Manipulation of seed oil content to produce industrial crops

Abstract Three examples are presented of the modification of seed oil quality by molecular genetics, in order to create potentially valuable industrial feedstocks. (a) Cloning of the desaturase gene responsible for the formation of petroselinic acid from the spice plant Coriander has allowed for the insertion of this gene into rapeseed, in an attempt to obtain transgenic rapeseed plants with a high seed petroselinic acid phenotype. The possibility that additional genes other than this desaturase may be required for petroselinic acid formation is discussed. (b) Attempts to achieve very high erucic acid phenotypes in rapeseed are concentrated around cloning an acyltransferase gene from meadowfoam. The transfer of this gene into rapeseed may result in the accumulation of a very high erucic acid seed oil, in contrast to current high erucic varieties which only contain between 45% and 55% of this useful industrial feedstock. (c) Attempts to clone the hydroxylase gene responsible for the accumulation of ricinoleic acid in castorbean are described. A biochemical strategy based upon solubilisation and purification of the hydroxylase protein was unsuccessful due to the loss of hydroxylase activity in the presence of detergents. A differential screening approach was therefore adopted. Antibodies were raised against microsomal membrane fractions from castorbean seeds at a late stage of development corresponding to their maximum oleate hydroxylase activity. The purified IgG fraction was then incubated in the presence of microsomal membrane fractions from either very young or mature castorbean seeds which had no oleate hydroxylase activity. This resulted in the isolation of a population of castorbean IgGs specific for microsomal membrane proteins which were only present at the stage of maximum oleate hydroxylase activity. The IgG fraction was then used to screen a λ-ZAP cDNA expression library prepared from poly A + -enriched RNA, extracted from seeds at the late developmental stage. From these antibody screens, 43 positive cDNA clones were isolated and sequenced from the 5′ end to an average of 250 bases each. All but two of the cDNA clones were eliminated due to (a) sequence similarity with previously known proteins or (b) inappropriate expression patterns of the corresponding mRNAs following Northern blot analysis. Potential sequence motifs in the oleate hydroxylase, which may be useful for the isolation or identification of candidate hydroxylase cDNA clones, such as the two clones isolated in this study, are discussed.