectives for micronucleus-mediated chromosome transfer = Inductie en karakterisering van microkernen in plantecellen : perspectieven voor chromosoom overdracht via microkernen
    Verhoeven, H.A. - \ 1989
    Agricultural University. Promotor(en): A. van Kammen; B. de Groot. - S.l. : Verhoeven - 117
    cytologie - plantenfysiologie - genetische modificatie - recombinant dna - celfysiologie - cytology - plant physiology - genetic engineering - recombinant dna - cell physiology

    In this thesis, micronucleation in plant cells has been investigated and systems for isolation and transfer of organelles have been established.
    The discovery, described in chapter two, that the phosphoric amide herbicide amiprophos-methyl induced micronuclei at a high frequency in cell suspensions of N.plumbaginifolia, has opened the possibility to develop a microcell-mediated chromosome transfer system analogous to that in mammalian cell lines. In mammalian cells, micronuclei are induced by prolonged exposure of cells to spindle toxins (colchicine, Colcemid), resulting in up to 60% micronucleated cells (Matsui et al., 1982). Micronucleated cells are isolated by the "shake-off' method, and subjected to high speed centrifugation, which results in fractionation of the cells into microcells, containing micronulei with one or a few chromosomes. Subsequently, microcells are fused to the recipient cells. The transferred chromosomes were found to remain intact and mitotically stable (Fournier, 1982). This technique has hitherto not been available for plant cells or protoplasts, due to the lack of efficient procedures to induce micronuclei. Gamma-irradiation is now often used in the construction of monochromosomal addition lines by somatic hybridization (Bates et al., 1987), to induce chromosome damage which promotes chromosome elimination from one of the fusion partners. As has been pointed out in the introduction (chapter one), ionizing radiation induces chromosome rearrage ments, deletions and insertions (Menczel et al., 1982). From research on mammalian cells, it is known that these phenomena occur with a lower frequency after microcell-mediated chromosome transfer (Fournier, 1981). If microprotoplasts would become available for plant genetic manipulation, transfer of a limited number of chromosomes by microprotoplast fusion would offer an alternative to the use of gamma-irradiation. With the finding that APM induces micronuclei at high frequency in plants, transfer of low numbers of chromosomes after micronucleation can now be tested for use in plant genetic manipulation. The APM treatment was found to be reversible, as was demonstrated by washing the cell suspension cultures free from APM. After washing, normal growth and cell division were soon resumed, with some abnormal, multipolar spindles in the first division after washing. This observation is in good agreement with the the reversible inhibition of microtubule polymerization by APM (Falconer and Seagull, 1987). This low cytotoxicity makes APM a useful tool in the induction of micronuclei in plants.

    The flow cytometric analysis of the nuclear DNA content of APM-treated cel suspension cultures of N.plumbaginifolia, revealed the presence of many micronuclei with a DNA content equivalent to one metaphase chromosome (which consists of both sister chromatids). Similar observations have been made in micronucleated rat kangaroo cells after treatment with Colcemid (Sekiguchi et al., 1978). Sorting of the micronuclei on the basis of the fluorescence of ethidium-bromide, followed by analysis of the DNA content by Feulgen staining (chapter three), shows that it is possible to separate micronuclei on the basis of their DNA content by flowcytometry, like it has been shown for isolated plant metaphase chromosomes. Chromosome identification is sometimes possible with isolated metaphase chromosomes (de Laat and Blaas, 1984; Conia et al., 1987a; 1987b). Identification of chromosomes present in a particular micronucleus is not possible. This is due to different degrees of chromosome decondensation in the micronuclei (which influences the fluorescence signal of the fluorochrome -DNA complex by quenching), and due to the various combinations of chromosomes in micronuclei containing more than one metaphase chromosome. This is illustrated by the DNA histograms of isolated micronuclei in chapter two, which lack the specific chromosome peaks, present in metaphase chromosome preparations (chapter four). When micronuclei are present in large numbers, the overall DNA histogram will show no appreciable contribution of a particular type of chromosome combination in micronuclei, since chromosome grouping appears to be a random process, as was shown by the analysis of the number of micronuclei per cell in chapter two, and by cytological data in chapter two and three. Furthermore, the reduction of the number of micronuclei per micronucleated cell, which appears to be the consequence of fusion of micronuclei into a lobed restitution nucleus, gives rise to even more combinations of chromosomes.

    The processes, involved in the formation of micronuclei, are studied in chapter three and four. The effects of the anti-microtubular herbicides APM, oryzalin and the alkaloid colchicine, used for metaphase arrest and induction of micronuclei in mammalian cells, on the mitotic index and micronueleus formation are compared. The disruption of the spindle by direct inhibition of microtubule assemble is responsible for the accumulation of cells at metaphase. The concentrations of the inhibitors required for complete metaphase arrest, vary from 3 μM for APM and oryzalin to 500 μM for colchicine, as a consequence of differences in binding specificity (Hertel et al., 1980; Dustin 1984). The differences in the percentage of ball metaphases indicate specific effects of the above mentioned inhibitors on chromosome scattering. Apart from the disruption of the microtubules, APM and oryzalin have been shown to influence the accumulation of calcium in the mitochondria (Hertel et al., 1981). Moreover, oryzalin disturbs the active excretion of calcium by the plasma membrane. These combined effects result in an increased cytoplasmic calcium concentration (Hertel et al., 1980), which will be higher after oryzalin treatment than after APM treatment, due to the reduction of active calcium excretion by oryzalin. Our 'data suggest that the APM or oryzalin induced increase of the cytoplasmic calcium concentration is involved in both formation and fusion of micronuclei. Colchicine, which does not influence the cytoplasmic calcium concentration, is not effective in the induction of micronuclei. The higher cytoplasmic calcium levels after oryzalin treatment, would increase the fusogenic properties of the nuclear membranes, which would explain why micronuclei exist for a shorter time after oryzalin treatment as compared to APM treatment. This hypothesis will be tested in future experiments by treatments with the calcium ionophore A23187 in combination with the calcium-specific chelator ethyleneglycolbis- (2-aminoethylether)-N,N'-tetra acetic acid (EGTA), with simultaneous measurements of the cytoplasmic calcium concentrations with the new calcium specific fluorochromes Fluo-3 and Rhod-2 (Haugland, 1989).

    In order to obtain both large numbers of micronucleated cells, and large numbers of micronuclei per micronucleated cell, the
    effect of DNA synthesis inhibitors was investigated. The results in chapter five show, that a considerable increase in the number of micronucleated cells can be achieved by HU or APH treatments, and that the time at which micronuclei appear can be controlled. The results further indicate that metaphases have to be exposed to APM for at least 12h, before micronucleation occurs, and that their lifetime is in the same order. These data demonstrate that it is possible to manipulate the conditions of the treatments in order to obtain either a high yield of metaphase chromosomes, or a high yield of micronuclei, with little contamination by micronuclei or chromosomes, respectively. In this way, it becomes possible to determine the moment at which the number of micronuclei per cell is at its maximal value.

    The isolation and characterization of microprotoplasts from micronucleated suspension cells is described in chapter six. Data obtained from DNA content measurements and flow cytometry demonstrate the presence of up to 40% of subprotoplasts with a DNA content less than the G1-level of the APM treated suspension cells. This indicates that genome fractionation has occurred, and the data on the FDA-staining show that most of the subprotoplasts still possess an intact plasma membrane, since FDA can not be retained by vacuolar membranes only (Lesney, 1986). The viability of the microprotoplasts and other types of subprotoplasts is indicated by the successful culture after gradient fractionation. As it is impossible to measure the DNA-content of microprotoplasts in a non-destructive way, no preselection could be performed to use only microprotoplasts for fusion. In a mass fusion system, the smallest microcells will be the least likely to fuse when electrofusion is used, because their small diameter will prevent alignment and membrane breakdown, which are both related to particle diameter (Zimmermann et al., 1982). Individual selection and fusion could overcome this problem (Koop et al., 1983). This control is essential for the efficient application of microprotoplasts, since the DNA content per microprotoplast will depend upon the DNA content per micronucleus in the cell suspension. Microprotoplast fusion will result in transfer of a part of the total number of chromosomes, directly followed by spontaneous chromosome elimination when two distantly related species are fused, since chromosome elimination seems to be directed by genome dose effects (Graves, 1984; Gilissen et al., 1989). Sofar no successful fusion experiments have been performed, which makes it impossible at the moment to comment on the usefulness of microprotoplasts in chromosome transfer. However, fusion experiments with karyoplasts indicate that it is possible to perform fusions in a controlled way (Spangenberg et al., 1987).

    In addition to the microprotoplast fusion, microinjection was developed for transfer of organelles and micronuclei. Glass needles with a large orifice (5pM) were prepared, along with a pressure system, based on the application of mercury. With the injection system, described in chapter seven, it is possible to suck donor material from a donor protoplast, and inject this directly into the recipient. The data on the complementation of the albino tobacco by injection of mature green chloroplasts or etiolated plastids, indicate that protoplasts can survive the injection treatment, and that the injected plastids can be replicated by the recipient. In this way, the organelles to be transferred are not subjected to damaging isolation procedures and they can be preselected visually. Selective transfer of organelles offers a number of advantages when compared to fusion techniques, or transfer of isolated genes. One of the advantages is the protective nature of the membranes associated with chloroplasts, mitochondria and nuclei. Although structural integrity and functionality has been demonstrated for isolated chloroplasts and mitochondria, it is not known whether isolated organelles are still physiologically intact. The isolation of intact nuclei from plant cells has also been described, with data indicating their structural integrity, as well as their ability to transfer genes into recipient protoplasts (Saxena et al., 1986). Transfer of marker genes does not necessarily implicate the functional integrity of isolated nuclei, since transfer of marker genes can be achieved by uptake of isolated genomic DNA. Preliminary results obtained from experiments with microinjection of micronuclei, indicate that it is possible to remove micronuclei from the donor by suction. Sofar, transfer into a recipient has not been achieved. The kanamycine- resistance, which was introduced into N.plumbaginifolia by transformation with Agrobacterium tumefaciens , will be used as selectable marker after transfer of micronuclei. The transfer of chromosomes will be tested with species specific repetitive DNA probes, which are able to discriminate between the donor genome N.plumbaginifolia and the recipient (either Lycopersicon esculentum or Solanum tuberosum ) . Several probes with the required specificity have already been characterized, from a series of highly repetitive sequences, isolated from N.plumbaginifolia (data not shown).

    With the methods, described in this thesis, the transfer of chromosomes via micronuclei has come within reach. Future work will focus on achieving transfer, and study the fate of the introduced micronuclei. This should provide an answer whether micronuclei can be used as chromosome carriers in plants, as has already been shown in mammalian somatic cell genetics.

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