Browsing by Author "Smith, GR"

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Australian component Genetic diversity within Sugarcane yellow leaf virus : ICSB final report 99-15
(2001) Borg, Z; Braithwaite, KS; Smith, GR
Sugarcane yellow leaf virus (SCYLV) is widespread in many sugarcane growing countries of the world. The aim of this study was to determine the extent of genomic variation in SCYLV isolates from various countries. Isolates were obtained from Mauritius, Reunion, South Africa, Australia, Papua New Guinea, Hawaii, U.S.A. and Brazil. Four regions of the SCYLV genome (ORF 1, Replicase, Coat Protein and Readthrough) were analysed for variation using both RFLP analysis and sequence comparisons. Of the fifty isolates used, several did not produce RT-PCR amplicons in one or more of the regions covered, suggesting variation in regions where the primers bind. Of the amplicons produced across the four regions studied, RFLP analysis and sequencing results revealed the Coat Protein (CP) region to be the most conserved, followed by the Replicase (REP) region. These areas should be targeted for diagnosis of SCYLV and for use in viral mediated transgenic resistance in sugarcane varieties. Phylogenetic studies of the nucleotide and protein sequences of the four regions covered were performed using eighteen isolates. Representative isolates from each country were used where possible. In a second round of phylogenetic studies, covering the REP and CP regions, nine isolates from North, South and Central America, sequenced by our American collaborators on this project, were included. Variation in nucleotide and protein sequences revealed that many haplotypes of SCYLV exist across the countries tested. Most isolates from the various countries are very closely related across all four regions. Phylograms produced from REP and CP sequences in Phylogeny study #2 revealed that isolates from Hawaii and Australia tend to group together as do isolates from Mauritius and Reunion. Phylogenetic analyses on the sequences used in this study have not identified any significant variants of SCYLV. In conclusion, we recommend that the YLS111 and 462 RT-PCR primers originally developed by Mike Irey and colleagues continue to be used for routine SCYLV diagnosis. Also, the viral coat protein and replicase regions being the most conserved, should be targeted in research to genetically engineer resistance to SCYLV.
Canegrub resistant plants containing antimetabolic compounds : SRDC final report BSS163
(BSES, 2000) Smith, GR; Nutt, KA; Allsopp, PG
Transgenic sugarcane plants engineered to express either the potato proteinase inhibitor II or the snowdrop lectin gene show increased antibiosis to larvae of Antitrogus consanguineus in pot-based glasshouse trials.Canegrubs feeding on the transgenic line UP87, transformed with the potato gene, gained as little as 4.2% of the weight of canegrubs fed on untransformed control plants. Similarly, larvae feeding on the roots of transgenic line G87, transformed with the snowdrop gene, gained only 20.6% of the weight of grubs feeding on the non-transgenic control plants. Overall, 22% of the tested transgenic plant lines engineered with either the potato or the snowdrop constructs resulted in a statistically significant reduction in gain of weight by canegrubs feeding on roots. Weight gains of insects were compared to those of larvae feeding on the roots of either non-transgenic control plants, or non-transgenic plants regenerated after passage through the tissue culture system.Plants transformed with a proteinase inhibitor from an ornamental tobacco showed no statistical effect on the weight gain of the grubs. This result was unexpected, as the proteinase inhibitor from tobacco was predicted to be as effective as the PI from potato. Further analysis of the gene construct by sequencing established that the gene construct was faulty in contrast to the original restriction analysis that had indicated that the plasmid was correctly constructed. Protein could not be produced by the faulty construct, and this is reflected in the negligible effect on weight gain of the grubs. The plasmid was re-constructed and transformed into sugarcane. Plants containing this construct are being grown for testing.Having now established that canegrub resistance can be engineered into sugarcane, the next phase is to test the efficacy of these transgenes against other species of canegrub larvae and to further develop a commercial product.
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.
Enhancement of the sensitivity of gene probes for viral diseases of sugarcane by the polymerase chain reaction : SRDC final report BS48S
(BSES, 1994) Smith, GR; Leonard, GJ
The sensivity of gene probes for two important viral pathogens of sugarcane, sugarcane mosaic virus (SCMV) and Fiji disease virus (FDV), has been significantly enhanced by a version of the polymerase chain reaction (PCR). This version, known as RT-PCR, involves a reverse transcription (RT) step to synthesise DNA from RNA template prior to the PCR amplication. RT-PCR increased the level of sensitivity of detection by 10(3)-fold for SCMV and by up to 10(6)-fold for FDV. The RT-PCR conditions for routine detection of SCMV and FDV in sugarcane tissues were determined, but the glasshouse trial was not completed.
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.
Identification of resistance mechanisms in sugarcane to infection by Pachymetra : SRDC final report BS79S
(BSES, 1999) McGhie, TK; Maclean, D; Smith, GR; Croft, BJ
Project objectives- Develop techniques for studying the biochemical reactions of sugarcane to infection by Pachymetra.- Identify biochemical mechanisms of infection by Pachymetra.- Identify biochemical changes produced in sugarcane by infection with Pachymetra.- Compare the effect of chemical constituents of different sugarcane varieties on Pachymetra oospore germination and hyphal growth.- Determine which resistance mechanisms and responses of sugarcane are present in resistant varieties, ranked by glasshouse screening, as an aid to future breeding programs.
Maintaining access to foreign germplasm by developing methods to detect unidentified viruses in sugarcane in quarantine : SRDC final report BS116S
(1998) Braithwaite, KS; Smith, GR
Four types of gneeric tests for detecting unidentified viruses in plants were trialed for their suitability for sugarcane. The tests were; double stranded RNA (dsRNA) analysis, sap inoculation of indicator plants, sap examination by electron microscopy and polymerisation chain reaction (PCR) amplification using 'group-specific' primers. The aim of this project was to develop and implement general diagnostic tests for unidentified viruses in sugarcane germplasm held in quarantine. The availability of these general tests has provided an opportunity to significantly improve the security of sugarcane quarantine.
Meristem transformation for sugarcane genetic engineering : SRDC final report BSS209
(1999) Smith, GR; Lakshmanan, P; Elliott, AR; Grof, CPL
Production and evaluation of SCMV resistant transgenic sugarcane pests derived from transformed callus : SRDC final report BS94S
(1997) Smith, GR; Joyce, PA
We have successfully demonstrated that sugarcane can be genetically engineered for resistance to sugarcane mosaic virus, using the coat protein gene of the virus to mediate resistance. At present the frequency of plants showing moderate to high transgene-mediated resistance to viral infection is low, but the second generation of constructs with stronger promoters should address this issue. Transgenic sugarcane plants of cultivars Q95, Q153 and Q155 that contained a sugarcane mosaic coat protein gene construct under control of the Emu promoter were produced. These plants were regenerated from embryogenic callus which had been co-transformed by microprojectile transformation with the coat protein gene construct and the neomycin phosphotransferase II (NPT II) selection gene. The plants were established in the PC2 glasshouse at the Pathology Farm, Eight Mile Plains and were then mechanically inoculated with an inoculum of sugarcane mosaic virus prepared from infected sugarcane grown at the Pathology Farm. One transgenic line of Q155 did not develop mosaic symptoms over the time course of the trial, while another 6 lines of cultivars Q95, Q153 and Q155 showed a delay in the development of symptoms. Molecular analysis of these lines by PCR indicated the presence of both the NPT II and CP genes. Further analysis of selected lines by Southern blot hybridisation indicated the presence of one to three copies of the CP gene.
Selection of a non-sugarcane gene for control of canegrubs : SRDC final report BS95S
(1996) Allsopp, PG; McGhie, TK; Shepherd, K; Smith, GR
Four types of non-sugarcane genes were investigation for potential toxictiy to canegrubs. They were: toxins from the bacterium Bacillus thuringiensis (Bt); plant proteinase inhibitors; plant lectins; and avidin. Each of these was tested in an artificial diet based on the wheatgerm and casein and developed within the project.A Bt toxin with toxicity to canegrubs was not identified. The Bt isolates tested, which had activity against New Zealand grassgrub, bound to the gut lining of larvae of Antitrogus consanguineus. This binding proved to be not sufficient for toxicity.
The production and evaluation of gene probes for diagnosis of sugarcane mosaic virus and Fiji disease virus (Final report SRDC Project BS10S)
(BSES, 1991) Smith, GR; Leonard, GJ
Specific and sensitive cDNA probes have been developed for detection of both FDV and SCMV in diseased sugarcane plants. Results from glasshouse evaluation indicate that FDV probes are not sensitive enough to be used for the detection of the virus in asymptomatic diseased plants. Results are not yet available from the SCMV probe glasshouse trial.
The production of transgenic sugarcane plants : SRDC final report BS44S
(1994) Smith, GR
Transgenic sugarcane plants, which expressed the coat protein of SCMV at a very low level, were produced by micoprojectile bombardment of sugarcane meristems. Transgenic plants expressing the luciferase (Luc) and B-glucuronidase (GUS) reporter genes were also produced. A paper describing regeneration of plantlets expressing the reporter gene GUS (Plant Cell Reports 12:343-346) was the first report of the use of sugar meristem tissues as a transformation target, and the second report of transgenic sugarcane plants. The level of expression of the coat protein gene in the regenerated plantlets was very low, possibly due to chimaerism, ie. mixtures of transformed and non-transformed cells in the same tissues. We have established that sugarcane meristems are a useful target for microprojectile transformation of sugarcane, although more research is necessary before this target can be routinely used for sugarcane genetic engineering.