Staff Publications

Staff Publications

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

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

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

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Micropropagation of Solanum quitoense var. quitoense by apical bud, petiole and hypocotyl culture
Gutirrez, Bernardo ; Cobo, María Mercedes ; Orellana, Miguel ; Vega, Joely ; Arahana, Venancio ; Jaramillo, Viviana ; Lourdes Torres, María de - \ 2019
Plant Biotechnology Reports 36 (2019)2. - ISSN 1342-4580 - p. 91 - 97.
1-naphtaleneacetic acid (NAA) - 6-benzylaminopurine (BAP) - Andean crop - gibberellic acid (GA3) - in vitro culture - Solanum quitoense

The development of in vitro propagation methods can improve the current commercial use and conservation of plants like naranjilla (Solanum quitoense), a distinctive Andean crop and key emerging agricultural product. In the present study, we report in vitro culture protocols for naranjilla apical buds, hypocotyls and petioles. In apical bud culture, MS medium supplemented with 0.10 mg l−1 1-naphtaleneacetic acid (NAA) produced longer plantlets with greater number of leaves. Hypocotyl culture yielded higher number of shoots when using older explants in MS medium supplemented with different combinations of NAA, 6-benzylaminopurine (BAP) and gibberellic acid (GA3). Petiole culture produced a significantly higher number of shoots per explant, with more abundant and bigger leaves, when using MS medium supplemented with 0.02 mg l−1 NAA, 4.50 mg l−1 BAP and 1.00 mg l−1 GA3. A factorial analysis reveals that the interaction between GA3 and NAA/BAP plays an important role in shoot regeneration. These results provide new tools for the in vitro regeneration of naranjilla plants, improving on previously reported protocols for this species by using alternative explant types and regeneration protocols.

BABY BOOM-induced somatic embryogenesis in Arabidopsis
Horstman, A. - \ 2015
Wageningen University. Promotor(en): Gerco Angenent, co-promotor(en): Kim Boutilier. - Wageningen : Wageningen University - ISBN 9789462572317 - 233
arabidopsis - somatische embryogenese - in vitro kweek - cellen - weefselkweek - plantengroeiregulatoren - somatische embryo's - transcriptie - arabidopsis - somatic embryogenesis - in vitro culture - cells - tissue culture - plant growth regulators - somatic embryos - transcription

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.

Microspore embryogenesis: reprogramming cell fate from pollen to embryo development
Hui Li, - \ 2014
Wageningen University. Promotor(en): Gerco Angenent, co-promotor(en): Kim Boutilier. - Wageningen : Wageningen University - ISBN 9789462570702 - 224
stuifmeel - embryogenese - embryonale ontwikkeling - biologische ontwikkeling - plantenontwikkeling - in vitro kweek - plantenembryo's - brassica napus - pollen - embryogenesis - embryonic development - biological development - plant development - in vitro culture - plant embryos - brassica napus

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.

Tegengaan van kwaliteitsverlies door stress bij weefselkweek
Krens, F.A. ; Klerk, G.J.M. de - \ 2013
Wageningen : Plant Research International - 29
vermeerderingsmateriaal - weefselkweek - kweektechnieken - stress - arabidopsis thaliana - in vitro kweek - hypoxie - oxidatieve stress - hyperhydriciteit - landbouwkundig onderzoek - gewaskwaliteit - propagation materials - tissue culture - culture techniques - in vitro culture - hypoxia - oxidative stress - hyperhydricity - agricultural research - crop quality
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.
Zonder transport staat alles stil, ook in weefselkweek : oplossing ligt in bevordering verdamping
Klerk, G.J.M. de; Kierkels, T. ; Heuvelink, E. - \ 2013
Onder Glas 10 (2013)9. - p. 18 - 19.
glastuinbouw - plantenvermeerdering - weefselkweek - kweektechnieken - in vitro kweek - transpiratie - tca - plantenontwikkeling - greenhouse horticulture - propagation - tissue culture - culture techniques - in vitro culture - transpiration - plant development
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.
Integrated in vitro-in silico models for predicting in vivo developmental toxicity : facilitating non-animal based safety assessment
Louisse, J. - \ 2012
Wageningen University. Promotor(en): Ivonne Rietjens; Bas Blaauboer, co-promotor(en): M. Verwei. - S.l. : s.n. - ISBN 9789461732415 - 256
embryonale ontwikkeling - foetale ontwikkeling - in vitro kweek - toxiciteit - biomarkers - computational science - alternatieven voor dierproeven - risicoschatting - embryonic development - fetal development - in vitro culture - toxicity - biomarkers - computational science - animal testing alternatives - risk assessment

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.

Apical dominance and growth in vitro of Alstroemeria
Pumisutapon, P. - \ 2012
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Geert-Jan de Klerk. - S.l. : s.n. - ISBN 9789461732484 - 118
alstroemeria - vegetatieve vermeerdering - in vitro kweek - microvermeerdering - plantenfysiologie - apicale dominantie - groeimedia - stress - plantengroeiregulatoren - alstroemeria - vegetative propagation - in vitro culture - micropropagation - plant physiology - apical dominance - growing media - stress - plant growth regulators

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.

The development of improved and new in vitro assays for detecting the genotoxic and non-genotoxic carcinogenic potential of chemicals in the discovery phase of drug development
Westerink, W.M.A. - \ 2011
Wageningen University. Promotor(en): Ivonne Rietjens; John Groten, co-promotor(en): W.G.E.J. Schoonen. - [S.l. : S.n. - ISBN 9789085858478 - 288
genotoxiciteit - carcinogenese - nieuwe geneesmiddelen - geneesmiddelenontwikkeling - in vitro - in vitro kweek - genotoxicity - carcinogenesis - new drugs - drug development - in vitro - in vitro culture
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-vitrovlees: yuck!(?) : een eerste verkenning van een eerste reactie
Weele, C.N. van der - \ 2010
Den Haag : LEI Wageningen UR (LEI-nota 10-179) - 23
houding van consumenten - kunstvlees - in vitro kweek - kweektechnieken - productontwikkeling - consumentengedrag - consumer attitudes - meat analogues - in vitro culture - culture techniques - product development - consumer behaviour
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.
Growth and metabolism of sponges
Koopmans, M. - \ 2009
Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): Dirk Martens. - [S.l. : S.n. - ISBN 9789085854418 - 192
sponsen - seizoengroei - biologische productie - metabolisme - groeitempo - in vitro kweek - geneesmiddelen - bioactieve verbindingen - sponges - seasonal growth - biological production - metabolism - growth rate - in vitro culture - drugs - bioactive compounds
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<0.01) was found for growth rate with temperature, algal biomass (measured as chlorophyll a), and carbon and nitrogen content in suspended particulate matter. Thus growth rate seems to be dependent on these factors. No correlation was found with dissolved organic carbon, suggesting that Haliclona oculata is more dependent on particulate organic carbon. To obtain more knowledge about the carbon requirements for growth by sponges, respiration rate and clearance rate were measured in situ in Haliclona oculata and compared to the earlier measured growth rate (Chapter 3). The net growth efficiency, being the ratio of carbon incorporated in biomass and the total carbon used by the sponge for respiration and growth, was found to be 0.10 ± 0.013. Thus, about 10% of the total used carbon was fixed in biomass and over 90% was used for generating energy for growth, maintenance, reproduction and pumping. H. oculata had 2.5 μmol C available for every μmol O2 consumed. A value of 0.75 for the respiratory quotient (RQ in μmol CO2 μmol O2 -1) is the average value reported in literature for different marine invertebrates. Thus, carbon was available in excess to meet the respiratory demand. We found that only 34% of the particulate carbon pumped through the sponge was used for both respiration and growth. Oxygen was not the limiting factor for growth, since only 3.3% of the oxygen pumped through the sponge body was used. Our results indicate that both oxygen and carbon availability are not limiting. The low growth efficiency agrees with the low growth rates found for many sponges.
In order to produce drugs by culturing sponges their growth must be improved. To improve growth, basic knowledge about how food sources are used by the sponge is needed. To find the exact relation between food retained and food converted to sponge biomass we need to be able to distinguish between feed components and sponge biomass, which means we need biomarkers for the feed and for the sponge. The fatty acid (FA) composition of organisms is specific and can therefore be used as biomarkers. We identified and compared fatty acid profiles of five different sponges in three habitats with those in the suspended particulate matter (SPM) in the surrounding water (Chapter 4). Haliclona oculata and Haliclona xena from the Oosterschelde, Haliclona xena and Halichondria panicea from Lake Veere, both in The Netherlands and Dysidea avara and Aplysina aerophoba from the Mediterranean were studied. In the SPM we found comparable FAs to the FAs of sponges up to chain lengths of 28 C-atoms. Different species of sponges showed similarities, but also very different FA profiles, while they were collected from the same habitat at the same moment. The biomarkers for diatoms and dinoflagellates were abundantly found in all sponges except A. aerophoba as this sponge relies mostly on bacterial food sources based on the many bacterial FAs found in this sponge. In all species, except A. aerophoba, C26:3(5,9,19) and C26:2(5,9) were very abundantly present. These FAs were also abundant in the SPM, while it was stated in literature that these compounds are very typical for sponges. Several FA biomarkers were found for the different sponges.
Fatty acid composition is dependent on different factors like food availability and temperature and thus the composition will change in the different seasons. We have studied fatty acid composition and stable isotope 13C natural abundance of suspended particulate matter (SPM) from seawater and sponges in different seasons in the same locations as in chapter 4 (Chapter 5). 13C natural abundance can be used to find the origin of compounds, as the 13C values of compounds are similar to the values from their original producers. The FA concentration variation in sponges was related to changes in fatty acid concentration in SPM. 13C natural abundance in sponge specific FAs showed very limited seasonal variation at all sites. Algal FAs in sponges were mainly acquired from the SPM through active filtration in all seasons. Sponge specific FAs had similar 13C ratios as algal FAs in May at the two Dutch sites, suggesting that sponges were mainly growing during spring and probably summer. During autumn and winter, they were still actively filtering, but the food collected during this period had little effect on sponge 13C values suggesting limited growth. The bacterial sponge A. aerophoba relies mostly on the symbiotic bacteria. In all sponges we found that the ω7 longer chain FAs, C24:1(17) and C26:3(5,9,19) could be traced back to be of bacterial origin. Using a 13C pulse-chase approach metabolic rate can be studied inside organisms. The carbon metabolism of two marine sponges, Haliclona oculata from the Oosterschelde (The Netherlands) and Dysidea avara from the Mediterranean (Spain), has been studied (Chapter 6). The sponges were fed 13C labelled diatom (Skeletonema costatum) for 8 hours in a closed system during which they took up between 75 and 85 % of the diatoms added. At different times whole sponges were sampled for total 13C enrichment, fatty acid composition and 13C enrichment in these fatty acids. During the first day the level of 13C label inside the sponges stayed the same after which the 13C label was metabolized and excreted. Algal biomarkers present in the sponges were highly labeled after feeding and their labeling levels decreased from the second day until no label was left 10 days after enrichment. The sponge specific long chain C26 fatty acids incorporated 13C label already during the first day and the amount of 13C label inside these FAs kept increasing until 3 weeks after labeling. Thus, the algae fed to the sponges were taken up by the sponges within 8 hrs and first conversion started during the first day. Conversion of label occurred at least until at least 3 weeks after feeding.
In different studies it was shown that sponges grow slow, but are able to regenerate damaged tissue fast. Moreover, it has been found that damaged tissue coincides with higher secondary metabolite production. Therefore, we were interested in carbon metabolic rate changes after damaging sponge tissue. We have examined the change of carbon metabolic rate of fatty acid synthesis due to mechanical damage of sponge tissue in Haliclona oculata and Dysidea avara (Chapter 7). Metabolic studies were performed by feeding sponges with 13C labeled biomass of diatom, Pheaodactylum tricornutum, either after or before damaging and tracing back the 13C content in the damaged and healthy tissue. Filtration and respiration rate in both sponges responded quickly to damage. For the finger-sponge H. oculata the rate of respiration was reduced immediately after damage. 6 Hours after damage the filtration rate increased to a level that was higher than the starting value, while the respiration rate returned to the initial value before damage. For the encrusting sponge D. avara the filtration rate also decreased directly after damage, but in this case it did not return to the value before damage after one day. Respiration was not measured for D. avara. The 13C data revealed that H. oculata has a higher metabolic rate in the tips where growth occurs compared to the rest of the tissue and that the metabolic rate is increased after damage of the tissue. For D. avara no differences were found between damaged and non damaged tissue. Thus far it is still not fully understood why, when, where and how bioactive metabolites are produced in sponges. For the near future sea-based sponge culture seems to be the best production method. However, for controlled production in a defined system it is better to develop in vitro production methods. This could be in vitro sponge culture or sponge cell culture, culture methods for symbionts or transfer production routes into another host. We still have insufficient information about the background of metabolite production in sponges. Before culture methods are developed we should focus on factors that induce metabolite production, which could be done in the natural habitat by studying the relation between stress factors (such as predation) and the production of bioactive metabolites. Next, the biosynthetic pathway of metabolite production should be unraveled, as well as the genes involved. The location of production within the sponge should be identified in order to choose between sponge cell culture and symbiont culture. Alternatively the biosynthetic pathways could be introduced into hosts that can be easily cultured in bioreactors. Chapter 8 discusses the current state of sponge metabolite production and the steps that need to be taken to develop commercial production techniques. The different possible production techniques are also discussed.
Weefselkweek in het donker : onderzoek naar nieuwe methodes van weefselkweekvermeerdering
Klerk, G.J.M. de - \ 2008
Wageningen : Plant Research International
bloembollen - weefselkweek - teeltsystemen - in vitro kweek - vermeerderingsmateriaal - wortels - donker - ornamental bulbs - tissue culture - cropping systems - in vitro culture - propagation materials - roots - dark
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.
Hydraulic properties of Zinnia elegans : from cellular development in vitro to performance in planta
Twumasi, P. - \ 2007
Wageningen University. Promotor(en): Anne Mie Emons; Olaf van Kooten, co-promotor(en): Wim van Ieperen; Jan Schel. - [S.l.] : S.n. - ISBN 9789085046585 - 160
zinnia elegans - water - xyleem - hydraulisch geleidingsvermogen - snijbloemen - plant-water relaties - temperatuur - osmolariteit - osmose - apoptose - in vitro kweek - zinnia elegans - water - xylem - hydraulic conductivity - cut flowers - plant water relations - temperature - osmolarity - osmosis - apoptosis - in vitro culture
Animal genetic resources conservation in The Netherlands and Europe: Poultry perspective
Woelders, H. ; Zuidberg, C.A. ; Hiemstra, S.J. - \ 2006
Poultry Science 85 (2006)2. - ISSN 0032-5791 - p. 216 - 222.
dierveredeling - pluimvee - kippen - genetische bronnen van diersoorten - conservering - cryopreservering - cryobeschermingsmiddelen - genenbanken - genetische diversiteit - germplasm - in vitro kweek - inseminatie - sperma - spermaconservering - europa - nederland - animal breeding - poultry - fowls - animal genetic resources - conservation - cryopreservation - cryoprotectants - gene banks - genetic diversity - in vitro culture - insemination - semen - semen preservation - europe - netherlands - fowl spermatozoa - frozen - sperm
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.
Novel in vitro, ex vivo and in vivo assays elucidating the effects of endocrine disrupting compounds (EDCs) on thyroid hormone action
Schriks, M. - \ 2006
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Tinka Murk; J.D. Furlow. - Wageningen : S.n. - ISBN 9085044847 - 159
in vitro kweek - schildklierhormonen - xenopus laevis - hormoonverstoorders - in vivo experimenten - in vitro culture - thyroid hormones - xenopus laevis - endocrine disruptors - in vivo experimentation
Toepassing van weefselkweek en embryogenese bij vermeerdering en veredeling van tulp
Langens-Gerrits, M.M. ; Bouman, H. ; Custers, J.B.M. - \ 2001
Lisse : PPO sector Bloembollen (PPO rapport 405) - 74
tulipa gesneriana - tulpen - weefselkweek - embryogenese - in vitro kweek - microvermeerdering - methodologie - bloembollen - tulipa gesneriana - tulips - tissue culture - embryogenesis - in vitro culture - micropropagation - methodology - ornamental bulbs
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
Biotechnology, a basket of options
Visser, B. - \ 2001
LEISA : ILEIA newsletter for low-external-input and sustainable agriculture 17 (2001)4. - ISSN 1569-8424 - p. 9 - 11.
plantenveredeling - vegetatieve vermeerdering - biotechnologie - in vitro kweek - dna - plant breeding - vegetative propagation - biotechnology - in vitro culture
Micropropagation technology in early phases of commercial seed potato production
Pruski, K. - \ 2001
Wageningen University. Promotor(en): P.C. Struik; J. Nowak; G.J. de Klerk. - S.l. : S.n. - ISBN 9789058084996 - 166
solanum tuberosum - aardappelen - microvermeerdering - vermeerderingsmateriaal - in vitro kweek - pootaardappelen - solanum tuberosum - potatoes - micropropagation - propagation materials - in vitro culture - seed potatoes

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.

Application of biotechnology and molecular biology and breeding : In vitro culture
Plas, L.H.W. van der; Klerk, G.J. de - \ 2000
cultuurmethoden - plantenvermeerdering - vegetatieve vermeerdering - microvermeerdering - in vitro kweek - orgaankweek - explantaten - plantenfysiologie - plantenveredeling - in vitro selectie - cultural methods - propagation - vegetative propagation - micropropagation - in vitro culture - organ culture - explants - plant physiology - plant breeding - in vitro selection
Manipulating the physiological quality of in vitro plantlets and transplants of potato
Mehari, T. - \ 2000
Agricultural University. Promotor(en): P.C. Struik; W.J.M. Lommen. - S.l. : S.n. - ISBN 9789058083302 - 230
solanum tuberosum - aardappelen - in vitro kweek - microvermeerdering - zaadproductie - transplantaten - groei - groeianalyse - bladoppervlakte-index - bladoppervlakte - lichtpenetratie - ophoping van drogestof - stikstof - temperatuur - acclimatisatie - solanum tuberosum - potatoes - in vitro culture - micropropagation - seed production - transplants - growth - growth analysis - leaf area index - leaf area - light penetration - dry matter accumulation - nitrogen - temperature - acclimatization

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.

Growth and morphogenesis of shoot initials of Douglas fir, Pseudotsuga menziesii (Mirb.) Franco, in vitro
Evers, P.W. - \ 1984
Landbouwhogeschool Wageningen. Promotor(en): R.A.A. Oldeman, co-promotor(en): R.L.M. Pierik. - Wageningen : Evers - 250
ongeslachtelijke voortplanting - knoppen - milieufactoren - bosbouw - bosbouwkundige handelingen - groei - in vitro kweek - plantenmorfologie - scheuten - houtteelt - stengels - bomen - vegetatieve vermeerdering - pseudotsuga menziesii - asexual reproduction - buds - environmental factors - forestry - forestry practices - growth - in vitro culture - plant morphology - shoots - silviculture - stems - trees - vegetative propagation - pseudotsuga menziesii
An optimalized method of micropropagation of Douglas fir is described. Seasonal changes were found in optima for nitrate and sucrose in the medium and in the optimum for the light intensity during the culture of shoot initials. Differences in morphogenesis were obtained from shoot initials that had been isolated from buds in 10 topophysical positions on 2-year-old trees. These differences between the shoots were influenced by the medium, the light intensity, and by forcing or topping the mother trees or treating them with growth regulators. Shoots from each of the topophysical positions had a characteristic photosynthesis. A first attempt is made to compare in vivo flushing with the morphogenesis of shoot initials in vitro.
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