AbstractThis project provides for the first time an in depth examination of oxidative stress and antioxidant protection in plant tissue cultures undergoing various stages of ageing and neoplastic progression towards a cancerous state. Two experimental systems were investigated: (1) Beta Vulgaris cultures comprising two fully habituated (cancerous), lines, one partially abituated line and a non-habituated cell line; (2) Glycine max cultures comprised of two similar aged (15 years) cell lines, one of which has lost the ability to produce pigments. Biochemical profiles of free radical activity and the sequential stages of lipid peroxidation were constructed, together with parallel assessments of primary antioxidants (catalase, peroxidase and superoxidase dismutase and the glutathione-ascorbate couple).
The main findings demonstrated that fully habituated cell lines of B. vulgaris , possessed activities and very low levels of primary antioxidants, that were on occasion at the assay limits of detection. In the partially habituated and nonhabituated cell lines antioxidant levels were low but not to the same extent. The redox status of the cultures decreased as abituation increased and the lowest GSH/GSSG redox ratio was observed in the fully habituated cell lines. Markers of lipid peroxidation increased inversely to decreased antioxidant protection; all B. vulgaris cultures had abnormal cytological manifesting morphological characteristics symptomatic of disturbed metabolism, oxidative stress and loss of totipotency.
In vitro aged callus cultures of G. max maintained in tissue culture for approximately fifteen years had lost their embryogenic capacity and one of the cell lines has lost the ability to produce pigments. Activities and levels of key antioxidants in these cell lines were greatly diminished and considerable disruption of the GSH/GSSG ratio concomitant with increased markers of lipid peroxidation was observed.
This study clearly demonstrates that ageing and habituation implying neoplastic progression to a cancerous state involves oxidative stress concomitant with a major and deleterious disturbance in antioxidant protection. The extent to which oxidative stress occurred increased with the age and the degree of neoplastic progression, such that the partially habituated cell line, was not demonstrably different to the normal B. vulgaris cell line. The loss of antioxidant protection associated with ageing and habituation involved the compromise of key antioxidant enzymes (catalase, peroxidase and Cu, Zn-SOD) and GSH/GSSG ratios decreased with age and the degree of habituation. This study concludes with an exploration of the role of oxidative stress in in vitro ageing and habituation and considers the importance of maintaining antioxidant status in tissue cultures used in plant biotechnology programmes, and emphasises the use of oxidative stress studies to help understand and ameliorate in vitro recalcitrance, the loss of totipotency and somaclonal variation.
|Date of Award||Jan 2006|
|Sponsors||Northwood Charitable Trust|
|Supervisor||David H. Bremner (Supervisor)|