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a Saskatchewan Wheat Pool Agricultural Research and Development, 201-407 Downey Road, Saskatoon, SK, S7N 4L8, Canada
b National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada
* Corresponding authors (David.Taylor{at}nrc.ca, vkatavic{at}pbi.nrc.ca)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: TAGs, triacylglycerols • HEA, high erucic acid • VLCFAs, very long chain fatty acids • FAE, fatty acid elongase • SLC, sphingolipid compensation • DAP, days after pollination • DW, dry weight
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The gene for an erucoyl-CoA preferring sn-2 acyltransferase (LPAT EC 2.3.1.51) from meadowfoam (Limnanthes douglasii L.) has been successfully cloned and used to alter seed oil sn-2 proportions of 22:1 in rapeseed (Brown et al., 1995; Hanke et al., 1995; Lassner et al., 1995; Brough et al., 1996). However, the overall proportions of erucic acid in the seed oil did not increase, nor were increases in total fatty acid and/or oil content reported. Recently, we (Zou et al., 1997) have confirmed that the yeast (Saccharomyces cerevisiae) SLC1-1 gene encodes an sn-2 acyltransferase capable of acylating sn-1-oleoyl-lysophosphatidic acid using a range of acyl-CoA thioesters, including 22:1-CoA. When the SLC1-1 LPAT gene was expressed under the control of a constitutive (double cauliflower mosaic virus 35S) promoter in A. thaliana and high erucic acid cultivars of B. napus Hero and Reston, the resulting transgenic plants showed increases in both overall proportions and amounts of VLCFAs in seed triacylglycerols (TAGs) and substantial increases in seed oil content (Zou et al., 1997). However, neither the meadowfoam nor the yeast LPAT transgene approach was successful in achieving targeted proportions of trierucin in the HEAR B. napus seed oil. These results together with the results from Weier et al. (1997) suggest that the level of trierucin depends not only on the activity of the introduced sn-2-acyltransferase but also on other biosynthesis or incorporation steps. For example, it is possible that the levels of erucoyl-CoA in the seed acyl-CoA pool may be too low to support high levels of trierucin biosynthesis. If this is the case, then over expression of genes regulating VLCFA biosynthesis may be required to boost very long-chain acyl-CoA availability for incorporation into seed TAGs.
VLCFAs are synthesized via the fatty acid elongation (FAE) pathway located in the cytosol. The initial substrate for elongation is oleic acid, synthesized in the plastids. Fatty acid elongation is achieved by the sequential addition of C2 moieties donated by malonyl-CoA to a long chain acyl-CoA primer. Each round of elongation involves four enzymatic reactions catalyzed by the FAE complex, a protein complex localized in the microsomal fraction. The FAE reactions are (i) condensation of malonyl-CoA with a long chain acyl-CoA to give a ß-ketoacyl-CoA; (ii) reduction to ß-hydroxyacyl-CoA; (iii) dehydration to enoyl-CoA, and (iv) reduction of the enoyl-CoA, resulting in an elongated acyl-CoA (Fehling and Mukherjee, 1991).
On the basis of the results of the expression of the A. thaliana fatty acid elongase gene (FAE1; encoding the seed-specific-elongase condensing enzyme) in Arabidopsis, tobacco (Nicotiana tabacum L.), and yeast, Miller and Kunst (1997) proposed that of the four enzymes involved in VLCFA biosynthesis, the two reductases and the dehydrase are expressed throughout the plant and are common to all microsomal FAE systems. In contrast, the condensing enzymes seem to be differentially expressed and probably unique to each FAE system.
Continuing our efforts to increase the amounts of VLCFA (and 22:1 in particular) in Canadian HEAR cultivars, we focused on two targets using a transgenic approach. To address the supply of erucoyl moieties for oil synthesis, we examined the role and function of the A. thaliana FAE1 by expressing it under the control of seed-specific (napin) promoter in both a low erucic acid (LEA), canola cultivar of B. napus and in HEAR germplasm, and analyzed the changes in VLCFA content in the seed oil of transgenic lines. The best FAE1 transgenic lines were also field tested. To examine the incorporation of erucoyl moieties into seed oil, we tested the performance of SLC1-1 B. napus cv. Hero transgenic progeny (Zou et al., 1997) in the field. Here we report detailed analyses of VLCFA content, erucic acid content, and oil content in the seed oil as well as seed yield of the field-grown FAE1 and SLC1-1 B. napus cv. Hero progeny.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Brassica napus cv. Hero SLC1-1 transgenic lines containing a yeast sn-2 lyso-phosphatidic acid acyltransferase (LPAT; EC 2.3.1.51) were produced and characterized as described previously (Zou et al., 1997). Polymerase chain reaction (PCR) and Southern (1975) analyses of the transgenic lines selected for further biochemical characterization and field testing showed that all of the lines contained a single SLC1-1 insert.
Lipid Substrates, Chemicals, and Biochemicals
[1-14C] oleic acid (2.15 x 109 Bq. mmol-1) was purchased from Amersham Canada, Ltd. (Oakville, ON) and [1-14C]-labeled oleic acid was converted to the corresponding labeled oleoyl-CoA by the method described by Taylor et al. (1990a)(b). Specific activity was adjusted as required by diluting with authentic unlabeled standard. Unlabeled oleoyl-CoA, malonyl-CoA, ATP, CoA-SH, NADH, NADPH, sodium acetate, and most other biochemicals were purchased from Sigma (St. Louis, MO). The 15:0 and 17:0 standards were supplied by Supelco Canada, Ltd. (Oakville, ON). HPLC-grade solvents (Omni-Solv, BDH Chemicals, Toronto, ON) were used throughout these studies.
FAE1 Transformation Vector
Drs. A. Millar and L. Kunst (from the Dept. of Botany, University of British Columbia, Canada) kindly provided the binary vector pNap:FAE1/NGKM (Fig. 1) containing the A. thaliana FAE1 coding region under the control of seed-specific, napin promoter. The binary vector was introduced by electroporation into the Agrobacterium tumefaciens strain GV3101 bearing helper plasmid pMP90 (Koncz and Schell, 1986) and used in transformation experiments.
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Hypocotyls were excised from 5 to 7-d–old seedlings of the HEAR cv. Hero, and cut into 5- to 7-mm segments. The hypocotyl explants were transformed by the method developed by DeBlock et al. (1989).
Experimental wild-type control plants were regenerated in vitro from the cotyledonary petioles and/or hypocotyl explants. Control explants were not cocultivated with A. tumefaciens. However, with this exception, control explants were subjected to all the other experimental procedures and conditions applied to explants that were cocultivated with Agrobacterium (and from which transformed shoots were developed).
Control and transformed shoots were rooted in vitro on rooting medium without kanamycin or with 25 µg mL-1 kanamycin, respectively. Plants with well-developed roots were transferred to soil and grown to maturity. Developing and mature seed from selfpollinated control and transgenic lines grown in the greenhouse, were harvested and subjected to molecular and biochemical analyses.
Molecular Analyses of Transgenic Plants
All molecular analyses (plasmid preparation, PCR, restriction digestion, DNA gel blot analyses etc.) were performed by methods prescribed by Sambrook et al. (1989) or Ausubel et al. (1995).
PCR Amplification of the Partial Expression Cassette NAP/FAE1/NOS
To check for integration of the napin:FAE1:nos transgene construct into the genome of putative transgenic plants, leaf tissue from T0 plants was collected and genomic DNA isolated. This DNA was used as a template to amplify the partial expression cassette NAP/FAE1/NOS by means of oligonucleotide primer NN-3 (5'-TTTCTTCGCCACTTGTCACTCC-3') which was designed according to the promoter region of the napin gene (position 948–969) and primer NN-4 (5'CGCGCTATATTTTGTTTTCTA-3') which was designed according to the nopaline-synthase 3' UTR sequence (position 1753–1773). The total size of the expected PCR product is about 2.0 kb (0.197 kb of napin promoter region + 1.608 kb FAE1 coding region + 0.204 kb of nopaline-synthase 3' UTR region).
Seed Lipid and Protein Analyses
The total fatty acid content and acyl composition of seed lipids was determined by gas chromatography (GC) of the fatty acid methyl esters (FAMEs) with either 15:0 or 17:0 free fatty acid added as an internal standard, as described previously (Taylor et al., 1995; Katavic et al., 1995; Zou et al., 1997).
For analyses of the FAE1 transgenic progeny, single seeds were cut with a scalpel into small pieces and an internal standard (15:0 free fatty acid) and 1 mL of 3 M methanolic-HCl (Supelco Canada, Ltd.) were added. Transmethylation was performed at 80°C for 2 h. Reaction mixtures were cooled on ice and 2 mL of 9 g L-1 NaCl was added. The mixture was extracted three times with 2 mL of hexane and then the hexane extracts were combined and taken to dryness under nitrogen. The acyl composition was determined by GC of the FAMEs on a Hewlett-Packard model 5890 gas chromatograph fitted with a DB-23 column (30 by 0.25 mm; film thickness, 0.25 µm; J & W Scientific, Folsom, CA). The GC conditions were as follows: injector temperature and flame ionization detector temperature, 250°C; running temperature program, 180°C for 1 min, then increasing at 4°C/min to 240°C and holding this temperature for 10 min. Data from 10 single seed runs of each FAE1 transgenic line were averaged.
For the SLC1-1 and FAE1 field trial progeny, a near-infrared reflectance (NIR) method was used to estimate oil and protein content on the basis of AOCS Procedure Am 1-92 (Firestone, 1998) using the NIR System 6500 (Foss North America) with software packages NEWISI and WINISI (Infrasoft International LLC). The sample size for NIR scanning was about 4.5 g, enough to fill the ring cup. The oil and protein contents as determined by NIR were calibrated against data obtained from NMR and Leco Protein Analyzer/Kjeldahl analyses (performed with a standard set of HEAR seed samples) (Tkachuk, 1981), respectively, and certified by the Canadian Grain Commission.
Elongase Assays of FAE1 Transgenics
Developing seeds were harvested 30 to 35 d after pollination (DAP) frozen immediately in liquid nitrogen and stored at -70°C until homogenized. Seeds (approx. 20) were ground in a cold mortar at 0°C in 2 mL grinding buffer {100 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) pH 7.4, 400 mM sorbitol, 2.5 mM EGTA [ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraaecetic acid], 2.5 mM EDTA (ethylenediaminetetraacedic acid), 5 mM MgCl2, 1 mM DTT (dithiothreitol), PVPP (polyvinylpyrrolidone) 150 mg mL-1}.
The slurried homogenate was filtered through 1 layer of Miracloth (Calbiochem, La Jolla, CA) and used to perform elongation assays as described by Taylor et al. (1992). In the standard reaction mixture, 0.2 to 0.5 mg of protein was incubated in shaking water-bath (100 rpm) at 30°C for 45 min at pH 7.2 with 90 mM HEPES-NaOH, 1 mM ATP, 1 mM CoA-SH, 0.5 mM NADH, 0.5 mM NADPH, 2 mM MgCl2, 1 mM malonyl-CoA + 18 µM [1-14C] oleoyl-CoA (3.7 x 102 Bq nmol-1) in final volume of 500 µL. In each set of reactions, the amount of homogenate protein added was normalized. Reactions were stopped by adding 3 mL of 100 g L-1 KOH in methanol and the mixtures were heated at 80°C for 1 h to saponify the acyl lipids and acyl-CoAs. The tubes were cooled on ice and two-2 mL hexane washes were performed to remove non-saponifiable material. These hexane washes were discarded, and 1 mL water was added to the reaction mixtures. The mixture was then acidified by adding 650 µL concentrated 12 M HCl, extracted twice with 2 mL hexane, the hexane extracts combined and dried under N2. Samples were transmethylated with 3 M methanolic-HCl at 80°C for 1 h. 2 mL of 9 g L-1 NaCl was added, samples were extracted 2x with 1 mL hexane, dried under N2, taken up in 110 µL of acetonitrile and quantified by radio-HPLC as described previously (Taylor et al., 1992).
Field Trials and Analysis of Progeny
All field trials were conducted by the Saskatchewan Wheat Pool at Rosthern, Saskatchewan (Saskatoon farm zone) in the summers of 1998 and 1999. The 1998 growing season (26 May–21 September) exhibited 1519 growing degree days, 2309 crop heat units and 172.4 mm of precipitation accumulation. The 1999 growing season (26 May–21 September) exhibited 757 growing degree-days, 1278 crop heat units and 167.5 mm of precipitation accumulation (http://www.farmzone.com).
In 1998, nineteen SLC1-1 T3 transgenic lines were field tested in a nursery trial. Transgenics or control lines were planted in a random block design in 3-m rows, with about 100 seeds per row with 60 cm between rows. Data werre collected from two to six rows of each transgenic line and 18 rows of non-transformed Hero control lines.
In the 1999 growing season, 37 B. napus cv. Hero FAE1 transgenic T2 lines were field tested in a nursery trial. Transgenics or control lines were planted in a random block design in 3-m rows, with about 100 seeds per row with 60 cm between rows. There were two rows of each line.
In 1999, 17 T4 SLC1-1 transgenic B. napus cv. Hero lines were selected for yield and quality assessment in the field. The SLC1-1 yield field trials were of a random block design. Each plot was about 6 m2 (five rows wide at 17.8-cm spacing, and 6 m long in size). The T4 field-grown lines (leaf material) were sampled and analyzed by PCR to confirm the presence of the 0.95-kbSLC1-1 insert, using the primers OM087 (5'-AGAGAGAGGGATCCATGAGTGTGATAGGTAGG-3') and OM088 (5'-GAGGAAGAAGGATCCGGGTCTATATACTACTCT-3') which were designed according to the 5' and 3' end sequences, respectively, of the SLC1-1 gene as described by Zou et al. (1997).
Analyses were conducted on the progeny (T5 seed) from triplicate plots. The oil content data collected for each line in this trial were analyzed by the Anova-Fisher's LSD method (P 
0.05) and Tukey's pairwise comparison method in the Minitab Statistical Software Suite Release 12 (Minitab, Inc. State College, PA).
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To analyze functional expression of the A. thaliana FAE1 gene in transgenic lines, the capacity for VLCFA biosynthesis (elongase activity) was assayed in vitro. Reverse-phase HPLC analyses of radiolabeled fatty acid methyl esters from wild-type Westar control plants and FAE1 Westar transgenic line WS-2-10 are shown in Fig. 4A and 4B, respectively. In the wild-type Westar control, low amounts of 20:1 product were detected, but no 22:1 was detected. In contrast, transgenic Westar line WS-2-10 contained substantial amounts of 20:1 and low amounts of 22:1 elongation product. The assay data revealed that the elongase complex activity in developing transgenic seeds (about 30–35 DAP) was up to 180 to 245 pmol min-1 mg-1 protein in our best lines, an increase of 180 to 245-fold over the low background activity (1.0 pmol min-1 mg-1 protein) found in the non-transformed wild-type control Westar, WS-WT line [Table 1 (A)]. This further confirms that the significant increases in the proportions of VLCFAs in our transgenic lines compared with the wild-type canola cv. Westar, are the result of functional restoration of the activity of an elongase complex condensing enzyme. The very strong increase in eicosenoic acid (20:1) proportions in our transgenic lines and the results from elongase assays indicate that the Arabidopsis seed-specific condensing enzyme encoded by the FAE1 gene, prefers 18:1 as a substrate and thus produces more 20:1 than 22:1. Thus, our transgenic experiments with FAE1 expressed in a canola background provide independent supporting evidence to explain the fatty acyl phenotype of Arabidopsis seed oil, wherein 20:1 is the most predominant VLCFA.
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The results of the GC analyses of fatty acid composition in seed oil of T2 Hero FAE1 transgenic lines and wild-type controls are shown in Fig. 5. Proportions of erucic acid and VLCFAs are shown as grams per kilogram of total fatty acids. In our best Hero T2 transgenic lines, proportions of erucic acid were 480 to 530 g kg-1 of total extractable fatty acids while wild-type control lines average about 430 g kg-1 erucic acid.
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Field-Trials with SLC1-1 and FAE1 B. napus cv. Hero Transgenic Progeny: Field Trial with FAE1 B. napus cv. Hero
To test the performance of transgenic FAE1 plants in the field, 37 T2 B. napus cv. Hero FAE1 transgenic lines were grown in nursery row trials. Mature T3 seed was collected from these field-grown plants and subjected to analyses of oil content and erucic acid proportions. The best Hero FAE1 transgenic lines showed 80 to 110 g kg-1 increases in erucic acid proportions and 20 to 48 g kg-1 increases in oil content, when compared with the wild-type controls (Table 2).
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The highlights of the 1999 SLC1-1 B. napus field trial for yield and quality are depicted in Table 3. Nineteen lines of T4 SLC1-1 transgenic B. napus cv. Hero were selected for field assessment. The T5 progeny in the best six lines showed 28 to 56 g kg-1 increases (representing net increases of 6.7–13.5% overall) in oil content on a DW basis. For controls, the oil content single plot values (n = 5) were: 413, 419, 426, 429, and 387 g kg-1 oil on a DW basis. The oil content data were analyzed by a one-way analysis of variance using Fisher's LSD (P = 0.05) and Tukey's pairwise comparisons in the Minitab program of suites, and both statistical tests found the SLC1-1 transgenic oil content to be significantly different in comparison to the non-transformed Hero control plots. There were also 24 to 70 g kg-1 increases in erucic acid proportions (representing net increases of 5.2–15% overall). Yield (g/plot) was consistently higher in these transgenic lines. As shown in Fig. 6, calculating the erucic acid yield based on these plot trials revealed that the five best lines (H-5-1-4, H-5-4-8, H-8-6-8, H-8-10-2A, and H-8-10-5) exhibited a 53 to 121% increase (see Table 3) in 22:1 yield (expressed as g 22:1/plot). In the specific case of line H-5-4-8, a "nearest neighbor analysis" of control lines in yield plot rows immediately adjacent to the SLC1-1 transgenic line, showed a dramatic 73% increase in average plot yield (Fig. 7).
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In parallel, we did complementation analyses in B. napus canola cv. Westar with the same gene construct. Our complementation data suggest that the other three enzymes of the elongase complex are functional in B. napus canola cultivars, which further confirms the important role of the condensing enzyme in determining the quantity and the chain length of VLCFA synthesized by the acyl-CoA elongase complex. The results from several other studies support this hypothesis. Lassner et al. (1996) expressed 3-ketoacyl-CoA synthase cDNA from jojoba in B. napus low erucic acid (canola) cultivar. Transformation of low erucic acid rapeseed with a jojoba cDNA partially restored the activity of microsomal condensing enzyme in developing canola seed. Transgenic seed oil had altered fatty acid composition and contained medium levels of VLCFAs. Similarly, when a gene encoding a condensing enzyme from Lunaria annua L. (containing 24:1—nervonic acid, as a major component of its seed oil) was introduced into high erucic acid rapeseed, the resulting oil contained approximately 200 g kg-1 24:1 (Lassner et al., 1996). The important role of the condensing enzyme was recently established by Millar and Kunst (1997). By expressing the A. thaliana FAE1 gene in yeast and tobacco where no significant quantities of VLCFA are found, they demonstrated that the introduction of the FAE1 gene alone is sufficient for the production of VLCFAs.
The seed-specific expression of A. thaliana FAE1 gene in B. napus canola cv. Westar resulted in a significant increase of VLCFA content in the seed oil with the amounts of eicosenoic acid being high in all transgenic lines. Data from lines in which the increase in erucic acid was very low (e.g , line WS-1 with 6 g kg-1 erucic acid and 105 g kg-1 eicosenoic acid, experimental control WT line had 0.2 g kg-1 erucic acid, and 14 g kg-1 eicosenoic acid in extractable seed oil) could indicate that A. thaliana condensing enzyme FAE1 prefers 18:1 over 20:1 as a substrate for elongation. On the other hand, our results correlate well with the studies of genetic control of very-long-chain-monounsaturated fatty acid biosynthesis in rapeseed by Downey (1964), in which a classical breeding approach was used to develop canola cultivars (low erucic acid, low glucosinolate) of B. napus from wild-type HEA B. napus. When selecting for rapeseed plants containing no erucic acid in their seed oil, it was observed that the percentage of eicosenoic acid remained relatively constant with decreasing erucic acid, except in the zero erucic acid genotype where the level of eicosenoic acid in the seed oil was only 10 g kg-1 instead of approximately 120 g kg -1. Furthermore, the genetic analyses of populations segregating for seed oil erucic acid content indicated that the 22:1 content is controlled by two genes which show no genetic dominance, but instead, act in an additive manner (Downey, 1964). These two genes (designated E1 and E2) have been mapped in rapeseed by a QTL approach (Ecke et al., 1995; Jourdren et al., 1996; Thormann et al., 1996). Recently, Fourmann et al. (1998) amplified two condensing enzymes in B. napus which correspond to those found in the parental species B. rapa L. and B. oleracea L. They mapped these two genes and found that they cosegregate with E1 and E2 loci. It has been hypothesized that different alleles at the E1 and E2 loci are leading to different levels of erucic acid content in the seed. (Jönsson, 1977). It is still not known whether the suspected differences among alleles are based on differences in a regulatory region or in the coding region of the genes.
Seed-specific expression of A. thaliana FAE1 gene in B. napus HAE cv. Hero led to increases in proportions of 22:1 from 480 to 530 g kg-1 in our best green-house grown transgenic lines. When grown in the field, all selected transgenic lines showed higher proportions of erucic acid than the field grown wild-type control cv. Hero lines. In the T3 progeny from our best line, H-10-2, erucic acid level in the extractable seed oil was 591 g kg-1 which is about 110 g kg-1 more erucic acid than that found in the field-grown wild-type control (481 g kg-1 erucic acid). As reported previously for A. thaliana (Millar and Kunst, 1997), overall, our results confirm that (i) the level of active condensing enzyme is somewhat rate limiting for the biosynthesis of erucic acid and other VLCFAs in HEA B. napus cultivars and (ii) in the elongase complex, it is the specificity of the condensing enzyme that determines which VLCFAs will accumulate. As discussed previously, the FAE1 gene from A. thaliana encodes a condensing enzyme that prefers 18:1 over 20:1 as a substrate for elongation. Thus, in the current experiments wherein we have over expressed the FAE1 gene in HEA B. napus cv. Hero, it is very likely that the observed increases in the seed proportions of erucic acid are at least, in part, due to increases in the proportions of 20:1 available for further elongation during the storage lipid deposition phases of seed development.
For the SLC1-1 transgenic rapeseed lines, the field data from both the nursery row and yield plot trials, in 1998 and 1999, respectively, are extremely encouraging, even though they are from only one location. These data indicate that under two very different growing seasons with respect to growing degree days/crop heat units, the SLC1-1 lines clearly and consistently out-perform the Hero controls in terms of erucic acid and oil yield, and confirm the earlier results obtained in greenhouse studies as reported by Zou et al. (1997).
Thus far, we have expressed separately, two main target genes: (i) a gene encoding an sn-2-acyltransferase from S. cerevisiae (SLC1-1) and (ii) a gene encoding a seed-specific condensing enzyme from A. thaliana (FAE1) in the HEAR cv. Hero. Individually expressed, both genes contributed to the increases in VLCFAs in seed oil. However, neither gene alone could boost the amounts of erucic acid to the target level of 80 mol% (828 g kg-1) or more erucic acid in seed oil. We have demonstrated that in rapeseed, the proportion of 22:1 is limited by both the rate of 22:1 synthesis and its subsequent incorporation into triacylglycerols.
Accordingly, we are in the process of returning to breeding and selection, initiating crosses between our best SLC1-1 cv. Hero transgenic lines and the best FAE1 cv. Hero transgenic lines. By combining both traits we will eventually produce lines which will be superior to parental transgenic lines with respect to both 22:1 content and oil content. The SLC1-1 gene is expressed under the control of constitutive promoter (cauliflower mosaic virus 35S), while FAE1 gene is expressed under the control of seed specific napin promoter in our cv. Hero transgenics. The results of our earlier experiments in which we expressed SLC1-1 gene under the seed specific napin promoter revealed that there was no advantage in expressing SLC1-1 gene under the control of napin promoter when compared with the level of expression of SLC1-1 gene under the control of 35S promoter in transgenic plants. The range of increases in overall amounts and proportions of erucic acid and VLCFAs were similar in both groups of transgenic cv. Hero lines (35S/SLC1-1 and napin/SLC1-1). However, when it comes to crossing transgenic lines, it could be advantageous to combine two transgenes under the control of two different regulatory sequences (35S/SLC1-1 and napin/FAE1) (it could be easier to follow the expression of the transgenes in the progeny) instead of having both transgenes being driven under the same (napin) promoter.
The application of these two transgenic technologies (FAE1 and SLC1-1) can improve the yield of 22:1 and seed oil considerably, and therefore offer new approaches to be combined with others (e.g., expression of the Limnanthes LPAT) to focus on the target of producing trierucin in rapeseed.
NOTE ADDED IN PROOF
Yield trials were conducted on the SLC1-1 B. napus transgenic lines for a second year at Rosthern, SK, from May through September 2000. The growing season was significantly different from the 1999 yield trial, with 1139 growing days, 1853 crop heat units, and 200.5 mm of precipitation. Nonetheless, the SLC transgenics performed extremely well once again. Compared with nontransformed control lines grown in a random block design, the SLC transgenic lines showed 2 to 4% increases in oil content on a mature seed weight basis, erucic acid increases of 8 to 13 g kg-1, average plot yield increases of 4 to 10 kg ha-1, all of which resulted in an increase of 200 to 450 kg ha-1 in the average erucic acid yield.
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ACKNOWLEDGMENTS |
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2 This is National Research Council of Canada Paper No. 43792.
Received for publication July 10, 2000.
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