Rachael Scarth

Discrimination among cultivars of rapeseed (Brassica napus L.) using DNA polymorphisms amplified from arbitrary primers.

RAPDs (Randomly Amplified Polymorphic DNAs) were used to discriminate among 23 cultivars of oilseed rape (Brassica napus) selected from several breeding programs. A set of 100 random sequence 10-mer primers were tested, of which 70 produced bands and 22 showed evidence of polymorphism. A selection of six primers produced 23 polymorphic bands of between 300 to 2200 base pairs in size, sufficient to distinguish between the cultivars. An analysis of seed of five cultivars obtained from four different sites showed stability of banding pattern over source of seed. The analysis was repeated using four different thermocyclers, each of which produced the same band pattern. UPGMA cluster analysis indicates that the relationships among some of the cultivars is closer for those from the same breeding program than for those from different programs. The results of this study show that RAPDs can be used as a method of identification for oilseed rape cultivars.

Altering Fatty Acid Composition in Oil Seed Crops

World consumption of vegetable oils increased steadily in the last decade, from 62.6 million metric tons (MMT) in 1993 to 87.8 MMT in 2000 (Goblitz, 2002). This demand has been primarily due to increased use of edible oils in food preparation. Yet, vegetable oils are being used in many industrial products including fuels. Part of this has resulted from alteration of the fatty acid composition of vegetables oils making them more versatile in their uses. The four major oilseed crops are soybean (Glycine max L. Merr.), sunflower (Helianthus annuus L.), rapeseed (Brassica), and peanut (Arachis hypogaea L.). The seed oil of these has been genetically altered through standard plant breeding methodology and molecular genetic engineering. The following is a review of recent developments in the genetic manipulation of these crop plants to change seed oil quality.

Stereospecific analyses of seed triacylglycerols from high-erucic acid brassicaceae: Detection of erucic acid at thesn-2 position inBrassica oleracea L. Genotypes

Stereospecific analyses of triacylglycerols from selected high-erucic acid breeding lines or cultivars ofBrassica napus L. andB. oleracea L. have been performed. Initial lipase screening revealed that while allB. napus lines contained little or no erucic acid at thesn-2 position, several of theB. oleracea lines had significant proportions of erucic acid at this position. Detailed stereospecific analyses were performed on the triacylglycerols from these lines by using a Grignard-based deacylation, conversion of thesn-1,sn-2 andsn-3 monoacylglycerols to their di-dinitrophenyl urethane (DNPU) derivatives, resolution of the di-DNPU-monoacylglycerols (MAGs) by high-performance liquid chromatography on a chiral column, transmethylation of eachsn-di-DNPU MAG fraction and analysis of the resulting fatty acid methyl esters by gas chromatography. The findings unequivocally demonstrate for the first time that, within the Brassicaceae, there existsB. oleracea germplasm containing seed oils with substantial erucic acid (30–35 mol%) at thesn-2 position. This has important implications for biotechnology and breeding efforts designed to increase the levels of erucic acid in rapeseed beyond 66 mol% to supply strategic industrial feedstocks. In the first instance, the germplasm will be of direct use in retrieving a gene encoding aBrassica lyso-phosphatidic acid acyltransferase with an affinity for erucoyl-CoA. In a breeding program, the germplasm offers promise for the introduction of this trait intoB. napus by interspecific hybridization and embryo rescue.

Proportions of C18∶1n−7 and C18∶1n−9 fatty acids in canola seedcoat surface and internal lipids

Lipids of canola seedcoats (Brassica napus L. andB. rapa L.) were prepared by surface washing and by complete extraction of seed coats with toluene. The major fatty acyl-containing triacylglycerols, wax esters and free fatty acids were separated by thin-layer chromatography prior to transesterification and analysis by gas-liquid chromatography. The proportion of C18∶1n−7 to C18∶1n−9 was higher in the extracted lipids than in the surface-washed lipids for all three classes.

Characterization of chlorophyll pigments in ripening canola seed (Brassica napus)

This study characterizes the chlorophyll pigments in ripeningBrassica napus seed. Seed samples, collected weekly as the crop ripened, were analyzed by high-performance liquid chromatography to characterize chlorophyll pigment composition. Chlorophyll A, chlorophyll B, pheophytin A and pheophytin B were the predominant pigments, while pheophorbide A, methylpheophorbide A and pyropheophytin A were minor components. No differences in pigment composition were observed between the three cultivars tested or between early and late seeding dates. There were differences in pigment composition between the two years of the study, which may result either from seed aging during storage or from environmental influences. Pigment composition was dependent on seed maturity, with physiologically mature green seeds containing both chlorophylls and pheophytins, but fully mature seeds containing only chlorophylls. Pheophytins and the minor components appeared transiently, presumably formed from the chlorophylls and subsequently degraded. The ratio of chlorophyll A/B increased during seed ripening, with fully mature canola seed having a chlorophyll A/B ratio twice that of physiologically mature green seed. The “B” derivatives degraded faster than the “A” derivatives, suggesting enzymatic reactions. The initial steps in the chlorophyll breakdown pathway in canola seed appear to be:

Effects of processing and storage on chlorophyll derivatives in commercially extracted canola oil

This study characterizes the chlorophyll pigments present in canola oil immediately after commercial extraction and following oil storage to determine the best storage conditions for analytical samples and to examine the changes that chlorophyll derivatives undergo during oil processing and storage. Samples of pressed, solvent-extracted, crude and degummed canola oils, obtained from a commercial crushing plant, were stored for one month under four different conditions—in the freezer, in a refrigerator and at room temperature both in the light and in the dark. Chlorophyll derivatives (chlorophylls, pheophytins, pyropheophytins) were measured by high-performance liquid chromatography immediately after sampling and then on a weekly basis. The main pigments present in commercially extracted canola oil were pheophytin a, pyropheophytin a, chlorophyll a and chlorophyll b. The “a” derivatives comprised 81 to 100% of total chlorophyll pigments in the fresh oil samples. During degumming, the remaining chlorophylls were converted to pheophytins and pyropheophytins. During oil storage, exposure to light at room temperature affected the composition of chlorophyll derivatives as chlorophyll b was converted to pheophytin b and chlorophyll a was converted first to pheophytin a, then to pyropheophytin a.

Interspecific hybridization between diploid Fagopyrum esculentum and tetraploid F. homotropicum

The wild diploid species Fagopyrum homotropicum (2n = 2x = 16) has been used for buckwheat improvement, but, prior to this study, the tetraploid form (2n = 4x = 32) had not been hybridized with the cultivated species F. esculentum. The objective of this study was to hybridize F. esculentum with tetraploid F. homotropicum to increase the genetic variability. Forty-one interspecific F1 hybrids were obtained through ovule rescue in vitro, with hybridity confirmed using morphological characters, chromosome numbers (2n = 3x = 24) and DNA analysis. The F1 plants were mainly sterile. However, seven seeds were set spontaneously on two hybrid plants, and a large number of seeds were obtained after colchicine treatment. The F2 plants were divided into two groups based on chromosome numbers and morphology. The first group was hexaploid plants (2n = 6x = 48) or hypohexaploid plants (2n = 44–46), partially fertile with “gigas” features including increased height, dark green leaves, and large seeds with thick seed hull...

Genotypic and environmental effects on saturated fatty acid concentration of canola grown in Manitoba

The low saturated fatty acid concentration of canola oil relative to other vegetable oils has resulted in a favourable market share for canola. Understanding the effects of genotype and environment on saturated fatty acid concentration will facilitate Brassica napus breeding efforts aimed at maintaining or reducing saturate levels in the seed oil. Canola-quality B. napus samples from the Manitoba Crop Variety Evaluation Team (MCVET) trials in 1999, 2000 and 2001 were tested for fatty acid composition, oil concentration and protein concentration. Weather data were obtained from nearby weather stations. The majority of the variation in total saturates for the cultivars studied was attributed to variation in palmitic acid (C16:0) due to the genotype main effect and variation in stearic acid (C18:0) due to the genotype and environment main effects. The variation due to the genotype × environment interaction was small relative to the main effects for the individual saturated fatty acids. C18:0 and arachidic (C...

A comparison of high-performance liquid chromatography and spectrophotometry to measure chlorophyll in canola seed and oil

There are several methods available to measure chlorophyll in canola oil and seed, and these will not necessarily yield the same results and should not be used in terchangeably. Total chlorophyll was determined for samples of canola seed and commercial canola oil by recognized spectrophotometric methods and by high-performance liquid chromatography (HPLC). The HPLC method, which summed all chlorophyll-related pigments detected, found approximately 1.4 times more total chlorophyll per sample than did the spectrophotometric methods. The spectrophotometric methods are calibrated with only chlorophyll a and underestimate other chlorophyll pigments, which have lower extinction, coefficients and different absorption maxima. The HPLC method detects each pigment at its absorption maxima and applies the appropriate absorptivity factor. Care must be taken when comparing results obtained by different methods. There appears to be a need for a standardized method of chlorophyll pigment measurement by HPLC.

The effect of freezing on the analysis of chlorophyll content of canola seed (Brassica napus L.)

High levels of chlorophyll in harvested canola seed result in loss of revenue to producers and problems for processors. Studies on chlorophyll degradation often require plant material to be stored for some time prior to measuring the chlorophyll content. Storage of unripe canola seed in a freezer for up to one month prior to measuring the chlorophyll content did not alter the chlorophyll level in the seed. Seeds were frozen while still in the pods as well as after removal with no change in chlorophyll content over time.