Advancing recombination towards precision breeding in plants

Abstract

During the stable genetic transformation of plants, introduced DNA predominantly inserts into random locations in the genome. The incidence of endogenous genes being substituted with DNA molecules of homologous sequence (Homologous Recombination, HR) is rare in plants compared to the incidence in certain lower eukaryotes. This thesis is concerned with elevating HR frequencies in higher plants by altering the enzymes involved in recombination, thereby enabling targeted gene replacement applications. Dual-LuciferaseR expression assays in Nicotiana leaves were used to screen candidate genes and chemicals for their effects on recombination frequencies. Measurements of Iuciferase activity, normalised against transient infiltration, were used to estimate recombination frequencies. Levels of non-HR were estimated using a LUC gene as a trap for; (1) integration near transcriptionally active genomic promoters, and (2) activation of an extra-chromosomal T-DNA construct in planta via end-joining. Levels of HR were estimated using a construct that required recombination within a T-DNA to obtain LUC activity. Experiments have shown that a chemical poly (ADP-ribose) polymerase (PARP) inhibitor (3-MB) decreased non-HR signal when infiltrated 1 day after transformation. The over-expression of genes that code for enzymes involved in recombination processes in Yeast and/or Arabidopsis produced reproducible enhancement of relative LUC activity, indicating a possible increase in HR. The induction of double-strand DNA breaks by treating DNA with endonucleases also appeared to enhance HR. The preliminary testing of these candidates is described using a novel strategy for the chromosomal gene replacement of an elongation factor l-alpha (EF-Ioi) gene in Arabidopsis plants. This strategy directs recombination to the 5' untranslated region (5’ UTR) intron, and uses selective enrichment, PCR and DNA sequence analysis to detect EF-Ia gene-replaced plants. This research suggests ways that the frequencies of HR can be increased in both research and commercial applications.