Induction and assessment of plant cell membrane permeability
This thesis describes the isolation, immobilisation and permeabilisation of Digitalis lanata (foxglove) and Nicotiana tabacum (tobacco) plant cells and protoplasts. Protoplasts were isolated from leaves of Digitalis lanata and Nicotiana tabacum 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 Nicotiana tabacum 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. Nicotiana tabacum 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 14C-Sucrose or 86Rb+ 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 86Rb+ 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 86Rb+ tracer ion during the efflux experiment. Thus, immobilised Nicotiana tabacum 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.