Promoter analysis in transgenic sugar beet
Crop improvement by gene manipulation will often require subtle regulation of expression of foreign genes in the transgenic plants. A strategy has been defined for production of high sucrose yield ! high juice purity / low environmental impact varieties of sugar beet. This requires modification of the phytohormone levels in the outer cambial rings of the storage roots. Control at the transcriptional level should provide the most efficient system for gene regulation since it will prevent the loss of energy involved in making unnecessary mRNAs. The hairy root transformation system was used in combination with the Escherichia coli derived uidA reporter gene which encodes P-D-glucuronidase (GUS) to examine promoters in transgenic sugar beet. GUS activities were measured in eight replicates of each of eight sugar beet hairy root clones which had been obtained after inoculation of material grown from a single seed with a CaMV 35S promoted uidA transcriptional fusion. Tukey test analysis showed that 19/28 and 16/28 inter-clone paired comparisons were significantly different when normalized for protein concentration and DNA concentration respectively. Plant transformation binary vectors with the Agrobacterium mannopine synthase {mas') and nopaline synthase {nos) promoters fused to the uidA coding region were constructed. GUS activities measured for eight replicates of each of eight clones transformed with the uidA coding region driven by the mas, CaMV 35S, nos and rolC promoters gave a ratio of 41:11:3:1 respectively. Histochemical localization of GUS showed that the mas, CaMV 35S and rolC promoters all caused expression in vascular tissue while expression in the apical meristem was also found with the CaMV 35S promoter construct. The hairy root/reporter gene system used was shown to be suitable for testing promoters in transgenic sugar beet.
Networked Digital Library of Theses and Dissertations