THE ROLE AND POTENTIAL CONTROL OF RETROTRANSPOSONS IN SOMACLONAL VARIATION OF BARLEY TRANSGENICS

Bradley Campbell (2011). THE ROLE AND POTENTIAL CONTROL OF RETROTRANSPOSONS IN SOMACLONAL VARIATION OF BARLEY TRANSGENICS PhD Thesis, School of Agriculture and Food Sciences, The University of Queensland.

       
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Author Bradley Campbell
Thesis Title THE ROLE AND POTENTIAL CONTROL OF RETROTRANSPOSONS IN SOMACLONAL VARIATION OF BARLEY TRANSGENICS
School, Centre or Institute School of Agriculture and Food Sciences
Institution The University of Queensland
Publication date 2011-12
Thesis type PhD Thesis
Supervisor Professor Ian Godwin
Dr George Piperidis
Total pages 226
Total colour pages 50
Total black and white pages 176
Language eng
Subjects 060405 Gene Expression (incl. Microarray and other genome-wide approaches)
060407 Genome Structure and Regulation
060702 Plant Cell and Molecular Biology
Abstract/Summary Barley (Hordeum vulgare) ranks as the world’s fourth major cereal crop after maize, wheat and rice, both in terms of quantity produced and in area of cultivation. It has received considerable research attention as a model for genetic analyses, and breeding programs globally are working towards improved varieties with better quality, disease-resistance and agronomic traits. Like many other food staples, the use of transgenic technology has become an important tool available for modern barley improvement programs. However, transformation technology relies upon in vitro plant tissue culture, which has been proven to generate spontaneous and heritable genetic changes termed somaclonal variation (SCV). To be of practical value, genetic engineering techniques should produce genetically elite plants with one or more novel characteristics. Extensive SCV can result in many undesirable changes in the genetic background of transgenic plants, affecting their potential as cultivars or elite parents. Many factors have been described as potentially responsible for SCV, and generally most can be classified as stress factors. Tissue culture-induced mobilisation of transposable-elements (TEs) is one cause of SCV based on the knowledge that TEs can be responsible for generating significant genomic change and are one of the driving forces of evolution. To elucidate the extent of TE participation in SCV, barley was chosen as a genetic model because of its importance and its well characterised genome. Retro-element BARE-1 (for barley retroelement 1) is part of an active family of copia-like retroelements, dispersed on all chromosomes. Hence, BARE-1 has been used as a case study in understanding the role of retro-elements in barley SCV. A retrotransposon-based marker system, inter-retrotransposon amplified polymorphism (IRAP), and inter-simple sequence repeats (ISSRs) were used to detect SCV induced by tissue culture. It was proposed that a greater frequency of polymorphisms emanating from the IRAP marker system in comparison to the ISSR marker system, would suggest that activation and transposition of BARE-1 elements, and thus retrotransposon activity, were making a significant contribution to the extent of SCV. However, there was no significant difference in the frequency of novel, non-parental bands. Cluster analysis also revealed that the level of polymorphism and genetic variability detected was comparable between IRAP and ISSR markers. The effect of transformation protocols on BARE-1 expression levels was then assessed. Treatments included osmoticum, bialaphos, kanamycin, hygromycin and cefotaxime. Anti-retroviral compounds were applied to tissue culture media to evaluate the effectiveness of reducing BARE-1 expression and, indirectly, SCV. It was observed that hygromycin B, osmoticum and cefotaxime had the greatest impact on BARE-1 up-regulation, with kanamycin and bialaphos having a low, observable impact on retrotransposon activity. The response of barley to anti-retroviral drug compounds differed according to the class of drug applied and the gene evaluated. A marked reduction in BARE-1 reverse transcriptase (RT) expression was observed in ‘Mackay’ explants treated with the nucleoside analog, reverse transcriptase inhibitor (NRTI), Zidovudine, over a period of hours. However, over a period of weeks, no discernible down-regulation of BARE-1 RT was observed and some treatments actually showed up-regulation. A transgenic approach using RNAi methodologies to silence a conserved domain of the BARE-1 RT and, indirectly, BARE-1 transposition was explored. All T0 lines tested positive for transgene sequences using PCR, but only 4 plants were according to Southern blot hybridisation. Relative expression of BARE-1 RT in T2 generation lines showed up-regulation compared to non-transgenic controls, possibly as a result of the inadvertent detection of the hairpin construct. Further investigation is required to evaluate if it is possible to down regulate or knockout BARE-1 RT and assess the level of SCV amongst transgenic progeny. Such experiments would be highly valuable in elucidating the biology of retrotransposons in cereals. Integration of IRAP markers, derived from a barley recombinant inbred (RI) line population, into a high density genetic consensus map and evaluation of their potential to be used for basic and applied genetic diversity analyses and marker-assisted selection programs was assessed. IRAP markers were shown to be distributed over all chromosomes, and significant associations with phenotypic QTLs for quality, agronomic and disease traits were found. Further evidence supporting retrotransposon activity is a substantial component of SCV was provided through the comparison between ISSR and IRAP markers to indicate the degree of genomic instability in barley explants as well as through the quantification of the BARE-1 retrotransposon family and its response to different stress inducing treatments sourced from transformation protocols. Assessment of BARE-1 activity response to anti-retroviral drug compounds provided some evidence of the efficacy of the compounds to reduce BARE-1 RT expression. As stated above, further work is required to conclusively demonstrate their capacity to reduce retrotransposon proliferation during tissue culture, as certain anti-retroviral compounds actually exacerbated BARE-1 expression above controls. Pursuit of such an intervention seems likely to be complicated by the complex interaction between retro-elements and their hosts combined with the multifarious cellular environment.
Keyword somaclonal variation
Retrotransposon
Hordeum vulgare L.
irap
Transformation
Additional Notes Colour Pages: 32, 51-52, 54, 56, 73, 77-78, 82-83, 85-86, 88-90, 92, 96-101, 107, 111-112, 114-116, 118-119, 124-125, 140, 145-146, 150, 152-153, 155, 166-167, 169-172, 182-183, 187, 189, 191-192 Landscape: 96-101, 114, 118-119, 145-146, 150, 182, 191-192

 
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Created: Fri, 29 Jun 2012, 15:21:10 EST by Mr Bradley Campbell on behalf of Library - Information Access Service