Browsing by Author "Harding, RM"

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Construction of synthetic Fiji virus resistance genes for use in sugarcane : SRDC final report BS86S
(1998) Smith, GR; Handley, JA; Dale, JL; Harding, RM
Fiji disease of sugarcane, caused by Fiji disease fijivirus (FDV), is one of the most important diseases affecting sugarcane in Australia. FDV is a member of the reovirus family and has a multipartite genome consisting of ten segments of double stranded (ds)RNA ranging in size from 1.8 to 4.4 kilobasepairs (KBP). The total genome size of FDV is approximately 30 kbp. Approximately 80% of the fdv genome has now been clones and sequenced. The majority of the FDV segments characterised to date encode a single protein product, indicated by the presence of a single open reading frame (ORF). Two of the segments, 7 and 9, were found to contain two ORFs each and hence encode two proteins. The predicted functions of segments 1, 3, 5, 7, 8, 9 and 10 have been assigned based on homology to equivalent segments in related reoviruses, and\or protein expression studies. A construct containing ORF 1 from segment 9 has been prepared and used to transform the sugarcane cultivars, Q117 and Q124. Transgenic plants have been produced for each of these cultivars and are currently being prepared for challenge experiments with FDV.
Development of transformation cassettes for sugarcane : SRDC final report QT002
(2000) McMahaon, JA; Harding, RM
There are currently few gene regulation sequences (promoters/terminators) which have been shown to be effective in sugarcane. At present, the industry standard gene regulatory sequences used for genetic engineering of sugarcane are the maize polyubiquitin (ubi) promoter and the nopaline synthase ( nos) terminator. The overall aim of this project was to develop further promoter and terminator sequences for use in the generation of transgenic sugarcane by testing whether the regulatory sequences derived from banana bunchy top nanovirus (BBTV) would function in sugarcane. Initially, a number of different native BBTV promoters were assessed for expression of the green fluorescent protein (GFP) reporter gene in transient assays, and several of the BBTV promoters were found to express GFP as well as the ubi control. However, when stably transformed mature sugarcane plants were analysed, expression of GFP was almost always abolished. In an attempt to enhance expression, the BBTV promoter sequences were modified by the inclusion ofthe intron from the ubi promoter. In transient assays, the BBTV promoters performed as well as, or better than, the ubi control. When stably transformed plants were analysed by western blot, GFP expression levels equal to that of the ubi control were observed. These results indicated that (i) the inclusion of the ubi intron enhanced the activity of the BBTV-derived promoters and (ii) the promoter sequences derived from BB TV could be used to drive trans gene expression in sugarcane. The BBTV promoters were assessed for their ability to drive the selectable marker gene, neomycin phosphotransferase (nptII). Initially, several native BBTV promoters were tested but none of the transformed sugarcane callus survived selection, indicating that these promoters were unable to drive sufficient levels of nptII expression. When several intron-enhanced promoters were tested, transformed callus was able to survive the selection process, but plantlets could not be regenerated due to problems with the callus. Although preliminary, these results indicated that intron-enhanced promoter sequences from BBTV could be used to drive the nptH selectable marker gene in sugarcane. Several BBTV terminator sequences were assessed for their ability to terminate transcription in sugarcane using nos as a control. These studies revealed that the BBTV termination sequences were as effective as nos in terminating transcription in both transiently and stably transformed sugarcane, thus providing alternative termination sequences for the industry. Constructs containing the promoters from BBTV DNA-4, 5 or 6, the ubi intron and the terminator from BBTV DNA-6 have been prepared. These constructs also contain a multiple cloning site to facilitate use in a variety of systems in sugarcane. Further, two cassettes for selection of transformed tissue have also been prepared which contain the promoter from either BBTV DNA-6 or -4, the ubi intron, the NPTII selectable marker gene and the terminator from BBTV DNA-3.
Field performance of transgenic sugarcane plants carrying genes for resistance to SCMV : final report BSS154
(2000) Smith, GR; Taylor, GO; Harding, RM; Stringer, JK; Cox, MC; Yoyce, PA
The field resistance of transgenic sugarcane plants to sugarcane mosaic potyvirus was successfully demonstrated, and a number of transgenic lines are available for consideration for agronomic evaluation. Some of the transgenic lines yielded significantly more tonnes sugar per hectare in this trial, but a firm conclusion about the overall performance of the transgenic lines compared to the parental clone can not be concluded due to the limitations of this trial. These plants contain the coat protein gene of sugarcane mosaic virus and prove that pathogen-derived resistance can be engineered into a genetically complex monocot. The precise molecular basis of the resistance appears to be RNA mediated. More research is necessary to prove this as a number of the resistant lines do not exhibit the usual RNA profiles of transgenic plants from other species which are virus resistant. A second pathogen-derived resistance gene, based on the virus replicase gene, is also capable of conferring virus resistance in sugarcane.Analysis of sugar and syrup produced from transgenic cane has revealed that no genes, native or transgenic, survive the laboratory production process. There is every confidence that this result would also be found with mill produced sugar, when the opportunity to mill transgenic sugarcane eventuates. There is now good scientific evidence to contribute to the debate that sugar manufactured from transgenic sugarcane plants is indistinguishable or substantially equivalent to sugar produced from non-transgenic plants.