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Under appropriate tissue culture conditions, somatic plant cells can be induced to form embryos in a process called somatic embryogenesis (SE). SE provides a way to clonally propagate desirable plants and is therefore an important plant breeding tool. SE has also fascinated scientists for decades as an expression of plant ‘totipotency’, the ability to regenerate a whole new individual through embryogenesis. This thesis aims to obtain a deeper understanding of somatic embryo induction in Arabidopsis by the transcription factor BABY BOOM (BBM), through identification and functional analysis of BBM-binding proteins and BBM target genes. Chapter 1 introduces the concept of somatic embryogenesis, describes the different SE systems in Arabidopsis, and discusses the role of the plant hormone auxin and chromatin modifying proteins in this process. An overview is presented on the current knowledge on SE-induction through ectopic overexpression of certain transcription factor genes. These include BBM, as well as other genes that are studied in this thesis in relation to BBM. BBM is part of the eight member AIL subfamily of AP2/ERF domain transcription factors. Chapter 2 reviews the role of AIL proteins during embryogenesis, stem cell niche specification, meristem maintenance and organ positioning and growth. We summarize the gene regulatory networks in which AILs function and describe how these transcription factors integrate multiple hormonal inputs, with special emphasis on the interactions between AILs and auxin. Finally, we conclude that although the functions of AILs in plant development are well described, knowledge on the molecular mode of action of AIL proteins and the identity of AIL target genes is still limited. Transcription factors function in protein complexes and in Chapter 3 we show that members of the HOMEODOMAIN GLABROUS (HDG) transcription factor family physically interact with BBM and other AILs. HDG genes are expressed in the epidermis, the outer cell layer of the plant, where they promote differentiation of cells into specialized epidermal cell types, such as trichomes or stomata. We show that ectopic overexpression of HDG1 leads to loss of root and shoot meristems, phenotypes that had previously been reported for loss-of-function ail mutants. Conversely, down-regulation of HDG genes led to reduced cell differentiation, enhanced cell proliferation and SE phenotypes, phenotypes that resemble those found in AIL overexpression lines. Moreover, we found that co-overexpression of BBM and HDG1 reduces the overexpression phenotypes of both proteins. These results suggest opposite functions of AIL and HDG transcription factors, with AILs stimulating cell proliferation and HDGs stimulating cell differentiation, with the ratio between the two proteins determining the developmental outcome. Finally, we show that HDGs and AILs regulate each other on a transcriptional level and that they share common target genes. A variety of AIL overexpression phenotypes has been described in the literature, with BBM and PLT5/AIL5 being the only known AILs that induce SE upon overexpression. We show in Chapter 4 that all AIL proteins except AIL1 and ANT are able to induce SE, but that this phenotype relies on a high AIL protein dosage. Using BBM and PLT2 as AIL representatives, we show that an intermediate AIL concentration induces organogenesis (ectopic root and shoot formation) and that a low concentration inhibits cellular differentiation. In addition, we show that BBM and PLT2 induce direct SE when activated at seed germination, while post-germination activation leads to indirect SE from callus. The LEAFY COTYLEDON (LEC)/LAFL genes, which also encode SE-inducing transcription factors, are direct targets of BBM/PLT2 during direct SE, showing that these two SE pathways are linked. Using LAFL gene mutants, we show that the LAFL pathway is an important downstream component of BBM-mediated SE. Chapter 5 presents the in vivo, genome-wide analysis of BBM DNA binding sites in somatic embryos using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Our ChIP-seq and gene expression analysis reveal that BBM binds and positively regulates auxin biosynthesis genes and the recently discovered positive regulators of SE, the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) genes. Knock-out of either pathway reduced BBM-mediated SE, showing that auxin biosynthesis and the AHL genes are important components of the BBM pathway. We also show that BBM binds to a consensus DNA motif that resembles the reported ANT binding motif. Chapter 6 reviews methods for identifying the direct target genes of a plant transcription factor using microarrays, as was done for HDG1 (Chapter 4). We describe which different systems can be used to control transcription factor activity, and how these can be combined with microarray analysis to identify target genes. In addition, we provide guidelines for the statistical analysis of microarray data and for the confirmation of candidate target genes. In plant biology, protein-protein interactions are often studied using bimolecular fluorescence complementation (BiFC) or split-YFP. In my BBM-HDG interaction studies I encountered problems using this method, which lead to the cautionary note on the use of BiFC presented in Chapter 7. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. However, because the fluorescent protein halves are prone to self-assembly, it is crucial to use proper controls and a quantitative read-out of fluorescence to avoid false positive interactions. We present a guideline for the setup of a BiFC experiment, discussing each step in the protocol. Chapter 8 discusses how the results presented in this thesis contribute to our knowledge on AIL transcription factors and somatic embryo induction, as well as the questions that still remain. An extended model of dose-dependent AIL function is proposed, as well as mechanisms by which the AIL-HDG interaction could function at the molecular level. Finally, an overview is provided of the molecular-genetic intersection between the different transcription factor-induced SE pathways.

Under appropriate tissue culture conditions, somatic plant cells can be induced to form embryos in a process called somatic embryogenesis (SE). SE provides a way to clonally propagate desirable plants and is therefore an important plant breeding tool. SE has also fascinated scientists for decades as an expression of plant ‘totipotency’, the ability to regenerate a whole new individual through embryogenesis. This thesis aims to obtain a deeper understanding of somatic embryo induction in Arabidopsis by the transcription factor BABY BOOM (BBM), through identification and functional analysis of BBM-binding proteins and BBM target genes. Chapter 1 introduces the concept of somatic embryogenesis, describes the different SE systems in Arabidopsis, and discusses the role of the plant hormone auxin and chromatin modifying proteins in this process. An overview is presented on the current knowledge on SE-induction through ectopic overexpression of certain transcription factor genes. These include BBM, as well as other genes that are studied in this thesis in relation to BBM. BBM is part of the eight member AIL subfamily of AP2/ERF domain transcription factors. Chapter 2 reviews the role of AIL proteins during embryogenesis, stem cell niche specification, meristem maintenance and organ positioning and growth. We summarize the gene regulatory networks in which AILs function and describe how these transcription factors integrate multiple hormonal inputs, with special emphasis on the interactions between AILs and auxin. Finally, we conclude that although the functions of AILs in plant development are well described, knowledge on the molecular mode of action of AIL proteins and the identity of AIL target genes is still limited. Transcription factors function in protein complexes and in Chapter 3 we show that members of the HOMEODOMAIN GLABROUS (HDG) transcription factor family physically interact with BBM and other AILs. HDG genes are expressed in the epidermis, the outer cell layer of the plant, where they promote differentiation of cells into specialized epidermal cell types, such as trichomes or stomata. We show that ectopic overexpression of HDG1 leads to loss of root and shoot meristems, phenotypes that had previously been reported for loss-of-function ail mutants. Conversely, down-regulation of HDG genes led to reduced cell differentiation, enhanced cell proliferation and SE phenotypes, phenotypes that resemble those found in AIL overexpression lines. Moreover, we found that co-overexpression of BBM and HDG1 reduces the overexpression phenotypes of both proteins. These results suggest opposite functions of AIL and HDG transcription factors, with AILs stimulating cell proliferation and HDGs stimulating cell differentiation, with the ratio between the two proteins determining the developmental outcome. Finally, we show that HDGs and AILs regulate each other on a transcriptional level and that they share common target genes. A variety of AIL overexpression phenotypes has been described in the literature, with BBM and PLT5/AIL5 being the only known AILs that induce SE upon overexpression. We show in Chapter 4 that all AIL proteins except AIL1 and ANT are able to induce SE, but that this phenotype relies on a high AIL protein dosage. Using BBM and PLT2 as AIL representatives, we show that an intermediate AIL concentration induces organogenesis (ectopic root and shoot formation) and that a low concentration inhibits cellular differentiation. In addition, we show that BBM and PLT2 induce direct SE when activated at seed germination, while post-germination activation leads to indirect SE from callus. The LEAFY COTYLEDON (LEC)/LAFL genes, which also encode SE-inducing transcription factors, are direct targets of BBM/PLT2 during direct SE, showing that these two SE pathways are linked. Using LAFL gene mutants, we show that the LAFL pathway is an important downstream component of BBM-mediated SE. Chapter 5 presents the in vivo, genome-wide analysis of BBM DNA binding sites in somatic embryos using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Our ChIP-seq and gene expression analysis reveal that BBM binds and positively regulates auxin biosynthesis genes and the recently discovered positive regulators of SE, the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) genes. Knock-out of either pathway reduced BBM-mediated SE, showing that auxin biosynthesis and the AHL genes are important components of the BBM pathway. We also show that BBM binds to a consensus DNA motif that resembles the reported ANT binding motif. Chapter 6 reviews methods for identifying the direct target genes of a plant transcription factor using microarrays, as was done for HDG1 (Chapter 4). We describe which different systems can be used to control transcription factor activity, and how these can be combined with microarray analysis to identify target genes. In addition, we provide guidelines for the statistical analysis of microarray data and for the confirmation of candidate target genes. In plant biology, protein-protein interactions are often studied using bimolecular fluorescence complementation (BiFC) or split-YFP. In my BBM-HDG interaction studies I encountered problems using this method, which lead to the cautionary note on the use of BiFC presented in Chapter 7. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. However, because the fluorescent protein halves are prone to self-assembly, it is crucial to use proper controls and a quantitative read-out of fluorescence to avoid false positive interactions. We present a guideline for the setup of a BiFC experiment, discussing each step in the protocol. Chapter 8 discusses how the results presented in this thesis contribute to our knowledge on AIL transcription factors and somatic embryo induction, as well as the questions that still remain. An extended model of dose-dependent AIL function is proposed, as well as mechanisms by which the AIL-HDG interaction could function at the molecular level. Finally, an overview is provided of the molecular-genetic intersection between the different transcription factor-induced SE pathways.

Increased global use of highly productive breeds of farm animals has been coupled to loss of genetic diversity in most species. In European countries, various governmental, non-governmental, and private organizations try to preserve genetic diversity of livestock in situ (e.g., by stimulating the use of indigenous, rare breeds by farmers; in nature reserves; or in noncommercial farms). In the case of poultry, maintaining in situ populations of the noncommercial (fancy) breeds largely relies on hobby farmers. In addition to in situ conservation, gene banks are being established for ex situ conservation. In at least 2 countries, France and The Netherlands, there are limited collections of frozen semen of rare poultry breeds. Since 2003, the CGN has started with a more systematic effort to collect, freeze, and store semen of indigenous Dutch poultry breeds. At present, the CGN gene bank contains semen of 11 Dutch rare poultry breeds. Also, CGN has performed research on the methodology for cryopreservation of fowl semen. This recent work was focused on finding a suitable replacement for glycerol, which is contraceptive in the hen, as a cryoprotectant. For reasons of hygiene and sample identification, we favored straw freezing, as opposed to the highly effective pellet freezing method. A significant interaction was found between cooling rate and cryoprotectant concentration. Best post-thaw sperm quality was obtained when combining 0.6 mol of dimethylacetamide/L with a cooling rate of +/- 200 degrees C/min. Inseminations, twice per week with 0.3 billion sperm per insemination resulted in 97 and 88% fertilized eggs with fresh and frozen semen, respectively. In 2005, CGN has used this straw freezing method to extend the collection of poultry semen in the Dutch gene bank.

Microspore embryogenesis is an expression of plant cell totipotency that leads to the production of haploid embryos. Besides being a widely exploited plant breeding tool, microspore embryogenesis is also a fascinating system that can be used to obtain a deeper mechanistic understanding of plant totipotency. This thesis aims to provide more insight into the process of microspore embryogenesis, from the formation of embryogenic cells to the outgrowth of differentiated embryos. In Chapter 1 background information is provided on the various aspects of Brassica napus microspore culture and plant development that intersect with the topics that are studied in this thesis. Emphasis is placed on the basic requirements and limitations for successful microspore embryo culture, as well as on the roles of the plant hormone auxin and epigenetic regulation in the development of plant embryos, during both zygotic and in vitro embryo development. Chapter 2 reviews the recent advances that have been made in understanding the developmental and molecular changes that take place during microspore embryogenesis in model systems. The commonly reported cellular changes associated with the establishment of embryo cell fate are summarized and evaluated. The subsequent differentiation of the embryo is also discussed, specifically, what is known about the establishment of polarity, with emphasis on the importance of exine rupture as a positional clue, and the processes that influence meristem maintenance during culture. Finally, the studies on the molecular changes during microspore embryo induction are put into context of male gametophytic development. Overall, the current perspective on microspore embryo initiation presents a landscape in which several routes can lead to the same final destination. Stress treatments are widely applied to induce embryogenic growth in microspore culture. Chapter 3 explores the role of histone acetylation status in stress-induced microspore embryogenesis in Brassica napus. Inhibition of histone deactylases (HDACs) using the HDAC inhibitor trichostatin A (TSA), phenocopies the heat stress treatment that is normally used to induce embryogenic cell proliferation in B. napus microspore culture. Arabidopsis is recalcitrant for haploid embryogenesis, yet treatment with TSA also induced embryogenic cell divisions in this model species. Our observations suggest that the totipotency of the male gametophyte is kept in check by an HDAC-dependent mechanism and that the stress treatments used to induce haploid embryo development in culture impinge on this HDAC-dependent pathway. The repression of HDACs or HDAC-mediated pathways by stress and the accompanying changes in histone acetylation status could provide a single, common regulation point for the induction of haploid embryogenesis. Chapter 4 builds on the knowledge developed in Chapter 3 on the role of HDAC proteins in plant totipotency. A wide variety of chemically distinct HDAC inhibitors was evaluated and additional inhibitors that enhance embryogenic cell induction and/or embryo yield were identified. One surprising observation was made during the course of this study: the initial donor microspore/pollen stage affects the quality of the embryo that is formed. In control cultures, embryos from progressively older stages of donor microspores/pollen became progressively compromised in their basal (axis region) region, characterized by a shift from normal embryos with apical (cotyledons) and basal (root) polarity to abnormal embryos with a reduced basal pole and ball-shaped embryos. These abnormal phenotypes could be partially complemented by treatment with HDAC inhibitors, which promoted growth of the basal region of the embryo. Progressive enhancement of embryo basal identity was accompanied by enhanced and broadened expression of the DR5 auxin response reporter. The embryo phenotypes observed in control and HDAC inhibitor treated microspore cultures are similar to the phenotypes induced by altered expression of the Arabidopsis TOPLESS (TPL)/HDAC19/BODENLOS (BDL) repressor complex, which acts to restrict expression of the AUXIN RESPONSE FACTOR ARF5/MONOPTEROS (MP) to the basal region of the embryo during zygotic embryo development. To understand why most embryogenic callus failed to develop further, we examined the transcriptome of globular-shaped embryos that have started to histodifferentiate and compared it with embryogenic callus. The transcriptome analysis showed that the expression of many genes that regulate (auxin-related) embryo patterning were downregulated in embryogenic callus compared to globular stage embryos. This result may simply reflect the lack of patterning in these embryos or might indicate a role of auxin-signalling in embryogenic callus formation. Chapter 5 examines how embryo identity and patterning is established in two B. napus microspore embryo pathways, a zygotic-like pathway, characterized by suspensor and then embryo proper formation, and a pathway characterized by initially unorganized structures that lack a suspensor. We specifically asked the question: how can embryo patterning be established in the absence of an initial asymmetric division and in the absence of a suspensor, two key events in zygotic embryo development. Analysis of embryo fate (GRP) and auxin (PIN1, PIN7 and DR5) markers showed that embryo fate was established prior to cell division, and independent of subsequent division pattern. The suspensorless embryo program was marked by a transient auxin maximum, followed by establishment of the apical and basal poles at the globular stage, coincident with release of the embryo from the pollen exine. Unlike zygotic embryo development, polar auxin transport (PAT) was not required for embryo initiation or polarity establishment in this system. Suspensor embryos developed in a similar fashion as zygotic embryos, PAT was required for specification of the embryo proper from the suspensor. Haploid embryogenesis therefore follows at least two programs, a PAT-dependent program that requires embryo proper specification from the suspensor, and an alternative PAT-independent program marked by an initial auxin maximum. In the final chapter, Chapter 6, the work presented in this thesis is put in context of the broader plant development field. The epigenetic regulation of developmental transitions that respond to stress and during pollen development are highlighted. A model is provided that histone acetylation levels mediated by HAT and HDAC regulate pollen fate.

Microspore embryogenesis is an expression of plant cell totipotency that leads to the production of haploid embryos. Besides being a widely exploited plant breeding tool, microspore embryogenesis is also a fascinating system that can be used to obtain a deeper mechanistic understanding of plant totipotency. This thesis aims to provide more insight into the process of microspore embryogenesis, from the formation of embryogenic cells to the outgrowth of differentiated embryos. In Chapter 1 background information is provided on the various aspects of Brassica napus microspore culture and plant development that intersect with the topics that are studied in this thesis. Emphasis is placed on the basic requirements and limitations for successful microspore embryo culture, as well as on the roles of the plant hormone auxin and epigenetic regulation in the development of plant embryos, during both zygotic and in vitro embryo development. Chapter 2 reviews the recent advances that have been made in understanding the developmental and molecular changes that take place during microspore embryogenesis in model systems. The commonly reported cellular changes associated with the establishment of embryo cell fate are summarized and evaluated. The subsequent differentiation of the embryo is also discussed, specifically, what is known about the establishment of polarity, with emphasis on the importance of exine rupture as a positional clue, and the processes that influence meristem maintenance during culture. Finally, the studies on the molecular changes during microspore embryo induction are put into context of male gametophytic development. Overall, the current perspective on microspore embryo initiation presents a landscape in which several routes can lead to the same final destination. Stress treatments are widely applied to induce embryogenic growth in microspore culture. Chapter 3 explores the role of histone acetylation status in stress-induced microspore embryogenesis in Brassica napus. Inhibition of histone deactylases (HDACs) using the HDAC inhibitor trichostatin A (TSA), phenocopies the heat stress treatment that is normally used to induce embryogenic cell proliferation in B. napus microspore culture. Arabidopsis is recalcitrant for haploid embryogenesis, yet treatment with TSA also induced embryogenic cell divisions in this model species. Our observations suggest that the totipotency of the male gametophyte is kept in check by an HDAC-dependent mechanism and that the stress treatments used to induce haploid embryo development in culture impinge on this HDAC-dependent pathway. The repression of HDACs or HDAC-mediated pathways by stress and the accompanying changes in histone acetylation status could provide a single, common regulation point for the induction of haploid embryogenesis. Chapter 4 builds on the knowledge developed in Chapter 3 on the role of HDAC proteins in plant totipotency. A wide variety of chemically distinct HDAC inhibitors was evaluated and additional inhibitors that enhance embryogenic cell induction and/or embryo yield were identified. One surprising observation was made during the course of this study: the initial donor microspore/pollen stage affects the quality of the embryo that is formed. In control cultures, embryos from progressively older stages of donor microspores/pollen became progressively compromised in their basal (axis region) region, characterized by a shift from normal embryos with apical (cotyledons) and basal (root) polarity to abnormal embryos with a reduced basal pole and ball-shaped embryos. These abnormal phenotypes could be partially complemented by treatment with HDAC inhibitors, which promoted growth of the basal region of the embryo. Progressive enhancement of embryo basal identity was accompanied by enhanced and broadened expression of the DR5 auxin response reporter. The embryo phenotypes observed in control and HDAC inhibitor treated microspore cultures are similar to the phenotypes induced by altered expression of the Arabidopsis TOPLESS (TPL)/HDAC19/BODENLOS (BDL) repressor complex, which acts to restrict expression of the AUXIN RESPONSE FACTOR ARF5/MONOPTEROS (MP) to the basal region of the embryo during zygotic embryo development. To understand why most embryogenic callus failed to develop further, we examined the transcriptome of globular-shaped embryos that have started to histodifferentiate and compared it with embryogenic callus. The transcriptome analysis showed that the expression of many genes that regulate (auxin-related) embryo patterning were downregulated in embryogenic callus compared to globular stage embryos. This result may simply reflect the lack of patterning in these embryos or might indicate a role of auxin-signalling in embryogenic callus formation. Chapter 5 examines how embryo identity and patterning is established in two B. napus microspore embryo pathways, a zygotic-like pathway, characterized by suspensor and then embryo proper formation, and a pathway characterized by initially unorganized structures that lack a suspensor. We specifically asked the question: how can embryo patterning be established in the absence of an initial asymmetric division and in the absence of a suspensor, two key events in zygotic embryo development. Analysis of embryo fate (GRP) and auxin (PIN1, PIN7 and DR5) markers showed that embryo fate was established prior to cell division, and independent of subsequent division pattern. The suspensorless embryo program was marked by a transient auxin maximum, followed by establishment of the apical and basal poles at the globular stage, coincident with release of the embryo from the pollen exine. Unlike zygotic embryo development, polar auxin transport (PAT) was not required for embryo initiation or polarity establishment in this system. Suspensor embryos developed in a similar fashion as zygotic embryos, PAT was required for specification of the embryo proper from the suspensor. Haploid embryogenesis therefore follows at least two programs, a PAT-dependent program that requires embryo proper specification from the suspensor, and an alternative PAT-independent program marked by an initial auxin maximum. In the final chapter, Chapter 6, the work presented in this thesis is put in context of the broader plant development field. The epigenetic regulation of developmental transitions that respond to stress and during pollen development are highlighted. A model is provided that histone acetylation levels mediated by HAT and HDAC regulate pollen fate.

Nuchter beschouwd is het opmerkelijk dat planten in staat zijn in kweekbuizen te groeien. Het is daarom niet verwonderlijk dat zich regelmatig problemen voordoen. De oplossing daarvan is van groot belang voor de praktijk. Dat maakt snelle vermeerdering door weefselkweek voor veel meer gewassen toegankelijk.

Het onderzoek in dit project betrof stress bij planten gerelateerd aan aspecten van weefselkweek. Door de invloed van deze stress op kwaliteit en kwantiteit van de geproduceerde planten leiden in vitro vermeerderingsbedrijven grote verliezen. In deze publicatie verslag van onderzoek bij de zandraket, Arabidopsis thaliana, hét modelgewas bij uitstek in het moleculair genetische onderzoek bij planten. Daarnaast werden enkele commerciële gewassen bestudeerd.

In Alstroemeria, micropropagation is achieved by axillary bud outgrowth. However, the multiplication rate is rather low (1.2–2.0 per cycle of 4 weeks) due to strong apical dominance. Even though several factors (i.e. culture media, growth regulators, and environmental conditions) have been studied, no significant improvements have been achieved. Basic research on apical dominance mechanism in Alstroemeria is therefore required. This may enhance the understanding how apical dominance controls axillary bud outgrowth in this plant and others. A second drawback in micropropagation of Alstroemeria is the slow growth of rhizomes. In this respect mineral nutrition and the effect of stress are significant. The former is implicated in storage organ growth to literature studies, in particular phosphate. The latter, the effect of stress, is based on observations of growers who noticed a strong increase of rhizome growth after moderate abiotic stress. This research deals with the backgrounds of growth of Alstroemeria in vitro. It deals with (1) the regulation of apical dominance, (2) the growth of rhizomes by applying abiotic stress and (3) the growth of rhizomes by adapting the formulation of inorganic nutrients. Alstroemeria is characterized by strong apical dominance. We observed that the outgrowth of axillary buds is inhibited byboth rhizome and aerial shoot tips. The hormonal regulation is like the regulation in model plants (Arabidopsis, pea, petunia) involving as promoting hormone cytokinin and as inhibiting ones auxin and the carotenoid-derived hormones namely strigolactones (SLs). The involvement of SLs is examined in more detail. Fluridone, an inhibitor of SL synthesis, promotes the outgrowth of lateral rhizomes. It is supposed that SLs act by inhibiting auxin transport. Our experiment to measure auxin transport seem to agree with this. We also found that the auxin transport inhibitors TIBA and NPA reverse the effect of fluridone. The moderate heat stress, and also cold stress and anaerobic stress strongly promote rhizome growth. The increase of growth is not reduced by stress-protective treatments. Therefore, we conclude provisionally that this phenomenon is itself a response of plantlets to protect them from future adverse conditions. Alstroemeria explants rapidly consume organics and inorganics from the nutrient medium but so far no relationship could be established with poor growth. There are marked differences with respect to mineral composition of the rhizome and the aerial shoot for Ca, P and Mg.

In chemical safety assessment, information on adverse effects after repeated dose and chronic exposure to low levels of hazardous compounds is essential for estimating human risks. At present, this information is almost solely obtained by performing animal experiments. Therefore, suitable methods to reduce, refine or replace (3Rs) repeated dose animal testing are urgently needed. At present, in vitro toxicity assays are able to screen compounds for toxicity, but since these tests result in in vitro concentration-response curves, whereas for the safety assessment of chemicals for human in vivo dose-response curves are needed, it is important that in vitro concentration-response curves can be translated to in vivo dose-response curves. The goal of the present project is to extrapolate in vitro concentration-response curves to in vivo dose-response curves with the help of physiologically based kinetic (PBK) models that describe the in vivo absorption, distribution, metabolism and excretion (ADME) processes. This is achieved by using the concentration-response curves, acquired in an appropriate in vitro toxicity test, as internal concentrations in the model, in order to calculate the in vivo dose levels that are needed to reach the internal (toxic) concentrations, by using the PBK-model. The predicted dose-response curves thus obtained can be used to determine safe exposure levels in chemical safety assessment. The endpoint used in the present study is developmental toxicity. The in vitro toxicity assay used is the differentiation assay of the embryonic stem cell test (EST). With the use of a rat PBK model, predicted dose-response curves for in vivo developmental toxicity for the rat are acquired, which are compared with experimental literature data on the in vivo developmental toxicity of these compounds in the rat. To obtain the dose-response curves for in vivo developmental toxicity in human, PBK-models describing the in vivo kinetics in human are used. The combined in vitro-in silico approach described is used for compounds belonging to the chemical class of glycol ethers or the chemical class of retinoids. This enables evaluation of whether the combined in vitro - in silico approach is able to predict dose-response curves for in vivo developmental toxicity for compounds belonging to the same chemical class, but with differences in toxic potency. The results of the research reveal the feasibility of translating in vitro concentration-response curves to in vivo dose-response curves using PBK modeling. This finding shows the possibility of using in vitro toxicity data in chemical risk assessment, which will, if applied in risk assessment, highly contribute to the 3Rs.

In Alstroemeria, micropropagation is achieved by axillary bud outgrowth. However, the multiplication rate is rather low (1.2–2.0 per cycle of 4 weeks) due to strong apical dominance. Even though several factors (i.e. culture media, growth regulators, and environmental conditions) have been studied, no significant improvements have been achieved. Basic research on apical dominance mechanism in Alstroemeria is therefore required. This may enhance the understanding how apical dominance controls axillary bud outgrowth in this plant and others. A second drawback in micropropagation of Alstroemeria is the slow growth of rhizomes. In this respect mineral nutrition and the effect of stress are significant. The former is implicated in storage organ growth to literature studies, in particular phosphate. The latter, the effect of stress, is based on observations of growers who noticed a strong increase of rhizome growth after moderate abiotic stress. This research deals with the backgrounds of growth of Alstroemeria in vitro. It deals with (1) the regulation of apical dominance, (2) the growth of rhizomes by applying abiotic stress and (3) the growth of rhizomes by adapting the formulation of inorganic nutrients. Alstroemeria is characterized by strong apical dominance. We observed that the outgrowth of axillary buds is inhibited byboth rhizome and aerial shoot tips. The hormonal regulation is like the regulation in model plants (Arabidopsis, pea, petunia) involving as promoting hormone cytokinin and as inhibiting ones auxin and the carotenoid-derived hormones namely strigolactones (SLs). The involvement of SLs is examined in more detail. Fluridone, an inhibitor of SL synthesis, promotes the outgrowth of lateral rhizomes. It is supposed that SLs act by inhibiting auxin transport. Our experiment to measure auxin transport seem to agree with this. We also found that the auxin transport inhibitors TIBA and NPA reverse the effect of fluridone. The moderate heat stress, and also cold stress and anaerobic stress strongly promote rhizome growth. The increase of growth is not reduced by stress-protective treatments. Therefore, we conclude provisionally that this phenomenon is itself a response of plantlets to protect them from future adverse conditions. Alstroemeria explants rapidly consume organics and inorganics from the nutrient medium but so far no relationship could be established with poor growth. There are marked differences with respect to mineral composition of the rhizome and the aerial shoot for Ca, P and Mg.

In drug development, toxicity is an important factor for attrition, resulting in a failure rate of 30%-40%. Hepatotoxicity, nephrotoxicity, cardiovascular safety, reproduction toxicity, developmental toxicity (teratogenicity), genotoxicity and carcinogenicity are the main causes for attrition in safety assessment. Screening on these aspects in the early discovery phase of drug development and using these data for compound optimization and deselection might result in drug development candidates with an improved success rate. The present thesis focused on early screening for genotoxicity and carcinogenicity. In recent years some progress has been made with assays to assess genotoxicity and non-genotoxic carcinogenicity at the end of the discovery phase. However, the time point at which these genotoxicity and carcinogenicity assays are performed is still relatively late, only a few assays for such a strategy are available, the throughput of these assays is in general still low and most of them have not yet been validated extensively. An additional drawback is that the currently used in vitro assays for the detection of genotoxicity give a high rate of false positive results, which makes application in the early discovery phase of drug development cumbersome. The goal of the present thesis was therefore to develop improved and new in vitro assays for detecting the genotoxic and non-genotoxic carcinogenic potential of chemicals, validate these assays with proper reference compounds, and to develop a strategy for application of these assays in the early discovery phase of drug development. High throughput assays based on bacteria, yeast and human/rodent cell lines were developed. In the case of human cell lines, the focus was on the HepG2 cell line as the properties of HepG2 cells are expected to give a good prediction for in vivo genotoxicity. The results in this thesis show that an early prediction can be made for bacterial mutagenicity (gene mutations), mammalian genotoxicity (chromosome damage), and non-genotoxic carcinogenic potential by aryl hydrocarbon receptor (AhR) activation. To develop a strategy for application of the HTS genotoxicity assays in the early discovery phase several combinations of assays were evaluated. The combination VitotoxTM + HepG2 p53 reporter assay was based on the presented results in this thesis the most useful for screening compounds for their genotoxic potential in the early drug discovery phase without the risk on high numbers of false positives. CYP1A induction assays in human HepG2 and rat H4IIE cells may be performed in parallel with these assays to be able to detect non-genotoxic carcinogenic potential by AhR activators. Further application of these assays may prove useful in future drug development strategies.

In drug development, toxicity is an important factor for attrition, resulting in a failure rate of 30%-40%. Hepatotoxicity, nephrotoxicity, cardiovascular safety, reproduction toxicity, developmental toxicity (teratogenicity), genotoxicity and carcinogenicity are the main causes for attrition in safety assessment. Screening on these aspects in the early discovery phase of drug development and using these data for compound optimization and deselection might result in drug development candidates with an improved success rate. The present thesis focused on early screening for genotoxicity and carcinogenicity. In recent years some progress has been made with assays to assess genotoxicity and non-genotoxic carcinogenicity at the end of the discovery phase. However, the time point at which these genotoxicity and carcinogenicity assays are performed is still relatively late, only a few assays for such a strategy are available, the throughput of these assays is in general still low and most of them have not yet been validated extensively. An additional drawback is that the currently used in vitro assays for the detection of genotoxicity give a high rate of false positive results, which makes application in the early discovery phase of drug development cumbersome. The goal of the present thesis was therefore to develop improved and new in vitro assays for detecting the genotoxic and non-genotoxic carcinogenic potential of chemicals, validate these assays with proper reference compounds, and to develop a strategy for application of these assays in the early discovery phase of drug development. High throughput assays based on bacteria, yeast and human/rodent cell lines were developed. In the case of human cell lines, the focus was on the HepG2 cell line as the properties of HepG2 cells are expected to give a good prediction for in vivo genotoxicity. The results in this thesis show that an early prediction can be made for bacterial mutagenicity (gene mutations), mammalian genotoxicity (chromosome damage), and non-genotoxic carcinogenic potential by aryl hydrocarbon receptor (AhR) activation. To develop a strategy for application of the HTS genotoxicity assays in the early discovery phase several combinations of assays were evaluated. The combination VitotoxTM + HepG2 p53 reporter assay was based on the presented results in this thesis the most useful for screening compounds for their genotoxic potential in the early drug discovery phase without the risk on high numbers of false positives. CYP1A induction assays in human HepG2 and rat H4IIE cells may be performed in parallel with these assays to be able to detect non-genotoxic carcinogenic potential by AhR activators. Further application of these assays may prove useful in future drug development strategies.

In de zoektocht naar nieuwe eiwitbronnen vormt in-vitrovlees één van de sporen. Het idee is om uit dierlijke stamcellen spierweefsel te laten groeien, ofwel vlees, met behulp van weefselkweektechnieken. Daarvoor zijn dierlijke cellijnen nodig, maar dieren komen er niet aan te pas. Daarnaast is de productie van in-vitrovlees potentieel efficiënter en duurzamer. Door deze mogelijke voordelen voor dieren en voor duurzaamheid bestaat er een grote maatschappelijke vraag om dit idee door nader onderzoek verder uit te werken. In biologisch opzicht zijn er nog veel obstakels te overwinnen. Vanuit maatschappelijk oogpunt springen enerzijds de voordelen in het oog, anderzijnds rijst de vraag of mensen in-vitrovlees ook echt zouden willen eten. Het komt nogal eens voor dat mensen met enige afschuw reageren op het idee van in-vitrovlees. Deze studie vormt een eerste verkenning van deze reactie, die in het Engels wordt aangeduid als 'yuck'-reactie.

In de zoektocht naar nieuwe eiwitbronnen vormt in-vitrovlees één van de sporen. Het idee is om uit dierlijke stamcellen spierweefsel te laten groeien, ofwel vlees, met behulp van weefselkweektechnieken. Daarvoor zijn dierlijke cellijnen nodig, maar dieren komen er niet aan te pas. Daarnaast is de productie van in-vitrovlees potentieel efficiënter en duurzamer. Door deze mogelijke voordelen voor dieren en voor duurzaamheid bestaat er een grote maatschappelijke vraag om dit idee door nader onderzoek verder uit te werken. In biologisch opzicht zijn er nog veel obstakels te overwinnen. Vanuit maatschappelijk oogpunt springen enerzijds de voordelen in het oog, anderzijnds rijst de vraag of mensen in-vitrovlees ook echt zouden willen eten. Het komt nogal eens voor dat mensen met enige afschuw reageren op het idee van in-vitrovlees. Deze studie vormt een eerste verkenning van deze reactie, die in het Engels wordt aangeduid als 'yuck'-reactie.

Sponges (phylum Porifera) are multi cellular filter-feeding invertebrate animals living attached to a substratum in mostly marine but also in freshwater habitats. The interest in sponges has increased rapidly since the discovery of potential new pharmaceutical compounds produced by many sponges. An enormous amount of different chemical structures have been found. Thus far no sustainable production technique has been developed for these marine natural products, because not sufficient knowledge is present about the needs of sponges for both growth and bioactive compound production. The aim of this thesis was to get a better understanding of the growth and metabolism of sponges and of their nutritional needs. Aquaculture is thus far the best method to produce these compounds, although also this technique is not fully developed. To gain more insight in the nutritional needs for growth, we studied the growth rate of Haliclona oculata in its natural environment, Oosterschelde, the Netherlands, and monitored environmental parameters in parallel (Chapter 2). A stereo photogrammetry approach was used for measuring growth rates. Stereo pictures were taken and used to measure volumetric changes monthly during 1 year. The volumetric growth rate of Haliclona oculata showed a seasonal trend with the highest average specific growth rate measured in May: 0.012±0.004 day−1. In our study a strong positive correlation (p

Sponges (phylum Porifera) are multi cellular filter-feeding invertebrate animals living attached to a substratum in mostly marine but also in freshwater habitats. The interest in sponges has increased rapidly since the discovery of potential new pharmaceutical compounds produced by many sponges. An enormous amount of different chemical structures have been found. Thus far no sustainable production technique has been developed for these marine natural products, because not sufficient knowledge is present about the needs of sponges for both growth and bioactive compound production. The aim of this thesis was to get a better understanding of the growth and metabolism of sponges and of their nutritional needs. Aquaculture is thus far the best method to produce these compounds, although also this technique is not fully developed. To gain more insight in the nutritional needs for growth, we studied the growth rate of Haliclona oculata in its natural environment, Oosterschelde, the Netherlands, and monitored environmental parameters in parallel (Chapter 2). A stereo photogrammetry approach was used for measuring growth rates. Stereo pictures were taken and used to measure volumetric changes monthly during 1 year. The volumetric growth rate of Haliclona oculata showed a seasonal trend with the highest average specific growth rate measured in May: 0.012±0.004 day−1. In our study a strong positive correlation (p

Een samenwerkingsverband van 8 weefselkweekbedrijven en Plant Research International heeft de mogelijkheden tot weefselkweekvermeerdering in het donker onderzocht. Er waren twee benaderingen. De eerste benadering, scheutcultuur in het donker, gaf goede resultaten en er zijn reeds proefproducties in bedrijven gedaan. Nadelige aspecten van groei in het donker, met name buitensporige strekking, konden worden tegengegaan. De tweede benadering, vermeerdering via wortelcultures, verliep minder voorspoedig: er waren bij veel gewassen problemen om een goedgroeiende wortelcultuur te verkrijgen. Verder verliep regeneratie van scheuten uit wortels matig of slecht.

Een samenwerkingsverband van 8 weefselkweekbedrijven en Plant Research International heeft de mogelijkheden tot weefselkweekvermeerdering in het donker onderzocht. Er waren twee benaderingen. De eerste benadering, scheutcultuur in het donker, gaf goede resultaten en er zijn reeds proefproducties in bedrijven gedaan. Nadelige aspecten van groei in het donker, met name buitensporige strekking, konden worden tegengegaan. De tweede benadering, vermeerdering via wortelcultures, verliep minder voorspoedig: er waren bij veel gewassen problemen om een goedgroeiende wortelcultuur te verkrijgen. Verder verliep regeneratie van scheuten uit wortels matig of slecht.

The water status in plants is dependent on the xylem hydraulic conductance. In cut flowers, for example, the preservation of continuous hydraulic conductance is important for maintaining longer vase life, an important index for cut flower quality. Many factors, such as stomata performance, root water uptake efficiency, temperature, light quality and humidity, influence the efficiency of water transport in the soil-plant-atmosphere continuum.It has also been shown that the dimensions of the xylem conduits in the plant, such as the conduit length, diameter and density, are important for plant water transport. Therefore, the regulation of the xylem conduit size during xylogenesis is necessary for providing a particular hydraulic efficiency in a given environment. To understand the regulation of xylogenesis and the influence of the processes to produce xylem conduits of particular dimensions with specific hydraulic capacity, this thesis focuses on the Zinnia elegans tracheary element (TE) differentiation at the cellular level. It further considers the regulation of xylem conduit dimensions in whole plants by using drought stress and temperature as variable environmental factors.In Chapter 1, the mechanisms of plant water transport, as regulated by xylem conduit dimensions with consequent effect on plant quality, are reviewed. Also, the chapter provides general literature on xylem formation, Zinnia elegans as model for xylogenic research, and finally gives the outline of the thesis.In Chapter 2 , two cultivars of the Zinnia elegans cut flowers, 'Envy' and 'Purple Prince', were grown in soils with different water contents with increasing water stress levels: 70, 50 and 20 %(v/v) soil water content (SWC). Plant length increased with SWC. The hydraulic efficiency in the stems of cut flowers decreased with increasing water stress. The xylem conduit length was not different between Purple Prince cut flowers from the three different water stress levels. In the Envy cultivar, a significant difference in conduit length was found between plants from 20 and 50% (v/v) SWCs. The proportion of large-diameter conduits increased with decreasing drought stress, i.e., in cut flowers from both cultivars the proportion of large-diameter vessels was highest in the 70% (v/v) SWC and least in the 20% (v/v) SWC. This means that the decrease in hydraulic conductivity with water stress was largely due to the decrease in the diameter of the conduits making the conduit length less important. The vase life of the cut flowers correlated negatively with the diameter of the xylem conduits, confirming that small-diameter conduits are more efficient in overcoming embolism and therefore maintaining efficient water transport in the stems.In Chapter 3 , the role of average day temperature (ADT) and difference in day and night temperature (DIF) on the length of xylem elements and conduits, and fusion of the individual elements into conduits in planta were investigated. Plant length increased significantly with ADT between 17 and 25 o C. Opposite DIF treatments resulted in different plant length at 4 and 8 o C absolute DIF. The responses to temperature differences were similar in both Zinnia cultivars, although Purple Prince cultivar plants were always bigger. The maximal vessel length (L max ) and half vessel lengths (τ) both increased with increasing ADT, about 110-130% increase from 17 to 25 o C ADT. However, DIF had no effect on these conduit parameters. Measurements of average lengths of individual vessel elements from plants grown at average temperatures 17 and 25 o C showed about 10% and 20% increase in Envy and Purple Prince respectively. This change in vessel element length constitutes a small part of the overall change in whole conduit length, and therefore, it can be deduced that the amount of vessel element fusion is more important during formation of longer xylem conduits in the plant.In Chapter 4 , a method for establishing an in vitroZinnia elegans cell culture for differentiation into tracheary elements (TEs) is described. The xylogenic Zinnia system is rather unique for studying xylogenesis at the cellular level. It offers possibilities to study in detail the effects of certain factors important for regulating the xylogenesis process. The in vitro culture has been well standardized to eliminate the inconsistencies in TE differentiation that have been observed in the past. Additional methods for monitoring and measuring the important hallmarks of TE differentiation, including DNA laddering, vacuolar rupture, cellulose bands and lignin synthesis, have been treated in this section.In Chapter 5 , the effect of leaf osmolarity (LO) induced by different light intensities and electrical conductivity (EC) of soil-less plant culture, on the differentiation of TEs in cell cultures from the two cultivars, Envy and Purple Prince, was investigated. Both light intensity and EC correlated with LO with a similar response, although it was always higher in the Envy cultivar. By increasing the LO, the percentage of TEs (%TE) formed also increased, with a similar trend in cultures from both cultivars. About 60% increase in %TE was realized in the Purple Prince cultivar as compared to earlier reports by different authors. The LO of the starting material also correlated positively with the final size of the TE. This implies that the efficiency of the xylogenic Zinnia system can be improved by adaptations in the growth conditions for the starting material.In Chapter 6 , we investigated the effect of osmotic stress and timed application of NAA and BA (xylem differentiating phytohormones) on the TE differentiation in cultures from the two Zinnia cultivars. The %TE increased with increasing extracellular osmotic stress until 300 mOsm, after which any additional increase resulted in a diminishing %TE. The size of the TEs increased with decreasing extracellular osmolarity within the working range of 100 to 700 mOsm. These results were similar in the cultures from both cultivars. Application of NAA/BA induced a significant increase in the rate of cell division corresponding to a similar increase in TE differentiation. It is proposed that the increase in cell division may be beneficial in providing certain bioactive materials for the development of the TEs. Delayed application of NAA/BA reduced the %TE, but with a significant increase in TE size.In Chapter 7 , the role of programmed cell death (PCD) in determining the final size of the TEs and the %TE was investigated. Application of two protease inhibitors, a general caspase inhibitor (Zasp) and a cysteine protease inhibitor (E64), to xylogenic cultures from the two cultivars, caused a significant increase in the size of TEs but 65-300% decrease in %TE. There proved to be a significant delay or complete inhibition of the hallmarks of PCD, such as DNA laddering and vacuole collapse, in the cultures treated with the inhibitors. It was also found that the second stream of TE differentiation, common in non-inhibited xylogenic Zinnia cultures, was absent in cultures treated with 10nM or higher of each of the two inhibitors.The research experiments and their outcomes in this thesis range from fundamental xylogenesis at the cellular level to whole plant water transport performance. The overall discussion of the experimental approaches, results, and their practical implications is presented in Chapter 8 . This chapter also offers suggestions for future research.

The water status in plants is dependent on the xylem hydraulic conductance. In cut flowers, for example, the preservation of continuous hydraulic conductance is important for maintaining longer vase life, an important index for cut flower quality. Many factors, such as stomata performance, root water uptake efficiency, temperature, light quality and humidity, influence the efficiency of water transport in the soil-plant-atmosphere continuum.It has also been shown that the dimensions of the xylem conduits in the plant, such as the conduit length, diameter and density, are important for plant water transport. Therefore, the regulation of the xylem conduit size during xylogenesis is necessary for providing a particular hydraulic efficiency in a given environment. To understand the regulation of xylogenesis and the influence of the processes to produce xylem conduits of particular dimensions with specific hydraulic capacity, this thesis focuses on the Zinnia elegans tracheary element (TE) differentiation at the cellular level. It further considers the regulation of xylem conduit dimensions in whole plants by using drought stress and temperature as variable environmental factors.In Chapter 1, the mechanisms of plant water transport, as regulated by xylem conduit dimensions with consequent effect on plant quality, are reviewed. Also, the chapter provides general literature on xylem formation, Zinnia elegans as model for xylogenic research, and finally gives the outline of the thesis.In Chapter 2 , two cultivars of the Zinnia elegans cut flowers, 'Envy' and 'Purple Prince', were grown in soils with different water contents with increasing water stress levels: 70, 50 and 20 %(v/v) soil water content (SWC). Plant length increased with SWC. The hydraulic efficiency in the stems of cut flowers decreased with increasing water stress. The xylem conduit length was not different between Purple Prince cut flowers from the three different water stress levels. In the Envy cultivar, a significant difference in conduit length was found between plants from 20 and 50% (v/v) SWCs. The proportion of large-diameter conduits increased with decreasing drought stress, i.e., in cut flowers from both cultivars the proportion of large-diameter vessels was highest in the 70% (v/v) SWC and least in the 20% (v/v) SWC. This means that the decrease in hydraulic conductivity with water stress was largely due to the decrease in the diameter of the conduits making the conduit length less important. The vase life of the cut flowers correlated negatively with the diameter of the xylem conduits, confirming that small-diameter conduits are more efficient in overcoming embolism and therefore maintaining efficient water transport in the stems.In Chapter 3 , the role of average day temperature (ADT) and difference in day and night temperature (DIF) on the length of xylem elements and conduits, and fusion of the individual elements into conduits in planta were investigated. Plant length increased significantly with ADT between 17 and 25 o C. Opposite DIF treatments resulted in different plant length at 4 and 8 o C absolute DIF. The responses to temperature differences were similar in both Zinnia cultivars, although Purple Prince cultivar plants were always bigger. The maximal vessel length (L max ) and half vessel lengths (τ) both increased with increasing ADT, about 110-130% increase from 17 to 25 o C ADT. However, DIF had no effect on these conduit parameters. Measurements of average lengths of individual vessel elements from plants grown at average temperatures 17 and 25 o C showed about 10% and 20% increase in Envy and Purple Prince respectively. This change in vessel element length constitutes a small part of the overall change in whole conduit length, and therefore, it can be deduced that the amount of vessel element fusion is more important during formation of longer xylem conduits in the plant.In Chapter 4 , a method for establishing an in vitroZinnia elegans cell culture for differentiation into tracheary elements (TEs) is described. The xylogenic Zinnia system is rather unique for studying xylogenesis at the cellular level. It offers possibilities to study in detail the effects of certain factors important for regulating the xylogenesis process. The in vitro culture has been well standardized to eliminate the inconsistencies in TE differentiation that have been observed in the past. Additional methods for monitoring and measuring the important hallmarks of TE differentiation, including DNA laddering, vacuolar rupture, cellulose bands and lignin synthesis, have been treated in this section.In Chapter 5 , the effect of leaf osmolarity (LO) induced by different light intensities and electrical conductivity (EC) of soil-less plant culture, on the differentiation of TEs in cell cultures from the two cultivars, Envy and Purple Prince, was investigated. Both light intensity and EC correlated with LO with a similar response, although it was always higher in the Envy cultivar. By increasing the LO, the percentage of TEs (%TE) formed also increased, with a similar trend in cultures from both cultivars. About 60% increase in %TE was realized in the Purple Prince cultivar as compared to earlier reports by different authors. The LO of the starting material also correlated positively with the final size of the TE. This implies that the efficiency of the xylogenic Zinnia system can be improved by adaptations in the growth conditions for the starting material.In Chapter 6 , we investigated the effect of osmotic stress and timed application of NAA and BA (xylem differentiating phytohormones) on the TE differentiation in cultures from the two Zinnia cultivars. The %TE increased with increasing extracellular osmotic stress until 300 mOsm, after which any additional increase resulted in a diminishing %TE. The size of the TEs increased with decreasing extracellular osmolarity within the working range of 100 to 700 mOsm. These results were similar in the cultures from both cultivars. Application of NAA/BA induced a significant increase in the rate of cell division corresponding to a similar increase in TE differentiation. It is proposed that the increase in cell division may be beneficial in providing certain bioactive materials for the development of the TEs. Delayed application of NAA/BA reduced the %TE, but with a significant increase in TE size.In Chapter 7 , the role of programmed cell death (PCD) in determining the final size of the TEs and the %TE was investigated. Application of two protease inhibitors, a general caspase inhibitor (Zasp) and a cysteine protease inhibitor (E64), to xylogenic cultures from the two cultivars, caused a significant increase in the size of TEs but 65-300% decrease in %TE. There proved to be a significant delay or complete inhibition of the hallmarks of PCD, such as DNA laddering and vacuole collapse, in the cultures treated with the inhibitors. It was also found that the second stream of TE differentiation, common in non-inhibited xylogenic Zinnia cultures, was absent in cultures treated with 10nM or higher of each of the two inhibitors.The research experiments and their outcomes in this thesis range from fundamental xylogenesis at the cellular level to whole plant water transport performance. The overall discussion of the experimental approaches, results, and their practical implications is presented in Chapter 8 . This chapter also offers suggestions for future research.

The last years, both scientific and public concern about the possible threat of compounds in the environment that may affect endocrine functions has increased. Thus far, the majority of endocrine disruptor research has focused on the interference of compounds with the sex hormone homeostasis. Less attention has been paid to disruption of the hypothalamus-pituitary-thyroid (HPT) axis, despite the fact that several lines of evidence suggest that this system is also susceptible to disruption by compounds present in the environment. Disturbances in thyroid hormone (TH) homeostasis may lead to mosaic effects on development, growth patterns and metabolism in vertebrates including mammals and amphibians. Recent studies now indicate that the Thyroid hormone Receptors (TRs) also are targets of industrial compounds, but the effects and mechanisms are difficult to establish since suitable test assays are very limited. In part, this may be because research has generally focused on the ability of compounds to affect TH transport, TH metabolism and TH blood plasma levels but not on disruption of TR mediated TH-action.The research presented in this thesis aims to enhance insight into the mechanisms underlying the effects of endocrine disrupting compounds (EDCs) on TR-mediated TH-action. To this end, newly developed and validated in vitro and in vivo assays are applied in addition to an ex vivo assay using isolated tail tissue. This ex vivo model more resembles the natural situation for cells than in vitro assays, but excludes the feedback mechanisms from the TH axis orextracellularmetabolism which may obscure TR-mediated responses. In the assays exposure is performed in combination with TH in order to closer approach the exposure of cells under physiological conditions, where TH is present during important vertebrate fetal developmental periods. The in vitro , ex vivo and in vivo assay responses also are compared to study to what degree the in vitro and ex vivo assays can predict the in vivo thyroid hormone disrupting potency of compounds. Apart from the development of newtestsystemsfor thyroid hormone disruption, the studies presented in this thesis also provide indications for the mechanism ofEDCson the level of the TR. Furthermore, the results illustrate thatthe in vitro results are highly predictive for the effects as observed in vivo . Finally, it can be concluded that testing of compounds for potential TH disrupting effects, should be carried out in presence and absence of TH at its EC 50 .

The last years, both scientific and public concern about the possible threat of compounds in the environment that may affect endocrine functions has increased. Thus far, the majority of endocrine disruptor research has focused on the interference of compounds with the sex hormone homeostasis. Less attention has been paid to disruption of the hypothalamus-pituitary-thyroid (HPT) axis, despite the fact that several lines of evidence suggest that this system is also susceptible to disruption by compounds present in the environment. Disturbances in thyroid hormone (TH) homeostasis may lead to mosaic effects on development, growth patterns and metabolism in vertebrates including mammals and amphibians. Recent studies now indicate that the Thyroid hormone Receptors (TRs) also are targets of industrial compounds, but the effects and mechanisms are difficult to establish since suitable test assays are very limited. In part, this may be because research has generally focused on the ability of compounds to affect TH transport, TH metabolism and TH blood plasma levels but not on disruption of TR mediated TH-action.The research presented in this thesis aims to enhance insight into the mechanisms underlying the effects of endocrine disrupting compounds (EDCs) on TR-mediated TH-action. To this end, newly developed and validated in vitro and in vivo assays are applied in addition to an ex vivo assay using isolated tail tissue. This ex vivo model more resembles the natural situation for cells than in vitro assays, but excludes the feedback mechanisms from the TH axis orextracellularmetabolism which may obscure TR-mediated responses. In the assays exposure is performed in combination with TH in order to closer approach the exposure of cells under physiological conditions, where TH is present during important vertebrate fetal developmental periods. The in vitro , ex vivo and in vivo assay responses also are compared to study to what degree the in vitro and ex vivo assays can predict the in vivo thyroid hormone disrupting potency of compounds. Apart from the development of newtestsystemsfor thyroid hormone disruption, the studies presented in this thesis also provide indications for the mechanism ofEDCson the level of the TR. Furthermore, the results illustrate thatthe in vitro results are highly predictive for the effects as observed in vivo . Finally, it can be concluded that testing of compounds for potential TH disrupting effects, should be carried out in presence and absence of TH at its EC 50 .

Nuchter beschouwd is het opmerkelijk dat planten in staat zijn in kweekbuizen te groeien. Het is daarom niet verwonderlijk dat zich regelmatig problemen voordoen. De oplossing daarvan is van groot belang voor de praktijk. Dat maakt snelle vermeerdering door weefselkweek voor veel meer gewassen toegankelijk.

Key words:Solanum tuberosum L., in vitro plantlet, in vitro tuberization, microtubers, minitubers, tuber bulking, photoperiod, in vitro storage, jasmonates, micropropagation, seed production.Micropropagation ( in vitro propagation) has been introduced to seed potato production programmes more than two decades ago. The research reported in this thesis studied possibilities of improvements to micropropagation methods commonly used in commercial laboratories. The focus was placed on seed potato growers in Western Canada who either produce the tissue culture plant material themselves and use it for the production of minitubers (nuclear tubers) in their own operations, or acquire it prior to planting.The autotrophic micropropagation (in CO 2 (1500 ppm) enriched atmosphere, no sucrose in the medium) of Russet Burbank variety showed its usefulness for the small commercial laboratories where often the full sterility is difficult to maintain. Autotrophically grown cultures produced stems very similar to conventionally grown in vitro cultures (no significant differences in length, number of nodes and dry weight). Also, the conventionally propagated cultures benefited from CO 2 enrichment during the 4 week growing period by a 20% increase in the number of nodes per stem and a 50% increase in stem dry weight (doubled stem length).The use of continuous low red light (690 nm) at 3μm-2s -1PPFD and 30 g l-1sucrose in the medium during the in vitro low temperature (4°C) storage of potato cultures were beneficial for maintenance of vigorous, high quality cultures with a high re-grow capacity. In small tissue culture laboratories, installation of a simple low light device in the regular refrigerator would improve maintenance of the high quality of the stored cultures.Significantly better production of microtubers (number of tubers and weight) was observed on solid (agar) than on liquid media, and under the 8 h photoperiod (SD) compared to no light. Microtubers derived from SD were greenish and seemed less juvenile than the tubers from 0 h light. Such microtubers performed better in the field or the greenhouse than microtubers produced in darkness. The 16 h photoperiod was inhibitory to the production of microtubers. The production of microtubers in all six commercial varieties tested in the studies benefited from SD. Tuber bulking rates were lower in the darkness than under the SD. Independently of the variety, fewer microtubers per explant with significantly lower weights, were produced under 0 h photoperiod than in SD. Production of microtubers in all three Russet varieties and in Sangre was superior to this of Shepody and Atlantic.Effects of jasmonic acid (JA) on in vitro tuberization of potato were also variety specific. Varieties Amisk, Russet Burbank, Sangre and Umatilla Russet produced the highest number of microtubers per nodal cutting (1.0 - 1.7) and their tubers were also the largest with 70 - 75% of microtubers in the> 0.1 g size category. Results with Shepody were inconsistent and Atlantic produced the lowest number of tubers per nodal cutting. Amisk, Atlantic and Umatilla Russet benefited (significantly higher total tuber weight, percentage and weight of microtubers > 0.1 g) from JA supplement to the tuberization medium (JAMed), but not from the pre-conditioning treatment (JAPret). However, none of these varieties clearly benefited from any JA treatments under light. Russet Burbank and Sangre also benefited from the JA supplement in the dark, although stock plantlet conditioning (JAPret) was more effective than the JA supplement to the tuberization medium (JAMed). Shepody did not show any benefits of JA on microtuberization, neither under dark or light conditions. The double treatment with JA (JAPret and JAMed) in light reduced production of microtubers comparing to the control and the individual JA treatments.The greenhouse/field performance of microtubers was highly dependent on JA conditioning of plantlets prior to in vitro tuberization, presence of JA in tuberization media, the photoperiod during tuberization and the dormancy release treatment. Although plantlets of all five tested cultivars performed well in the greenhouse, the microtuber performance was variety dependent, so were the responses to JA. JA conditioning of stock plants prior to taking explants for tuberization was beneficial for minituber production and it can be proposed as a treatment enhancing the quality of microtubers and their performance in the greenhouse production of minitubers. In the field, the results with JA were inconclusive; stock plantlets pre-treated with JA (JAPret) enhanced the Pre-elite tuber production in Russet Burbank by approximately 40% but significantly lowered it in Shepody by 17%. In the greenhouse, the three Russet varieties responded best to JA treatments producing results comparable to in vitro plantlets in production of minitubers whereas microtubers of Shepody produced inconclusive results and variety Atlantic performance was poor. The varietal responses were similar in the field studies, however, yield and the number of Pre-elite tubers produced from microtubers were less than 50% of these from plantlets. SD during in vitro tuberization was an important factor (irrespective of other treatments) in producing microtubers which then performed well in the greenhouse and in the field. Microtubers produced in dark performed poorly. Although Rindite proved to be very effective in microtuber dormancy release in greenhouse conditions, more studies are required to provide evidence that the product is safe to use with microtubers. No severe damage to microtubers treated with Rindite was observed when microtubers were from 8 h light tuberization treatment (minimal damage to tubers from the dark treatment). In the field situation, GA gave better results in dormancy release. In all three Russet varieties, the highest number and yield of Pre-elite tubers were obtained when the microtubers were soaked in 100 ppm solution of GA prior to field planting.Optimalization of commercial production of disease/virus-free propagules in the seed potato system encompasses various aspects of micropropagation technology. These include improvements in mass multiplication phase, storage of cultures and the use of microtubers in the system. Microtubers of the Russet varieties can be successfully used to speed up the multiplication in the seed potato system, in the greenhouse production of minitubers.

Key words:Solanum tuberosum L., in vitro plantlet, in vitro tuberization, microtubers, minitubers, tuber bulking, photoperiod, in vitro storage, jasmonates, micropropagation, seed production.Micropropagation ( in vitro propagation) has been introduced to seed potato production programmes more than two decades ago. The research reported in this thesis studied possibilities of improvements to micropropagation methods commonly used in commercial laboratories. The focus was placed on seed potato growers in Western Canada who either produce the tissue culture plant material themselves and use it for the production of minitubers (nuclear tubers) in their own operations, or acquire it prior to planting.The autotrophic micropropagation (in CO 2 (1500 ppm) enriched atmosphere, no sucrose in the medium) of Russet Burbank variety showed its usefulness for the small commercial laboratories where often the full sterility is difficult to maintain. Autotrophically grown cultures produced stems very similar to conventionally grown in vitro cultures (no significant differences in length, number of nodes and dry weight). Also, the conventionally propagated cultures benefited from CO 2 enrichment during the 4 week growing period by a 20% increase in the number of nodes per stem and a 50% increase in stem dry weight (doubled stem length).The use of continuous low red light (690 nm) at 3μm-2s -1PPFD and 30 g l-1sucrose in the medium during the in vitro low temperature (4°C) storage of potato cultures were beneficial for maintenance of vigorous, high quality cultures with a high re-grow capacity. In small tissue culture laboratories, installation of a simple low light device in the regular refrigerator would improve maintenance of the high quality of the stored cultures.Significantly better production of microtubers (number of tubers and weight) was observed on solid (agar) than on liquid media, and under the 8 h photoperiod (SD) compared to no light. Microtubers derived from SD were greenish and seemed less juvenile than the tubers from 0 h light. Such microtubers performed better in the field or the greenhouse than microtubers produced in darkness. The 16 h photoperiod was inhibitory to the production of microtubers. The production of microtubers in all six commercial varieties tested in the studies benefited from SD. Tuber bulking rates were lower in the darkness than under the SD. Independently of the variety, fewer microtubers per explant with significantly lower weights, were produced under 0 h photoperiod than in SD. Production of microtubers in all three Russet varieties and in Sangre was superior to this of Shepody and Atlantic.Effects of jasmonic acid (JA) on in vitro tuberization of potato were also variety specific. Varieties Amisk, Russet Burbank, Sangre and Umatilla Russet produced the highest number of microtubers per nodal cutting (1.0 - 1.7) and their tubers were also the largest with 70 - 75% of microtubers in the> 0.1 g size category. Results with Shepody were inconsistent and Atlantic produced the lowest number of tubers per nodal cutting. Amisk, Atlantic and Umatilla Russet benefited (significantly higher total tuber weight, percentage and weight of microtubers > 0.1 g) from JA supplement to the tuberization medium (JAMed), but not from the pre-conditioning treatment (JAPret). However, none of these varieties clearly benefited from any JA treatments under light. Russet Burbank and Sangre also benefited from the JA supplement in the dark, although stock plantlet conditioning (JAPret) was more effective than the JA supplement to the tuberization medium (JAMed). Shepody did not show any benefits of JA on microtuberization, neither under dark or light conditions. The double treatment with JA (JAPret and JAMed) in light reduced production of microtubers comparing to the control and the individual JA treatments.The greenhouse/field performance of microtubers was highly dependent on JA conditioning of plantlets prior to in vitro tuberization, presence of JA in tuberization media, the photoperiod during tuberization and the dormancy release treatment. Although plantlets of all five tested cultivars performed well in the greenhouse, the microtuber performance was variety dependent, so were the responses to JA. JA conditioning of stock plants prior to taking explants for tuberization was beneficial for minituber production and it can be proposed as a treatment enhancing the quality of microtubers and their performance in the greenhouse production of minitubers. In the field, the results with JA were inconclusive; stock plantlets pre-treated with JA (JAPret) enhanced the Pre-elite tuber production in Russet Burbank by approximately 40% but significantly lowered it in Shepody by 17%. In the greenhouse, the three Russet varieties responded best to JA treatments producing results comparable to in vitro plantlets in production of minitubers whereas microtubers of Shepody produced inconclusive results and variety Atlantic performance was poor. The varietal responses were similar in the field studies, however, yield and the number of Pre-elite tubers produced from microtubers were less than 50% of these from plantlets. SD during in vitro tuberization was an important factor (irrespective of other treatments) in producing microtubers which then performed well in the greenhouse and in the field. Microtubers produced in dark performed poorly. Although Rindite proved to be very effective in microtuber dormancy release in greenhouse conditions, more studies are required to provide evidence that the product is safe to use with microtubers. No severe damage to microtubers treated with Rindite was observed when microtubers were from 8 h light tuberization treatment (minimal damage to tubers from the dark treatment). In the field situation, GA gave better results in dormancy release. In all three Russet varieties, the highest number and yield of Pre-elite tubers were obtained when the microtubers were soaked in 100 ppm solution of GA prior to field planting.Optimalization of commercial production of disease/virus-free propagules in the seed potato system encompasses various aspects of micropropagation technology. These include improvements in mass multiplication phase, storage of cultures and the use of microtubers in the system. Microtubers of the Russet varieties can be successfully used to speed up the multiplication in the seed potato system, in the greenhouse production of minitubers.

In dit rapport worden de resultaten beschreven van twee projecten waarin weekfselkweekmethoden voor vermeerderding en veredeling van tulp zijn onderzocht. Vermeerdering van tulp op het veld gaat erg langzaam waardoor het lang duurt voordat nieuwe cultivars op de markt kunnen worden gebracht. Vermeerdering m.b.v. weefselkweek kan dit aanzienlijk versnellen. Het protocol dat bij aanvang van de twee projecten beschikbaar was, is aanzienlijk verbeterd. In het eerste project werd nagegaan of somatische embryogenese (vorming van embryo's uit niet-geslachtscellen) toepasbaar is voor een sneller vermeerderingssysteem van tulp. Het tweede project draaide om het verbeteren van de vermeedering van tulp in vitro, uitgaande van scheutvorming op bloemstengelplakjes

In dit rapport worden de resultaten beschreven van twee projecten waarin weekfselkweekmethoden voor vermeerderding en veredeling van tulp zijn onderzocht. Vermeerdering van tulp op het veld gaat erg langzaam waardoor het lang duurt voordat nieuwe cultivars op de markt kunnen worden gebracht. Vermeerdering m.b.v. weefselkweek kan dit aanzienlijk versnellen. Het protocol dat bij aanvang van de twee projecten beschikbaar was, is aanzienlijk verbeterd. In het eerste project werd nagegaan of somatische embryogenese (vorming van embryo's uit niet-geslachtscellen) toepasbaar is voor een sneller vermeerderingssysteem van tulp. Het tweede project draaide om het verbeteren van de vermeedering van tulp in vitro, uitgaande van scheutvorming op bloemstengelplakjes

In vitro techniques have been introduced in potato seed production systems in recent years. This research project aimed at studying the morphological and physiological changes in plants and crops in the last three phases of a seed production system that included an in vitro multiplication, an in vitro normalisation (growing cuttings to rooted plantlets), a transplant production, and a tuber production (field) phase.Leaf area was identified as an important plant parameter for plant growth in the normalisation and transplant production phases. Explants and plantlets with larger initial leaf area performed better than those with smaller initial leaf area. In vitro treatments mainly affected leaf area of transplants through their effects on early above-ground leaf area. Leaf area increase was better described by logistic than by exponential or expolinear curves in all phases of growth, suggesting restriction of leaf area increase in all phases.Low temperature decreased leaf and stem dry weights in all phases, and increased tuber fresh and dry yields, average tuber weight, leaf/stem ratio, specific leaf area and harvest index in the tuber production phase. Growing in vitro plants at low normalisation temperatures increased leaf and total plant dry weights early in the transplant production and tuber production phases. It resulted in higher tuber yields, heavier individual tubers and higher harvest index.Fertilising plants with higher nitrogen (40 versus 10 mg N per plant) during transplant production resulted in plants with higher groundcover in the field. This led to higher interception of solar radiation and higher tuber yield in one of the two experiments. Growing plants at higher temperature (26/20 versus 12/18 °C) during transplant production increased leaf area at the end of the transplant production phase. After transplanting to the field, it resulted in crops with higher groundcover, which intercepted more incoming solar radiation. Yield tended to be higher, but differences could not be assessed as statistically significant. A glasshouse experiment showed that high temperature during transplant production increased leaf and stem dry weights in the tuber production phase, but reduced tuber dry weights and harvest index when temperatures during tuber production were high. Thus, high temperature during transplant production may favour haulm growth and light interception in the field, but may also reduce dry matter partitioning to tubers.Conditions in the tuber production phase were found to be of greater importance for final yield than conditions and treatments in earlier phases.Strategies to optimise the production and use of propagules and transplants should focus on achieving leafy starting material, reducing stress during changes in environment and optimising conditions during tuber production. Production of transplants should be adjusted to the expected growth conditions in the tuber production phase.Key words:Solanum tuberosum L., in vitro plantlet, seed production, normalisation, transplant production, tuber production, acclimatisation, leaf area, groundcover, logistic growth, temperature, nitrogen, dry matter production, specific leaf area, harvest index, radiation interception, radiation use efficiency.

In vitro techniques have been introduced in potato seed production systems in recent years. This research project aimed at studying the morphological and physiological changes in plants and crops in the last three phases of a seed production system that included an in vitro multiplication, an in vitro normalisation (growing cuttings to rooted plantlets), a transplant production, and a tuber production (field) phase.Leaf area was identified as an important plant parameter for plant growth in the normalisation and transplant production phases. Explants and plantlets with larger initial leaf area performed better than those with smaller initial leaf area. In vitro treatments mainly affected leaf area of transplants through their effects on early above-ground leaf area. Leaf area increase was better described by logistic than by exponential or expolinear curves in all phases of growth, suggesting restriction of leaf area increase in all phases.Low temperature decreased leaf and stem dry weights in all phases, and increased tuber fresh and dry yields, average tuber weight, leaf/stem ratio, specific leaf area and harvest index in the tuber production phase. Growing in vitro plants at low normalisation temperatures increased leaf and total plant dry weights early in the transplant production and tuber production phases. It resulted in higher tuber yields, heavier individual tubers and higher harvest index.Fertilising plants with higher nitrogen (40 versus 10 mg N per plant) during transplant production resulted in plants with higher groundcover in the field. This led to higher interception of solar radiation and higher tuber yield in one of the two experiments. Growing plants at higher temperature (26/20 versus 12/18 °C) during transplant production increased leaf area at the end of the transplant production phase. After transplanting to the field, it resulted in crops with higher groundcover, which intercepted more incoming solar radiation. Yield tended to be higher, but differences could not be assessed as statistically significant. A glasshouse experiment showed that high temperature during transplant production increased leaf and stem dry weights in the tuber production phase, but reduced tuber dry weights and harvest index when temperatures during tuber production were high. Thus, high temperature during transplant production may favour haulm growth and light interception in the field, but may also reduce dry matter partitioning to tubers.Conditions in the tuber production phase were found to be of greater importance for final yield than conditions and treatments in earlier phases.Strategies to optimise the production and use of propagules and transplants should focus on achieving leafy starting material, reducing stress during changes in environment and optimising conditions during tuber production. Production of transplants should be adjusted to the expected growth conditions in the tuber production phase.Key words:Solanum tuberosum L., in vitro plantlet, seed production, normalisation, transplant production, tuber production, acclimatisation, leaf area, groundcover, logistic growth, temperature, nitrogen, dry matter production, specific leaf area, harvest index, radiation interception, radiation use efficiency.