Anthony, Paul
(1999)
Respiratory gas carriers in plant culture systems.
PhD thesis, University of Nottingham.
Abstract
A crucial pre-requisite in genetically manipulating higher plants involves systems for culturing plant protoplasts and cells under static conditions with an adequate oxygen supply. This is especially the case for cells from cryopreservation, where respiratory perturbations are known to occur during early post-thaw recovery. Therefore, studies were undertaken to assess the potential, and actual, beneficial effects involving culture of cells at an interface between inert, oxygen-gassed perfluorocarbon (PFC) liquid overlaid with liquid or semi-solidified media supplemented with or without the non-ionic surfactant, Pluronic F-68. Assessments were also made to compare the efficacy of PFC supplementation with other physical (medium implanted with glass rods to increase the surface area available for gaseous exchange) and chemical (haemoglobin; Hb) options, both alone and in combination, for gaseous manipulation of plant protoplast cultures.
Investigations involving novel PFC-mediated oxygen delivery to cultured protoplasts were carried out on a broad range of plant species, which included Petunia hybrida (a herbaceous species) and Passiflora giberti (a woody species), as model systems, together with cassava (Manihot esculenta) a relatively recalcitrant species in tissue culture. Studies revealed enhanced protoplast initial plating efficiencies (IPEs) as measured by increased mitotic division, thereby demonstrating no short-term detrimental effects of exposure to PFC. Similarly, supplementation of culture media with Hb, at 1:50 (v/v), increased the mean IPEs of both Petunia and Passiflora protoplasts over that of untreated controls. Additionally, supplementation of aqueous medium with 0.01% (w/v) Pluronic® F-68 not only lowered interfacial tension, but further enhanced mitotic activity over that stimulated by both oxygenated PFC and Hb.
In the context of cryopreservation, media supplementation with Pluronic F-68, at 0.01-1.0% (w/v), significantly improved the post-thaw viability and growth of embryogenic suspension cells of the rice (Oryza sativa L.) cultivars Taipei 309 and Tarom, together with non-embryogenic cells of Lolium multiflorum and Moricandia arvensis. Moreover, a more pronounced synergistic effect in terms of viability and growth was observed for Taipei 309 cells when 0.01% (w/v) Pluronic® F-68 was evaluated in conjunction with oxygenated PFC. Plants regenerated from such cryopreserved cells were morphologically normal with expected chromosome complements (2n = 2x = 24), thus confirming the long-term biocompatibility of PFCs, with no adverse effect up on cellular totipotency.
These results indicate, for the first time, that both oxygenated PFC and Hb provide options for enhancing cellular oxygen supply to cultured eukaryotic cells in vitro. However, the recoverability and, hence, recyclability of PFCs make them a commercially more attractive option, despite the high initial investment cost. Overall, PFC-facilitated improvements in cell culture technology will have increasingly important biotechnological implications in the context of plant micropropagation, somatic hybridisation, transgenic plant production and commercial exploitation of these technologies.
NB. This ethesis has been created by scanning the typescript original and may contain inaccuracies. In case of difficulty, please refer to the original text.
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