Among the economically important agronomic traits, the fatty acid (FA) composition of oilseed crops is one of the most likely to be modified by molecular breeding. In this proposal, we described experiments that will enable us to engineer a canola (rapeseed) oil with a lower content of linolenic acid (ALA), an undesirable component that, in addition to being of lower nutritional quality, causes rancidity and flavor instability due to its tendency to auto-oxidize. To effect this change the gene responsible for the formation of ALA needs to be isolated. However, since the gene encodes a microsomal enzyme that is difficult to purify, an alternative to the conventional biochemical approach to gene isolation must be sought. We intend to use a powerful genetic approach, transposon tagging, to isolate the corresponding gene. The small model plant Arabidopsis thaliana is a genetically simpler relative of rapeseed. We propose to isolate the fad3 gene, responsible for the formation of ALA in Arabidopsis, as a first step towards the modification of the FA composition in canola. We will use a streptomycin resistance assay (SPT) to visually detect transposition of the maize transposon Activator (Ac) in Arabidopsis. Somatic excision of Ac from an SPT:: Ac gene produces green spots on the white cotyledons of seedlings germinated on streptomycin and germinal excision results in green revertant seedlings in the same assay. Many of the green revertants carry a transposed Ac element. There, the SPT:: Ac system allows us to for germinal transposition events. By selecting green revertants from among the progeny of plants that carry an SPT:: Ac gene only 5 cM from fad3, we should be able to identify tagged fad3 mutations and to clone them by homology to Ac. The analogous gene from canola can be isolated by homology to the Arabidopsis gene and subsequently engineered to suppress the action of the endogenous fad3 genes when reintroduced into canola.
