Transgenic white clover for enhanced yield and performance: trait dissection, trait stacking and phenomics

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Master of Science to the Department of Botany, School of Life Science, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora, Victoria.

The public version of this thesis does not contain all material submitted for examination.

White clover (Trifolium repens L.) is one of the dominant dairy pasture legume species in temperate regions of Australia and is a key pasture legume in other temperate regions around the world. Efficient and profitable cultivation of this highly beneficial pasture crop is hampered by various abiotic and biotic stresses. Major problems associated with white clover cultivation in Australia are: rising soil acidity, aluminium toxicity, and maintenance of high biomass production in suboptimal environments. The focus of this research is to improve tolerance to soil aluminium and increase biomass production and utilization of phosphorus fertilisers through trait stacking in transgenic white clover. A construct directed at ameliorating aluminium toxicity was engineered by placing the white clover nodule enhanced malate dehydrogenase (ne MDH) coding region and terminator under the control of a root enhanced phosphate transporter (Tr PT1) promoter, also from white clover. For increased biomass an isopentenyl transferase (ipt) gene from A. tumefaciens was placed under the control of a developmentally regulated promoter from the Arabidopsis thaliana Myb32 gene and the ipt terminator. Transgenic white clover plants were developed using an Agrobacteriummediated transformation system, containing the above mentioned agronomically valuable transgenes and the selectable marker hygromycin phosphotransferase (hph). Molecular analysis of independent transgenic plants confirmed the presence of both TrPT1:TrneMDH:TrneMDH+atmyb32:ipt:ipt and atmyb32:ipt:ipt Transformation efficiency was higher with a single transgene as compared to multiple cassettes. Selected transgenic lines were analysed for delayed leaf xvii senescence and aluminium tolerance. A wide spectrum of phenotypes were observed ranging from below mean to high performance as compared to isogenic controls (wild type). A further detailed expression analysis of these plants may help explain these results. However this research provides evidence that transgene stacking in white clover is possible and can potentially help to enhance Aluminium tolerance and increased biomass production.