Isoflavonoids are a class of plant secondary metabolites which have multiple biological roles in plants as pest feeding deterrents, phytoalexins and signals to rhizobial microbes. Some isoflavonoids, or their breakdown products, are estrogenic when ingested by animals, and pastures with high levels of the isoflavonoid formononetin can cause sterility in ewes. White clover has low levels of isoflavonoids and is susceptible to pests like the clover root weevil. The overall aim of this project was to test whether isoflavonoids could be manipulated in white clover through metabolic engineering. The genes of the key isoflavonoid biosynthesis enzymes have been cloned from a range of legumes and three major genes, chalone reductase (CHR), isoflavone synthase (IFS) and isoflavonoid O-methyltransferase (IOMT), were cloned from white clover in this study. The white clover IFS2_12 gene was expressed in transgenic tobacco. Genistein, an isoflavonoid that is not naturally present in tobacco, was detected in the IFS-expressing tobacco, thus confirming the functionality of the IFS2_12 gene. Tobacco plants were transformed with ANT1, a transcription factor that induces the production of anthocyanins that share precursors with the isoflavonoid biosynthesis pathway. When IFS was expressed in red tobacco leaves, where anthocyanin biosynthesis was occurring, the levels of genistein were greater than in anthocyanin-free green leaves. White clover was transformed to overexpress the cloned IFS2_12 gene and some transformants had greater levels of IFS gene expression, up to 12.9 times the average wild type level. However, these transformants did not produce formononetin levels greater than the wild-type. A gene fusion of alfalfa chalcone isomerase (CHI), which produces the precursors naringenin and liquiritigenin, and soybean IFS, which converts the precursors to genistein and daidzein, respectively, was received from the Noble Foundation. Transgenic white clover plants expressing IFS/CHI were produced using a novel method that also regenerated wild-type clones of the transgenic plants. When compared with their wild-type clones, two IFS/CHI transformants produced higher levels of formononetin, thus supporting the suggestion that isoflavonoid levels can be increased in white clover through overexpression of isoflavonoid biosynthesis genes.

Isoflavone synthase (IFS; CYP93C) plays a key role in the biosynthesis of the plant secondary metabolites, isoflavonoids. These phenolic compounds, which are well-known for their multiple biological effects, are produced mostly in leguminous plants (family Fabaceae). However, at least 225 of them have also been described in 59 other families, without any knowledge of orthologues to hitherto known IFS genes from legumes (with the single exception of sugar beet - Beta vulgaris, from the family Chenopodiaceae). In view of these facts, this masters thesis has focused on two main objectives: (1) to identify isoflavone synthase genes in selected leguminous and non-leguminous plants exploiting the PCR strategy with degenerate and non-degenerate primers, and (2) to find a system for the verification of the correct function of these genes. Our methodology for the identification of IFS orthologues was successfully demonstrated in the case of two examined legumes - Phaseolus vulgaris L. and Pachyrhizus tuberosus (Lam.) Spreng, in the genomic DNA of which the complete IFS sequences have been newly identified. To design a procedure for ascertaining the correct function of these genes and others once they have been completely described, a pilot study with IFS from Pisum sativum L. (CYP93C18; GenBank number...