Production and excretion of secondary metabolites by plant cell cultures of Tagetes

R.M. Buitelaar

    Research output: Thesisinternal PhD, WU

    Abstract

    <p>In this thesis, the results are presented of several approaches to improve the production and excretion of thiophenes by cell cultures or hairy roots of <em>Tagetes spp.</em><p>In chapter one, most of the techniques to improve the production and/or excretion of secondary metabolites with plant cell cultures are discussed in a literature survey. From this chapter, it becomes clear that to date no general rules exist that apply for all plant species. The methods described in this chapter are the introduction of transformed cells in the form of hairy roots, the use of elicitors, the immobilization of plant cells, the use of two-phase cultures and the use of selected fermenters; all have as aim to improve the growth, production and excretion conditions for the plant cell cultures. All these methods have also been tested in the work described in this thesis, with the aim to obtain a higher production of thiophenes for less costs (meaning faster growth and better excretion).<p>Chapter two is the description of a side-step towards the extension of an immobilization technique for large scale operation that was introduced a few years before, the resonance nozzle technique. Until then, it had only been tested with alginate, but in this chapter, <em>K</em> -carrageenan, agar and gellan gum were tested as well. The gel formation with these materials is improved when the drops are caught in an organic phase, so the reactions of four cell types (bacteria, yeast, plant cells and insect cells) to several organic solvents and the immobilization were examined. The various cell types appeared to react differently to the applied conditions, but in general hydrophobic solvents proved to be more suitable than hydrophilic solvents.<p>The latter phenomenon was elaborated in more detail in the third chapter. Cells of <em>Tagetes minuta</em> , <em></em> in free or immobilized state, were grown in the presence of various organic solvents and their respiration activity was followed. The introduction of organic solvents can have advantageous effects, like the selective removal of the product from the cells or the aqueous phase, thereby combining production and downstream processing, while the removal of the product can relieve product inhibition effects. There appeared to be a relation between the hydrophobicity of the solvent and the residual activity of the cells - the more hydrophobic the solvent was, the more active the cells were in its presence. The immobilization of the cells provided a slight protection against harmful solvents. Although these <em>Tagetes minuta</em> cells were easy to work with (a fast growing, fine cell suspension), we changed to hairy root cultures of <em>Tagetes patula</em> , <em></em> transformed with <em>Agrobacterium rhizogenes</em> LBA 9402, because those cells did produce thiophenes, in contrast to the former. These hairy root cultures are more difficult to work with, because they grow as one clump of interconnected roots, so circulation is difficult and samples cannot be taken. Therefore, in chapter four methods are compared that determine the hairy root biomass present in a fermenter in an indirect way. From the three methods extensively tested, i.e. volume/weight balance, oxygen consumption and conductivity measurement, the latter proved to be the simplest and easiest to handle when the roots were in good condition.<p>In chapter five, the sensitivity of the hairy roots to organic solvents is described. The results were comparable to those obtained with the <em>Tagetes minuta</em> cell suspensions. An interesting phenomenon was observed with respect to the excretion of thiophenes. In one-phase cultures with only aqueous medium, the excretion is always practically zero. In the presence of some of the solvents tested, e.g. hexadecane, part of the thiophenes produced were excreted into the solvent. When experiments were performed with these two-phase cultures in bioreactors with volumes up to 6 litres, the excretion could rise to about 70% of the total thiophenes produced. Obviously, this is an important result, because it can facilitate the downstream processing of the thiophenes to a great extent. Of the two types of bioreactors tested, the stirred tank reactor and the bubble column, the latter gave the best results for this system.<p>In chapter six, similar experiments are described in aqueous two-phase systems. Again, the hairy roots were able to grow and to produce in the aqueous two-phase systems tested, i.e. in shake flasks and bioreactors. In this case, the excretion was stimulated up to 10% of the total thiophene production. In chapter seven, the results are presented of elicitation studies with the <em>Tagetes patula</em> hairy root cultures. The biotic elicitors applied were bacteria, yeast and fungi, while abiotic elicitors were used in the form of extreme pH, Ca <sup>2+</SUP>addition and the addition of Ethephon, a precursor of ethene. Nearly all elicitor treatments resulted in a higher thiophene production. The highest increase was obtained when the pH was adjusted to 12 (280% of the control production), but the cells were no longer viable after this treatment. The elicitation with extracts of <em>Aspergillus niger</em> led to a production that raised up to 185% of the control, without loss of viability.<p>In chapter eight, the combined effects of elicitor treatment and of the use of a solid second phase, XAD-7, is described. Here, the excretion was affected by the addition of the resin, while growth and total production were not influenced, either in the presence or the absence of elicitor and/or XAD.<br/>Finally, in the general discussion, the obtained results are shortly evaluated.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Awarding Institution
    Supervisors/Advisors
    • Tramper, J., Promotor, External person
    Award date13 Dec 1991
    Place of PublicationS.l.
    Publisher
    Publication statusPublished - 1991

    Keywords

    • asteraceae
    • tissue culture
    • cell culture
    • meristems
    • biochemistry
    • metabolism
    • secondary metabolites
    • tagetes

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