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Protocols in ecological and environmental plant physiology


Article << Previous     |     Next >>   Contents Vol 34(6)

Downregulation of neutral invertase activity in sugarcane cell suspension cultures leads to a reduction in respiration and growth and an increase in sucrose accumulation

Debra Rossouw A, Sue Bosch A, Jens Kossmann A, Frederik C. Botha A B, Jan-Hendrik Groenewald A C

A Institute for Plant Biotechnology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa.
B South African Sugarcane Research Institute, Private Bag X02, Mount Edgecombe 4300, South Africa.
C Corresponding author. Email: jhgr@sun.ac.za
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Suspension cultures were used as a model system to investigate sucrose metabolism in four sugarcane (Saccharum spp. interspecific hybrids) cell lines transformed with antisense neutral invertase (NI) constructs. Throughout a 14-day growth cycle two cell lines in which the antisense sequence was under the control of a tandem CaMV-35S: maize ubiquitin promoter showed a strong reduction in NI activity, as well as reduced hexose and increased sucrose concentrations in comparison to the control line. In lines where the antisense NI sequence was under the control of the weaker CaMV-35S promoter alone, changes in enzyme activity and sugar concentrations were intermediate to those of the more strongly inhibited lines and the control. In comparison to the control line, a higher sucrose to hexose ratio, i.e. increased purity, was obtained in all the lines with reduced NI activity. The in vivo rate of sucrose hydrolysis was reduced in the transgenic lines, suggesting a concomitant reduction in the flux through the ‘futile cycle’ of sucrose breakdown and re-synthesis. Differences between the transgenic cultures and the control were most pronounced during the early stages of the growth cycle and tapered off as the cultures matured. The transgenic cultures displayed impaired growth characteristics suggesting that the growth rate of these cells was retarded because of the reduced availability of hexoses for respiration.

Keywords: antisense, sucrose metabolism.

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