Manipulation of neutral invertase activity in sugarcane

Joubert, Debra (2006-12)

Thesis (MSc (Genetics. Institute for Plant Biotechnology))--University of Stellenbosch, 2006.


The main goal of this project was to elucidate the apparent role of neutral invertase (NI) in sucrose accumulation in sugarcane. In the first part of the study putative transgenic cell lines (transformed with antisense NI constructs) were characterised to confirm the stable integration and expression of the transgene. Batch suspension cultures were used to initiate replicate cultures of several of these transgenic lines as well as a control, and the metabolism of the cultures during a 14 day growth cycle was examined. The transgenic lines had substantially reduced levels of NI activity. While the activities of the other invertases remained unchanged, the activity of sucrose synthase (SuSy) was significantly higher in the transgenic suspension cultures relative to the control. Throughout the growth cycle, sucrose concentrations in the transgenic lines were consistently higher, and glucose and fructose concentrations lower, than the control. The transgenic cultures also exhibited a decreased growth rate in comparison to the control. Labelling studies confirmed a decrease in the in vivo rate of invertase-mediated sucrose hydrolysis in the transgenic lines, as well as indicating a decline in the partitioning of carbon to respiratory pathways in these cultures. In the second part of the study, which focussed on greenhouse-grown transgenic plants, similar results were reported. NI activity was significantly decreased, and SuSy activity increased in all of the tissues sampled. The sucrose concentration and purity were also higher in the transgenic tissues, while the in vivo sucrose hydrolysis rate was lower. Allocation of carbon to respiration was lower in the transgenic plants, suggesting that a decrease in sucrose breakdown reduces the availability of hexoses for growth and respiration. Overall, the results suggest that NI plays a key role in the control of sucrose metabolism, and that changes in the activity of this enzyme have far-reaching effects on cellular metabolism. The fact that the trends reported in the whole-plant studies parallel those of the suspension cultures confirms that suspension cultures can be used as a model system in metabolic engineering research in sugarcane. Thus the possibility now exists to analyse large numbers of transgenic lines in a quicker time frame and at a reduced cost in comparison to conventional methods.

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