Thesis (M.S.)--University of Wisconsin--Madison, 1967. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis--Copenhagen. / Published also without thesis note. "Résumé" (in Danish): p. [311]-315. Bibliography: p. [273]-305.

The popular use of in vitro systems for toxicity studies has increased dramatically over the past decade. Among the in vitro systems used, primary hepatocyte cultures are the most widely employed. However, in addition to being difficult to obtain and maintain in culture, the functional heterogeneity of liver is absent. Primary organ cultures of thin liver slices should overcome these limitations but the lack of a reproducible method for the rapid preparation of thin, consistent slices, combined with the difficulty in maintaining adult liver tissue in culture, has hindered their use for in vitro hepatotoxicity studies. Using a recently-developed tissue slicer, thin, consistent liver slices were prepared rapidly under minimally traumatic conditions. Subsequent culture of these slices in a novel dynamic organ culture system (DOCS) resulted in a maintenance of hepatocyte functional integrity. Slice adenosine triphosphate (ATP) and K⁺ content were maintained at in vivo levels, following an initial recovery period (2-4h) for up to 20h. Protein synthesis and secretion were linear for 20h and 16h respectively. Slices also synthesized glycogen between 4 and 12h in culture and were hormonally-responsive during the 20h culture period as demonstrated by a two-fold stimulation of glucose production by glucagon (10⁻⁷ M). Bromobenzene and allyl alcohol hepatotoxicity were studied in this system of organ culture. The slices retained their biotransformation ability for at least 6h based on maintenance of cytochrome P-450 content and O-deethylase activity. Either compound caused dose (.01-1.0 mM) and time (0-6h) dependent cytotoxicity as indicated by the loss of slice K⁺, inhibition of protein synthesis and leakage of lactate dehydrogenase (LDH). By 2h, a significant (p < 0.05) inhibition of protein synthesis was observed in allyl alcohol (.05 mM) treated slices. At 4h and 6h, significant loss of slice K('+), LDH, and inhibition of protein synthesis were evident in slices exposed to allyl alcohol (0.25 mM) or bromobenzene (0.5 mM). This toxicity was blocked by co-treatment with pyrazole (1.0 mM) or SKF 525-A (100 μM) in slices exposed to allyl alcohol or bromobenzene, respectively. Therefore, this system provides a new tool for the in vitro study of hepatotoxicity under conditions where hepatocellular functional integrity and biotransformation are maintained.

The objective of the present study was to develop a comprehensive and reproducible regeneration system for spinach (Spinacia oleracea L. ) from commercially important cultivars and to assess the potential use of spinach for Agrobacterium tumefaciensmediated transformation. Tissue cultures of spinach were initiated from seed material. Axenic shoot cultures of spinach were established on MS-based medium containing 1.0 VM NAA at a temperature of 15°C and under a 16 h photoperiod. These three parameters were found most suitable for the establishment of shoot cultures and the encouragement of axillary shoot growth. Attempts to enhance axillary shoot production of spinach were investigated by the use of a double phase culture system, employing semi-solid and liquid culture media. The application of liquid medium was feasable with a volume of 5 ml for a duration of 7 or 14 d or with a volume of 10 ml for a duration of 7 d, but the multiplication rate of spinach was not increased. Adventitious shoot production was initiated from cultured spinach root explants derived from axenic shoots or hypocotyl explants. Sections from root tips and middle segments exhibited the highest shoot regeneration capacity when cultured on Nitsch and Nitsch (1969) medium supplemented with 20 μM NAA and 5.0 QCM GA3. Histological analysis demonstrated that the regenerating shoots originated directly from the root explants. Adventitious shoots were rooted on MS-based medium containing 1.0 μM NAA and transferred to the glasshouse, where the plants were grown to maturity. Seeds collected from regenerated plants were 95 % viable, producing a homgenous, fertile Rl-generation. Flow cytometric analysis was used to determine ploidy levels of regenerated plants and their progenies and showed that spinach leaf tissue from all generations displayed an even proportion of Go/G1 cells and G2/M cells, which may be characteristic for this species. Transformation studies using in vitro derived spinach explants demonstrated a positive response using two strains of Agrobacterium tumefaciens. The highest transformation rate was achieved with 25 % of explants being GUS-positive, therefore confirming susceptibility of spinach to the binary vector containing both T-DNA border sequences. It was found that best results were obtained with root explants which had been incubated for 8 weeks prior to co-cultivation with Agrobacterium and in vitro material which had been maintained in culture for up to 2 years. This reproducible regeneration system for spinach and the demonstration that spinach is amenable to Agrobacterium-mediated transformation provides the basis for potential commercial application within spinach breeding, aiming to generate an improved crop plant.

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This thesis describes the isolation, immobilisation and permeabilisation of <i>Digitalis lanata</i> (foxglove) and <i>Nicotiana tabacum</i> (tobacco) plant cells and protoplasts. Protoplasts were isolated from leaves of <i>Digitalis lanata</i> and <i>Nicotiana tabacum</i> and cultured in conditions of varying osmotic potential, illumination and cell density in order to achieve maximum cell stability. The regeneration of cellulose cell walls in <i>Nicotiana tabacum</i> protoplasts could be inhibited by the addition of dilute concentrations of the herbicide 2,6-Dichlorobenzonitrile. At concentrations of 2-10 mg/l in culture medium cell wall regeneration could be prevented for up to 6-8 weeks. Isolated cells and protoplasts were stabilised by entrapment within a beaded agarose support matrix. <i>Nicotiana tabacum</i> protoplasts and cells, immobilised within an agarose beaded support matrix were used to develop a technique to permeabilise and destabilise the cell membranes. Cells or protoplasts pre-loaded with 6-Carboxy-Fluorescein, Neutral Red stains and <SUP>14</SUP>C-Sucrose or <SUP>86</SUP>Rb<SUP></SUP>+ radioactive tracers were employed as markers for cell permeability or leakiness. Efflux or Compartmental analysis was used to determine the influence of various selected permeabilising agents on the integrity and the leakiness of either protoplast or cell membranes. Immobilised protoplasts or cells were subjected to a three - step procedure involving initial loading with <SUP>86</SUP>Rb<SUP></SUP>+ tracer, a membrane permeabilisation step and finally a recovery step. Protoplast membrane stability could not be regained after the recovery step, following a 30 minute period of permeabilisation with 50 mM acetic acid in culture medium. Immobilised cells could, however, regain membrane integrity with good intracellular retention of <SUP>86</SUP>Rb<SUP></SUP>+ tracer ion during the efflux experiment. Thus, immobilised <i>Nicotiana tabacum</i> cells could be made reversibly permeable with the use of specific agents. It is anticipated that such techniques which encourage reversible permeabilisation of immobilised cells have potential in larger scale plant cell culture systems to effect the release of intracellularly stored, useful secondary metabolites. There are also possibilities for the inclusion of exogenous precursors, cofactors and foreign genetic material which would increase compound yield and may produce novel secondary metabolites.

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