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SummaryWe investigated whether termination of transcripts with a self-cleaving ribozyme can enhance nuclear retention and serve as a tool to decrease speci®c plant gene expression. Nuclear retention was ®rst monitored in tobacco using the b-glucuronidase gene terminated with either the 35S CaMV 3¢ untranslated sequence (UTR) or a cis-acting ribozyme. Northern blot analysis of nuclear RNA and total RNA, and in situ hybridizations showed that the ribozyme-terminated transcripts were preferentially retained in the nucleus of transgenic tobacco. Ribozyme-terminated transcripts were subsequently tested as a gene down-regulation strategy in soybean. The embryo-speci®c D-12 fatty acid desaturase FAD2-1 gene was targeted because its down-regulation elevates oleic acid content of seed storage lipids. Both ribozyme-terminated antisense and standard antisense constructs were capable of gene downregulation, producing over 57% oleic acid compared with less than 18% in wild-type seed. Ribozyme termination cassettes were also constructed to evaluate sense transcripts for single gene downregulation and the simultaneous down-regulation of two embryo-speci®c genes in soybean using a single promoter. Eight independent soybean transformants were screened that harboured standard plus sense or ribozyme terminated FAD2-1 cassette. Two of the eight ribozyme terminated transformants displayed oleic acids levels in the seed storage lipids of over 75%, while none of the standard plus sense FAD2-1 lines showed elevated oleic acid phenotypes. The dual constructs targeted FAD2-1 and the FatB gene encoding a palmitoyl-thioesterase. Five transgenic soybean lines harbouring the dual constructs had oleic acid levels, greater than 85%, and saturated fatty acids levels, less than 6%. Thus, ribozyme termination of transcripts can be utilized to speci®cally down-regulate endogenous gene expression in soybean.

Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks. Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids. An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of ␣-eleostearic (18:3⌬ 9cis,11trans,13trans ) and ␣-parinaric (18:4⌬ 9cis,11trans,13trans,15cis ) acids. Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of Momordica charantia and Impatiens balsamina, tissues that accumulate large amounts of ␣-eleostearic and ␣-parinaric acids, respectively. From ESTs of both species, a class of cDNAs encoding a diverged form of the ⌬ 12 -oleic acid desaturase was identified. Expression of full-length cDNAs for the Momordica (MomoFadX) and Impatiens (ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of ␣-eleostearic and ␣-parinaric acids, neither of which is present in untransformed soybean embryos. ␣-Eleostearic and ␣-parinaric acids together accounted for as much as 17% (wt͞wt) of the total fatty acids of embryos expressing MomoFadX. These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants. These findings also demonstrate a previously uncharacterized activity for ⌬ 12 -oleic acid desaturase-type enzymes that we have termed ''conjugase.'' H undreds of unusual fatty acid structures are known to occur in the seed oils of various plant species (1). The biosynthetic pathways of many of these fatty acids are unknown or have not been well characterized. One such class consists of fatty acids with double bonds that are conjugated. This structural configuration is in contrast to that of linoleic (18:2⌬ 9cis,12cis ) and ␣-linolenic (18:3⌬ 9cis,12cis,15cis ) acids, the typical polyunsaturated fatty acids of plant seed oils, which contain double bonds that are separated by methylene (ϪCH 2 Ϫ) groups (Fig. 1). Among the fatty acids with conjugated double bonds that occur in the plant kingdom are ␣-eleostearic acid (18:3⌬ 9cis,11cis,13trans) and ␣-parinaric acid (18:4⌬ 9cis,11trans,13trans,15cis ) ( Fig. 1). ␣-Eleostearic acid accounts for Ͼ65% (wt͞wt) of the total fatty acids of tung oil, a high-value drying oil obtained from seeds of Aleurites fordii (2). Other sources of this fatty acid include the seed oil of Momordica charantia, which contains approximately 65% (wt͞wt) ␣-eleostearic acid (2). In addition, ␣-parinaric acid composes 30 to 65% (wt͞wt) of the seed oils of plants such as Parinarium laurinum and Impatiens species (1, 3, 4).The presence of conjugated double bonds in fatty acids markedly increases their rate of oxidation relative to polyunsaturated fatty acids with methylene-interrupted double bonds (5). This property makes seed oils, such as tung oil, that are enriched in fatty acids with conjugated double bonds...

SummaryPhenotypic characterization of soybean event 335-13, which possesses oil with an increased oleic acid content (> 85%) and reduced palmitic acid content (< 5%), was conducted across multiple environments during 2004 and 2005. Under these conditions, the stability of the novel fatty acid profile of the oil was not influenced by environment. Importantly, the novel soybean event 335-13 was not compromised in yield in both irrigated and non-irrigated production schemes. Moreover, seed characteristics, including total oil and protein, as well as amino acid profile, were not altered as a result of the large shift in the fatty acid profile.The novel oil trait was inherited in a simple Mendelian fashion. The event 335-13 was also evaluated as a feedstock for biodiesel. Extruded oil from event 335-13 produced a biodiesel with improved cold flow and enhanced oxidative stability, two critical fuel parameters that can limit the utility of this renewable transportation fuel.

Two relatively rare fatty acids, gamma-linolenic acid (GLA) and stearidonic acid (STA), have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion efficiency in animals to eicosapentaenoic acid when compared with the more widely consumed omega-3 fatty acid, alpha-linolenic acid (ALA), present in most vegetable oils. Exploiting the wealth of information currently available on in planta oil biosynthesis and coupling this information with the tool of genetic engineering it is now feasible to deliberately perturb fatty acid pools to generate unique oils in commodity crops. In an attempt to maximize the STA content of soybean oil, a borage Delta(6) desaturase and an Arabidopsis Delta(15) desaturase were pyramided by either sexual crossing of transgenic events, re-transformation of a Delta(6) desaturase event with the Delta(15) desaturase or co-transformation of both desaturases. Expression of both desaturases in this study was under the control of the seed-specific soybean beta-conglycinin promoter. Soybean events that carried only the Delta(15 )desaturase possessed a significant elevation of ALA content, while events with both desaturases displayed a relative STA abundance greater than 29%, creating a soybean with omega-3 fatty acids representing over 60% of the fatty acid profile. Analyses of the membrane lipids in a subset of the transgenic events suggest that soybean seeds compensate for enhanced production of polyunsaturated fatty acids by increasing the relative content of palmitic acid in phosphatidylcholine and other phospholipids.

and black currents (Ribes nigrum L.) (Goffman and Galletti, 2001). The yield potential of these herbs is Through a single desaturation step, the Borago officinalis L. ⌬ 6 rather limited and levels of GLA and STA in the seed desaturase can convert linoleic acid and ␣-linolenic acid to ␥-linolenic acid (GLA) and stearidonic acid (STA), respectively. Both GLA and storage lipids constitute Ͻ16 and 5%, respectively. STA are of interest to the pharmaceutical and nutraceutical industries.In plants, GLA is produced by the desaturation of Production of these fatty acids is costly. One potential strategy to linoleic acid via a ⌬ 6 desaturase (Sayanova et al., 1997). reduce production cost would be to generate them in a major oilseed A cDNA clone of a ⌬ 6 desaturase from borage excrop. To this end, a cDNA of the B. officinalis ⌬ 6 -desaturase gene pressed in tobacco was capable of generating both GLA was cloned downstream of the embryo-specific promoter ␤-conglyciand STA in the transgenic plants at 12.9 and 8.5%, nin. The resultant cassette was assembled into a two T-DNA binary respectively, in young leaves (Sayanova et al., 1997). vector, in which the second T-DNA element harbored a selectable Accumulation of the novel fatty acids in the transgenic marker cassette. The final plasmid was subsequently used to transform tobacco lines varied across tissue, with the highest levels soybean [Glycine max (L.) Merr.]. The simultaneous delivery of two of 27.2% GLA and 8.7% STA being observed in stem T-DNA elements was used as a strategy to derive soybean progeny transgenic for the ⌬ 6 desaturase T-DNA and free of the marker gene tissue (Sayanova et al., 1999). Introduction of a fungal T-DNA. Twenty-nine transgenic soybean lines were recovered that (Mortierella alpina Peyronel) ⌬ 6 desaturase into canola harbored both T-DNA elements, of which 17 produced GLA and (Brassica napus L.) resulted in accumulation of GLA STA in the seed storage lipids. Average GLA levels ranged from 3.4 in the seed to approximately 13%, while dual expression up to 28.7%, while STA levels varied from just under 0.6 to 4.2% in of the fungal ⌬ 12 -and ⌬ 6 -desaturase genes resulted in the T 1 generation. Among the 17 lines that produced GLA and STA, elevated levels of GLA in seeds, 43%, and production four lines were identified that were free of the selectable marker of STA levels of ≈2% (Liu et al., 2001). The combination T-DNA element.of ⌬ 12 -and ⌬ 6 -desaturase genes was required to enhance GLA levels in canola since the predominate fatty acid in B. napus seed is oleic. Expression of the ⌬ 12 desaturase Univ. of Nebraska, Lincoln, NE 68583; B. Schweiger and A. Kinney, DuPont Exp. Stn., Wilmington, DE 19880. This publication is a contri-nology by implementing the identical marker gene for

Through a single desaturation step, the Borago officinalis L. Δ6 desaturase can convert linoleic acid and α‐linolenic acid to γ‐linolenic acid (GLA) and stearidonic acid (STA), respectively. Both GLA and STA are of interest to the pharmaceutical and nutraceutical industries. Production of these fatty acids is costly. One potential strategy to reduce production cost would be to generate them in a major oilseed crop. To this end, a cDNA of the B. officinalis Δ6‐desaturase gene was cloned downstream of the embryo‐specific promoter β‐conglycinin. The resultant cassette was assembled into a two T‐DNA binary vector, in which the second T‐DNA element harbored a selectable marker cassette. The final plasmid was subsequently used to transform soybean [Glycine max (L.) Merr.]. The simultaneous delivery of two T‐DNA elements was used as a strategy to derive soybean progeny transgenic for the Δ6 desaturase T‐DNA and free of the marker gene T‐DNA. Twenty‐nine transgenic soybean lines were recovered that harbored both T‐DNA elements, of which 17 produced GLA and STA in the seed storage lipids. Average GLA levels ranged from 3.4 up to 28.7%, while STA levels varied from just under 0.6 to 4.2% in the T1 generation. Among the 17 lines that produced GLA and STA, four lines were identified that were free of the selectable marker T‐DNA element.

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