CHAPTER 1

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one sucrose molecule to another to produce the trisaccharide 1-kestose. ... scolymus geen wat vir die ensiem sukrose: sukrose 1-fruktosieltransferase (1- SST; ...

CARBON TURNOVER AND SUCROSE METABOLISM IN THE CULM OF TRANSGENIC SUGARCANE PRODUCING 1-KESTOSE

Tarryn Louise Nicholson

Thesis presented in fulfillment of the requirements for the degree of Master of Science at the University of Stellenbosch

Supervisors: Professor F.C. Botha and Professor B.I Huckett December 2007

Declaration I, the undersigned, hereby declare that the work contained in this thesis is my own original work and that I have not previously, in its entirety or in part, submitted it at any university for a degree.

……………………… T.L. Nicholson December 2007

Copyright © 2007 Stellenbosch University All rights reserved ii

ABSTRACT Carbon partitioning was investigated in sugarcane (Saccharum spp. hybrids) that was genetically modified with sucrose: sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) from Cynara scolymus. This enzyme catalyses the transfer of a fructosyl moiety from one sucrose molecule to another to produce the trisaccharide 1-kestose. Molecular characterisation of four sugarcane lines, regenerated after transformation, confirmed that two lines (2153 and 2121) were transgenic, with at least one intact copy of 1-SST present in line 2153, and a minimum of five copies (or portions thereof) present in line 2121. The novel gene was successfully transcribed and translated in both lines, as confirmed by cDNA gel blot hybridisation and HPLC analysis respectively. Kestose production was stable under field resembling conditions and levels of this trisaccharide progressively increased with increasing internodal maturity from 7.94 ± 2.96 nmol.g-1 fresh mass (fm) in internode 6 to 112.01 ± 17.42 nmol.g-1 fm in internode 16 of 2153, and by 1.05 ± 0.93 nmol.g-1 fm from the youngest to the oldest internode in line 2121. Sugarcane line 2153 contained 100 times more 1-kestose than 2121 in the oldest sampled internode hence the lines were referred to as high- and low-1-kestose producers. The production of 1-kestose did not reduce sucrose levels in the transgenics, instead they contained significantly higher levels of sucrose than the control line NCo310 (p