Jet Neuf Research Articles

Generation of stable transgenic Brassica napus cv. Jet Neuf cell cultures as a tool to investigate in planta protein function

Generation of stable transgenic Brassica napus cv. Jet Neuf cell cultures as a tool to investigate in planta protein function

In-field screening for host plant resistance to Delia radicum and Brevicoryne brassicae within selected rapeseed cultivars and new interspecific hybrids.

Rapeseed (Brassica napus) can be attacked by a wide range of pests, for example, cabbage root fly (Delia radicum) and cabbage aphid (Brevicoryne brassicae). One of the best methods of pest management is breeding for insect resistance in rapeseed. Wild genotypes of Brassicaceae and rapeseed cultivars can be used as a source of resistance. In 2017, 2018, and 2019, field trials were performed to assess the level of resistance to D. radicum and B. brassicae within 53 registered rapeseed cultivars and 31 interspecific hybrid combinations originating from the resources of the Department of Genetics and Plant Breeding of Poznań University of Life Sciences (PULS). The level of resistance varied among genotypes and years. Only one hybrid combination and two B. napus cultivars maintained high level of resistance in all tested years, i.e., B. napus cv. Jet Neuf × B. carinata – PI 649096, Galileus, and Markolo. The results of this research indicate that resistance to insects is present in Brassicaceae family and can be transferred to rapeseed cultivars. The importance of continuous improvement of rapeseed pest resistance and the search for new sources of resistance is discussed; furthermore, plans for future investigations are presented.

A rapid nile red fluorescence-based method for triacylglycerol content in microspore-derived cell suspension cultures of Brassica napus.

Brassica napus (L. cv Jet Neuf) microspore-derived (BnaMD) cell suspension cultures were used to study the biochemistry and molecular biology of plant lipid metabolism. The cell suspension cultures may also be a potentially useful trait-testing tool for further improvement of oilseed rape, the world's third largest vegetable oil crop. Nile red fluorescence methods have been used to assess neutral lipid accumulation in microorganisms and mammalian oocytes but have not been established for plant cell suspension cultures. In this study, a Nile red method was established and optimized for triacylglycerol (TAG) content analysis in BnaMD cell suspension cultures. Nile red fluorescence was easily determined in 96-well plates using a 490nm excitation wavelength and a 595nm emission wavelength without any significant interference from other cellular components. The lipid content of the cell suspension cultures at different growth stages was determined using a conventional lipid analysis method and the Nile red fluorescence method. There was a strong positive correlation between the Nile red fluorescence and TAG content in the cell suspension cultures. Therefore, the TAG content of BnaMD cell suspension cultures can be rapidly and robustly determined using a Nile red fluorescence-based method that can potentially be adapted for application to other plant cell suspension cultures.

The effect of endo- and exogenous factors on growth and maturation of oilseed rape (Brassica napus cv. Jet Neuf) siliques cultured in vitro

The effect of developmental stage of siliques, growth regulators, agar and sucrose concentrations in the medium was investigated on the morphogenesis of the silliques and seed maturation. Regeneration of shoots, roots and callus occurred at the base of siliques during their growth. The developmental stage of the incubated siliques was the most important factor affecting the number of seeds, while the conditions of culture (agar and growth regulators) constituted a secondary factor. The reasons for the insignificant number of seeds produced could be the lack of fertilization of some of the ovules, limited ability of siliques to take up nutrients and the unspecific composition of the medium.

A 10‐kDa acyl‐CoA‐binding protein (ACBP) from Brassica napus enhances acyl exchange between acyl‐CoA and phosphatidylcholine

The gene encoding a 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus was over-expressed in developing seeds of Arabidopsis thaliana. Biochemical analysis of T(2) and T(3) A. thaliana seeds revealed a significant increase in polyunsaturated fatty acids (FAs) (18:2(cisDelta9,12) and 18:3(cisDelta9,12,15)) at the expense of very long monounsaturated FA (20:1(cisDelta11)) and saturated FAs. In vitro assays demonstrated that recombinant B. napus ACBP (rBnACBP) strongly increases the formation of phosphatidylcholine (PC) in the absence of added lysophosphatidylcholine in microsomes from DeltaYOR175c yeast expressing A. thaliana lysophosphatidylcholine acyltransferase (AthLPCAT) cDNA or in microsomes from microspore-derived cell suspension cultures of B. napus L. cv. Jet Neuf. rBnACBP or bovine serum albumin (BSA) were also shown to be crucial for AthLPCAT to catalyse the transfer of acyl group from PC into acyl-CoA in vitro. These data suggest that the cytosolic 10-kDa ACBP has an effect on the equilibrium between metabolically active acyl pools (acyl-CoA and phospholipid pools) involved in FA modifications and triacylglycerol bioassembly in plants. Over-expression of ACBP during seed development may represent a useful biotechnological approach for altering the FA composition of seed oil.

Molecular modification of triacylglycerol accumulation by over-expression ofDGAT1to produce canola with increased seed oil content under field conditionsThis paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

The final step in the Kennedy pathway for seed oil synthesis is catalyzed by an acyl-CoA-dependent diacylglycerol acyltransferase, DGAT1 (EC. 2.3.1.20). We have cloned DGAT1 genes from both Arabidopsis thaliana (L.) Heynh ecotype Columbia and Brassica napus ‘Jet Neuf’ and over-expressed them in canola under the control of the seed-specific promoter, napin. DGAT1 from A. thaliana was inserted into B. napus ‘Quantum,’ whereas DGAT1 from B. napus was introduced into the B. napus double haploid breeding line DH12075. Both sets of transgenic plants exhibited increased seed oil content in both greenhouse and in field trial settings, ranging from 2.5% to 7% of dried mass on an absolute basis. The ‘Quantum’ transgenic lines were field-tested in plots at Watrous, Saskatchewan, in 2006 and 2007. Larger scale field trials of the DH12075 transgenics were carried out in 2007 at Ellerslie and Vegreville, Alberta. This is the first study wholly dedicated to DGAT1 over-expression and the resultant oil-content increases in transgenic canola under field conditions. Collectively, the field trial results strongly support the hypothesis that the level of DGAT1 activity during seed development in an oilseed crop can have a substantial effect on the flow of carbon into seed oil. Therefore, the over-expression of DGAT1 is a positive strategy for increasing oil content and cultivar performance in canola.

Molecular and phenotypic characterization of near isogenic lines at QTL for quantitative resistance to Leptosphaeria maculans in oilseed rape (Brassica napus L.)

The most common and effective way to control phoma stem canker (blackleg) caused by Leptosphaeria maculans in oilseed rape (Brassica napus) is by breeding resistant cultivars. Specific resistance genes have been identified in B. napus and related species but in some B. napus cultivars resistance is polygenic [mediated by quantitative trait loci (QTL)], postulated to be race non-specific and durable. The genetic basis of quantitative resistance in the French winter oilseed rape 'Darmor', which was derived from 'Jet Neuf', was previously examined in two genetic backgrounds. Stable QTL involved in blackleg resistance across year and genetic backgrounds were identified. In this study, near isogenic lines (NILs) were produced in the susceptible background 'Yudal' for four of these QTL using marker-assisted selection. Various strategies were used to develop new molecular markers, which were mapped in these QTL regions. These were used to characterize the length and homozygosity of the 'Darmor-bzh' introgressed segment in the NILs. Individuals from each NIL were evaluated in blackleg disease field trials and assessed for their level of stem canker in comparison to the recurrent line 'Yudal'. The effect of QTL LmA2 was clearly validated and to a lesser extent, QTL LmA9 also showed an effect on the disease level. This work provides valuable material that can be used to study the mode of action of genetic factors involved in L. maculans quantitative resistance.

Microspore-derived cell suspension cultures of oilseed rape as a system for studying gene expression

Abiotic stress, such as extreme temperature, drought, or excessive salinity, is one of the leading causes of crop loss worldwide. Microspore-derived (MD) cell suspension cultures of Brassica napus L. cv. Jet Neuf have been shown to be a useful system for studying the biochemistry of developing oilseeds. In the present study, we describe the application of MD cell suspension cultures of B. napus as a system for studying gene expression in response to abiotic stress, and demonstrate emybryogenic competence in cultures that have been continuously subcultured for more than 20 years. MD cell suspension cultures of B. napus L. cv Jet Neuf were exposed to low temperature or osmotic stress and the expression profile of known stress responsive genes was evaluated. The gene expression profile of BN115, a known cold-responsive gene in B. napus, was similar to that described for intact cold-acclimated plants. Likewise, two late embryogenesis abundant (Lea) genes were shown to be up-regulated in response to low temperature or osmotic stress. The results demonstrate that B. napus MD cell suspension cultures are a useful system for the investigation of changes in gene expression in plants brought about by abiotic stress.

Combining Ability in Some Varieties of Winter Oil Rape (Brassica Napus L.)

ABSTRACTFive winter oil rape varieties (Falcon, Banaćanka, Sremica, Jet Neuf and Samuray) have been studied for general and specific combining abilities, mode of inheritance and gene effects for the number of leaves per plant and stem diameter. The varieties were examined in a diallel set of crosses excluding reciprocals. Falcon was the best general combiner for the number of leaves per plant, Banacanka for stem diameter. Samuray was the poorest general combiner for both traits. Dominance of the better parent and heterosis were expressed in the inheritance of the number of leaves per plant. Heterosis for stem diameter was registered in one cross. Genes with additive effect predominated in the inheritance of the number of leaves per plant, genes with dominant effect in the inheritance of stem diameter. The variety Falcon may be used as a component of synthetic varieties for improvement of the number of leaves per plant, the variety Jet Neuf for improvement of stem diameter.

Properties of lysophosphatidylcholine acyltransferase from Brassica napus cultures.

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT; EC 2.3.1.23) catalyzes the acyl-CoA-dependent acylation of lysophosphatidylcholine (LPC) to produce PC and CoA. LPCAT activity may affect the incorporation of fatty acyl moieties at the sn-2 position of PC where PUFA are formed and may indirectly influence seed TAG composition. LPCAT activity in microsomes prepared from microspore-derived cell suspension cultures of oilseed rape (Brassica napus L. cv Jet Neuf) was assayed using [1-14C]acyl-CoA as the fatty acyl donor. LPCAT activity was optimal at neutral pH and 35 degrees C, and was inhibited by 50% at a BSA concentration of 3 mg mL(-1). At acyl-CoA concentrations above 20 microM, LPCAT activity was more specific for oleoyl (18:1)-CoA than stearoyl (18:0)- and palmitoyl (16:0)-CoA. Lauroyl (12:0)-CoA, however, was not an effective acyl donor. LPC species containing 12:0, 16:0, 18:0, or 18:1 as the fatty acyl moiety all served as effective acyl acceptors for LPCAT, although 12:0-LPC was somewhat less effective as a substrate at lower concentrations. The failure of LPCAT to catalyze the incorporation of a 12:0 moiety from acyl-CoA into PC is consistent with the tendency of acyltransferases to discriminate against incorporation of this fatty acyl moiety at the sn-2 position of TAG from the seed oil of transgenic B. napus expressing a medium-chain thioesterase.

Sucrose-induced changes in the transcriptome of cell suspension cultures of oilseed rape reveal genes associated with lipid biosynthesis

Microspore-derived cell suspension cultures of Brassica napus L. cv Jet Neuf grown in increasing concentrations of sucrose have been shown to produce increasing amounts of triacylglycerol (TAG) that are accompanied by increasing acyl-CoA:diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity. Here, we show that the sucrose-induced increase in the TAG biosynthetic capacity of these cells is also associated with increased production of mRNA encoding DGAT. In addition, differential display (DD) of mRNA was used to evaluate the production of other transcripts that might be linked to lipid biosynthesis that were induced by increasing the sucrose concentration in the growth media. The PCR-based technique identified eight cDNA fragments representing upregulated transcripts. The deduced amino acid sequence of one of these fragments displayed 96% identity to a mitochondrial acyl carrier protein from Arabidopsis thaliana suggesting a possible association between fatty acid biosynthesis in the mitochondria and TAG biosynthesis in the endoplasmic reticulum. Two other cDNA fragments encoded polypeptide segments that exhibited a high level of identity to ethylene-inducible protein and a member of the thioredoxin superfamily from A. thaliana. Another cDNA fragment had homology to sequences of unknown function whereas the remaining five cDNA fragments had no homology to previously reported sequences, and therefore represented the expression of unknown genes. Collectively, these results suggest DD of mRNA was an effective method for monitoring sucrose-modulated gene expression and that the method has the potential to identify novel sequences that may be related directly or indirectly to plant lipid metabolism.

Field resistance of six cultivars of winter oilseed rape against Turnip yellow mosaic virus

The resistance of six cultivars of winter oilseed rape (SL 509, SL 507, Darmor, Solida, Jet Neuf, Silesia) against Turnip yellow mosaic virus was studied. The number of over-wintering plants and plants with symptoms of TYMV infection were monitored. All plants were tested by the double diffusion test in agar and by DAS-ELISA to prove infection. ELISA was the most sensitive method, revealing 32–76% of latent virus infections in individual cultivars. The results clearly illustrated that monitoring of plant symptoms is not sufficient to prove TYMV infection in the field and that sensitive, large scale methods like ELISA must be employed to obtain reliable data.

Characterization of cDNAs encoding diacylglycerol acyltransferase from cultures of Brassica napus and sucrose-mediated induction of enzyme biosynthesis

cDNAs encoding acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20), designated BnDGAT1 and BnDGAT2, were obtained from a microspore-derived cell suspension culture of oilseed rape ( Brassica napus L. cv Jet Neuf). BnDGAT2 shares a very high level of identity with BnDGAT1, but is a smaller protein lacking the relatively hydrophilic N-terminal segment found in BnDGAT1. Both transcripts were produced in the cell suspension cultures and the cDNAs were functionally expressed in transformed yeast ( Pichia pastoris) cells. Sucrose-mediated changes in triacylglycerol (TAG) metabolism and expression of BnDGAT1 were examined in the cell suspension cultures following transfer of cells from media containing 6% (w/v) sucrose to media containing 14% sucrose. TAG content and DGAT activity of the cells increased transiently within the first 12 h after transfer (HAT). The rapid decline in TAG content observed at 12 HAT was inversely related to an increase in TAG lipase (EC 3.1.1.3) activity. The transient increases in TAG content and DGAT activity correlated with the elevated amounts of BnDGAT1 polypeptide. Transcript levels were also induced, but levels of mRNA encoding BnDGAT1 were not tightly correlated with DGAT activity and amount of polypeptide suggesting some control of expression at the post-transcriptional level. In general, the rapid changes in TAG content were closely associated with the changes in the activity of TAG-metabolizing enzymes and expression of BnDGAT1.

Regulation and characterization of four CBF transcription factors from Brassica napus.

Four orthologues of the Arabidopsis CBF/Dreb transcriptional activator genes were isolated from the winter Brassica napus, cv. Jet neuf. All four BNCBF clones encode a putative DRE/CRT (LTRE)-binding protein with an AP2 DNA-binding domain, a putative nuclear localization signal and a possible acidic activation domain. Deduced amino acid sequences suggested that BNCBFs 5, 7and 16 are very similar to the Arabidopsis CBFI whereas BNCBF17 is different in that it contains two extra regions of 16 and 21 amino acids in the acidic domain. Transcripts hybridizing specifically to BNCBF17 and to one or more of the other BNCBFs accumulated in leaves within 30 min of cold exposure of the Brassica seedlings and preceded transcript accumulation of the cold-inducible BN28 gene, a Brassica orthologue of the cor6.6 or KIN gene from Arabidopsis. Cold-induced accumulation of BNCBF17 mRNA was rapid but was short-lived compared to transcripts hybridizing to BNCBF5/7/16. Transcripts hybridizing to one or more of BNCBF5/7/16 accumulated at low levels after the plants were subjected to prolonged exposure to salt stress. BNCBF17 was not responsive to salt stress. BNCBF transcript accumulation was similar in both spring and winter Brassica but the persistence of the transcripts in the cold were generally shorter in the spring than in the winter type. BNCBF5 and 17 proteins bind in vitro to the LTRE domains of the cold-inducible BN115 (cor15a orthologue) or BN28 promoters. Differential binding preferences, however, to LTREs between BNI 15 and BN28 were observed. Mutation of the core CCGAC sequence of the LTRE indicated that BNCBF17 had a lower sequence binding specificity than BNCBF5. Furthermore, experiments indicated that the LTREs were able to drive BNCBF5 and 17 trans-activation of the Lac-Z reporter gene in yeast. We conclude that the BNCBFs reported here could function as trans-acting factors in low-temperature responses in Brassica, controlling the expression of cold-induced genes through an ABA-independent pathway.

Genetic Control and Host Range of Avirulence Toward Brassica napus Cultivars Quinta and Jet Neuf in Leptosphaeria maculans

ABSTRACT Leptosphaeria maculans causes blackleg of oilseed rape. Gene-for-gene interactions between race PG3 and Brassica napus cv. Quinta were related to interaction between the fungal avirulence (Avr) gene AvrLm1 and the corresponding resistance gene Rlm1. AvrLm1 isolates were aviru-lent on cvs. Doublol, Vivol, Columbus, and Capitol, and no recombinant phenotypes were observed in the progeny of two AvrLm1 x avrLm1 crosses, suggesting that all of these cultivars may possess Rlm1 or genes displaying the same recognition spectrum, or that a cluster of Avr genes is present at the Avrlm1 locus. In one cross, segregation distortion was observed at the AvrLm1 locus that could be explained by interaction between AvrLm1 and one unlinked deleterious gene, termed Del1. Incompatibility toward cvs. Jet Neuf and Darmor.bzh was governed by a single gene, unlinked to AvrLm1 or Del1. This avirulence gene was termed AvrLm4. Preliminary plant genetic analysis suggested the occurrence of a corresponding dominant resistance gene, termed Rlm4, present in the Quinta line analyzed and linked to Rlm1.

Expression and properties of diacylglycerol acyltransferase from cell-suspension cultures of oilseed rape

The expression of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) with predicted molecular mass of 56.9. kDa (BnDGAT1) was examined using microspore-derived cell suspension cultures of oilseed rape (Brassica napus L. cv Jet Neuf). As well, a recombinant histidine-tagged N-terminal fragment of BnDGAT1 [BnDGAT1((1-116))His(6)], which was relatively hydrophilic, was partially characterized. A temporal increase in DGAT activity occurred within a 24 h period following transfer of cells from 6% (w/v) sucrose to 14% (w/v) sucrose. Western blotting indicated that the abundance of BnDGAT1 protein was closely correlated with DGAT activity. BnDGAT1 mRNA also exhibited a temporal increase within the 24 h period following transfer of cells into higher sucrose concentrations, but the transcript level was not closely associated with DGAT activity as BnDGAT1 protein. The fragment BnDGAT1(1-116)His(6) interacted with [1-(14)C]oleoyl-CoA, suggesting that the N-terminal region of BnDGAT1 may have a role in binding cellular acyl-CoA.

Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape.

Several factors, including an unidentified endogenous component, were found to stimulate microsomal diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) from a microspore-derived cell-suspension culture of oilseed rape (Brassica napus L. cv. Jet Neuf). At a concentration of 25 mM, MgSO4 and MgCl2 stimulated microsomal DGAT 25- and 10-fold, respectively. ATP and CoA at concentrations of 2 and 1 mM stimulated the enzyme 2.4- and 12-fold, respectively, although the effects were lessened in the presence of higher Mg2+ concentrations. Although microsomal DGAT activity was increased only slightly by the addition of exogenous sn-1,2-diacylglycerol to the reaction mixture, it was increased substantially by the addition of exogenous phosphatidate. sn-Glycerol-3-phosphate and other phospholipids tested did not have this stimulatory effect. DGAT activity did not decrease when microsomes were incubated with ATP in the presence of the cytosolic fraction. This fraction, however, contained a small organic compound(s) that stimulated microsomal DGAT activity.

Co-ordination of photosynthetic gene expression during low-temperature acclimation and development in Brassica napus cv. Jet Neuf leaves

The effect of low-temperature acclimation and leaf age on the expression of two photosynthetic genes was examined in mature and young leaves of Brassica napus cv. Jet Neuf. Nuclear-encoded ( ssu) and chloroplast-encoded ( lsu) genes were used to examine the changes at the DNA, mRNA and protein level. After acclimation, expression of lsu and ssu genes was low in mature leaves and high in young leaves. Corresponding Western blots showed a similar pattern for polypeptide accumulation with lower amounts of SSU and LSU polypeptides in mature leaves and higher amounts in young leaves when compared to their non-acclimated counterparts. However, in acclimated mature leaves relatively more LSU and SSU polypeptides accumulated than did the corresponding transcript. Despite the disparities between protein and message levels during acclimation, protein turnover kinetics showed that LSU and SSU had similar stability in acclimated and non-acclimated leaves of both ages. From Western blots, the ratio of LSU:SSU showed that both polypeptides accumulated in proportion during cold acclimation. Methylation studies at various restriction sites of lsu revealed alteration at PstI and HindIII sites with acclimation.

Identification of N-ethylmaleimide-sensitive and -insensitive phosphatidate phosphatase activity in microspore-derived cultures of oilseed rape

Microsomal phosphatidate (PA) phosphatase (EC 3.1.3.4) from microspore-derived (MD) cultures of Brassica napus L. was characterized based on sensitivity to the thiol-reactive agent, N-ethylmaleimide (NEM) and dependence on magnesium ions. Both membrane-bound and solubilized PA phosphatase from MD embryos of B. napus L. Topas were inhibited by 50% following preincubation with 0.5 mM NEM. The activity of PA phosphatase was also partially dependent on the inclusion of magnesium ions in the assay buffer. The stability properties of solubilized PA phosphatase were compared to solubilized diacylglycerol acyltransferase (EC 2.3.1.20). PA phosphatase was considerably more labile than diacylglycerol acyltransferase during storage at 4°C. Bovine serum albumin, however, increased the stability of PA phosphatase during storage. PA phosphatase activity was resolved into two major fractions by anion-exchange chromatography on Macro-Prep 50Q. The activity of the enzyme in both fractions was partially inhibited following preincubation with NEM. Microsomal PA phosphatase from MD cell suspension cultures of B. napus L. cv Jet Neuf was also partially inhibited by NEM. The results suggest plant tissues, as in mammalian systems, may contain different forms of PA phosphatase involved in both glycerolipid synthesis and signal transduction.

Triacylglycerol biosynthesis and gene expression in microspore-derived cell suspension cultures of oilseed rape

developing seeds of oilseed rape is fundamental to the rational development of biotechnological strategies for The effect of sucrose concentration on triacylglycerol increasing seed oil content and manipulating oil composibiosynthesis and associated gene expression was tion. In oilseeds, the biosynthesis of TAG in developing examined in a microspore-derived cell suspension cul- seeds is catalysed by the sequential addition of fatty acyl ture of oilseed rape (Brassica napus L. cv. Jet Neuf). groups to a glycerol backbone derived from sn-glycerol The triacylglycerol content of the cells increased about 3-phosphate (Stymne and Stobart, 1987; Stobart and 5-fold on a fresh weight basis when the sucrose con- Stymne, 1990). Diacylglycerol acyltransferase (DGAT; centration in the growth medium was raised from 2% EC 2.3.1.20) catalyses the acylation of sn-1, to 22% (w/v). The specific activity of microsomal 2-diacylglycerol to yield TAG (Stymne and Stobart, diacylglycerol acyltransferase (EC 2.3.1.20) and its 1987). In developing seeds of Brassica napus L., maximum activity per unit fresh weight increased about 2.5-fold DGAT activity occurs during the rapid phase of oil and 6-fold, respectively, when sucrose concentration accumulation ( Tzen et al., 1993; Weselake et al., 1993a). was increased from 2% to 14%. mRNA encoding the The enzyme may also exert significant flux control in major oleosin also appeared to increase in abundance TAG bioassembly (Perry and Harwood, 1993a, b). The over the 2‐14% sucrose concentration range when enzymes of TAG bioassembly are associated with the RNA fractions were analysed by the reverse transcrip- endoplasmic reticulum ( ER) (Stobart et al., 1986). tion-polymerase chain reaction. The sucrose-mediated The apparently hydrophobic and labile nature of these increases in diacylglycerol acyltransferase activity and enzymes appears to have impeded progress on their oleosin mRNA indicated that the cell suspension could purification and characterization (Gibson et al., 1994; be a useful research tool for the identification of Little et al., 1994; Eccleston and Harwood, 1995; Kocsis cDNAs encoding triacylglycerol biosynthetic enzymes et al., 1996). TAG accumulates in developing seeds of B. and associated proteins. napus as small droplets, termed oil bodies, which are incapsulated by a single layer of phospholipid (Murphy,