ABSTRACT

Oil crops are important sources of energy, both for human consumption and feeding livestock. They are also sources of many non-edible purposes providing raw material for a wide range of industrial products. Rapeseed (Brassica napus) with Canola quality, i.e. low-glucosinolate, low-erucic varieties, nowadays represent one of the world’s major sources of vegetable oil. The value of rapeseed for food and feed uses can be further improved by increasing desirable traits, e.g. oil content, and reducing undesired characteristics, e.g. fiber content or anti-nutritional compounds. Alternatives for industrial processing and non-food purposes are, for example, high-erucic acid low-glucosinolate rapeseed varieties or laurate Canola.

Biotechnological approaches combined with classical breeding schemes offer a wide spectrum of methods, such as generation of doubled-haploid lines, interspecific hybridization and molecular marker techniques, which are altogether involved in developing improved basic stocks and cultivars possessing novel desirable traits. However, further adjustments of rapeseed quality will not be realized satisfactorily without the assistance of genetic engineering. A variety of novel traits have already been introduced into rapeseed and are evaluated in field trials, including novel pollen control systems for hybrid breeding, herbicide tolerance, modified seed quality, and many others. It is expected that so-called conventional and modern methods will be used in a synergistic way in future rapeseed breeding.

INTRODUCTION

The value and utility of an oilseed crop for both nutritional and industrial purposes primarily depends upon the fatty acid composition of the seed oil. During the last decade the aim of breeding programs for improved quality and versatility in oil crops has been the acquisition and application of knowledge on the variation as well as the genetic and biochemical control of fatty acid composition in order to obtain tailor-made raw materials suitable for either nutritional or industrial purposes.

STRATEGIES IN BREEDING FOR QUALITY

Together with soybean (Glycine max), rapeseed (Brassica napus) benefits from molecular genetics and is amenable to biotechnological methods (Fig. 1). Consequently, a couple of different breeding procedures, i.e., haploid techniques such as microspore culture for the production of doubled-haploid lines, wide hybridizations using embryo rescue techniques, or protoplast fusion are involved in creating novel genetic variation. Once a useful property has been identified in a basic breeding stock, e.g., a mutant line or germplasm from a wild relative, it may take many years to accomplish the development of cultivars possessing this novel desirable trait. Marker-assisted selection has shown a significant impact on the efficiency of plant breeding routines such as backcrossing programs. Out of the wide range of currently available biochemical and molecular markers techniques, RFLPs, RAPDs, AFLPs, and microsatellites (single sequence repeats) are likely to have the greatest effect in crop breeding programs (cf. Lydiate et al. 1995, Cheung and Landry 1996).

Furthermore, in those cases where conventional approaches have not been sufficient further improvements of oil composition can be achieved by genetic engineering, e.g., used for transferring specific foreign gene(s) between distant species or for efficiently inhibiting inherent genes. A variety of novel traits have already been introduced into rapeseed and other crop plants and have been evaluated in field trials since the early 1990s including genetically engineered hybrid systems, tolerance to broad-spectrum herbicides, modified oil and protein composition, and many other traits increasing the economic value of the rapeseed crop. In comparison to the more restrictive situation in Europe, field testing and admission to the market of novel agricultural products in North America

continues its rapid pace (cf. Friedt and Lühs 1998).

IMPROVING THE NUTRITIONAL AND HEALTH VALUE OF RAPESEED OIL

Regarding the nutritional use of vegetable oils the development of new oilseeds, using either biotechnology or traditional plant breeding methods has always been driven by consumer and food industry demands, which were the result of current scientific nutritional recommendations. These needs have led to the commercialization of several oilseeds with modified fatty acid profiles, including double-low rapeseed (Canola), low-linolenic soybean and linseed as well as high-oleic sunflower. These opportunities broaden the use of commodity crops and allow the exploitation of high-value niche markets.

Quality breeding in rapeseed (B. napus) is one of the best examples of the influence of nutritional concerns upon fatty acid modifications in oilseeds. Feeding high-erucic acid rapeseed (HEAR) oil - containing about 45-50% of its total fatty acids as erucic acid (22:1n-9) - to rats resulted in myocardial damage being characterized by fatty deposits and tissue scarring. Despite firm evidence that this fatty acid was more a threat to rats than to man, these nutritional concerns led to the development of rapeseed oil with low 22:1n-9 levels by using traditional selection breeding techniques (cf. Ackman 1990, Lühs and Friedt 1994a).

Today, the oil of modern rapeseed varieties almost lacking nutritionally undesirable long chain fatty acids is highly appreciated due to its fatty acid profile, which meets new health recommendations to reduce total dietary saturated fat intake. The current interest in the nutritional and health effects of fatty acids, such as laurate (12:0), myristate (14:0) and palmitate (16:0), relates to evidence associating high intake of these fatty acids in the diet with increased levels of blood cholesterol, arteriosclerosis and high coronary heart disease risk (Grundy and Denke 1990, Gurr 1992, Hu et al. 1997). With regard to commonly consumed vegetable oils and fats low-erucic acid rapeseed or Canola oil contains the lowest level (ca. 6-7%) of saturated fatty acids (Table 1). Furthermore, containing 58-60% of its total fatty acids as oleic acid (18:1n-9) it is an important source of this monounsaturated fatty acid (MUFA), the nutritional significance of which has increased during the last 15 years (Mudson and Grundy 1985, Dupont et al. 1989, Valsta et al. 1992, Marsic et al. 1992, McDonald 1995, Trautwein 1997). These new insights into the high nutritional quality of Canola oil was pushed by its introduction in the United States market by receiving the GRAS (Generally Recognized as Safe) status in 1985, and is continuing today by the admission to the market of low-saturated fatty acid oilseeds, often accompanied by the low-linolenic and/or the high-oleic fatty acid trait(s).

Contrary to the nutritional necessity of certain n-6 and n-3 polyunsaturated fatty acids (PUFA), Canola or soybean oil containing 8-10% α-linolenic acid (18:3n-3), are more liable to rapid oxidative damage than oils with little or no 18:3n-3 (Table 1). For example oxidation of linoleate (18:2n-6) and linolenate is approximately 10 and 25 times higher, respectively, than that of oleic acid (Frankel 1991, Kinsella 1991, Carlson 1995, Horrobin 1995, Lands 1997). The low-linolenic acid trait was created in rapeseed (B. napus) using chemical mutagenesis and subsequent selection for altered ratios of linoleic/linolenic acid facilitated by rapid screening methods like the thiobarbituric acid(TBA)-test. Above the release of several Canadian spring rapeseed varieties (‘Stellar’, ‘Apollo’ and ‘Allons’) it was only recently achieved to transfer this trait into suitably adapted winter rapeseed lines (Rakow 1973, Rücker and Röbbelen 1996, Scarth et al. 1997). To increase the oleic acid content to above 80% and, concomitantly, lower the level of PUFA different breeding procedures have been utilized, including mutagenesis applied to seeds (Auld et al. 1992, Rücker and Röbbelen 1995) or microspore-derived embryos (Wong et al. 1991).

Table 1: Fatty acid profiles of specific rapeseed genotypes in comparison to soybean

Oil type/seed variety	Origin/method	12:0*	14:0	16:0	18:0	18:1 n-9	18:2 n-6	18:3 n-3	20:1 n-9	22:1 n-9	Others
Rapeseed
High-erucic acid rapeseed	Traditional	-	-	3	1	11	12	9	8	52	4
Double-low / Canola	Spontaneous mutant	-	-	4	2	60	21	10	1	1	1
Low-linolenic Canola	Mutagenesis	-	-	4	2	61	28	3	1	-	1
Laurate Canola	Genetic engineering	37	4	3	1	33	12	7	-	-	3
High myristate /palmitate	Genetic engineering	-	18	23	2	34	15	4	-	-	4
High-oleic Canola	Mutagenesis / transgenic			4	1	84	5	3	1	-	2
Soybean
Conventional	Traditional	-	-	11	4	23	54	8	-	-	-
Low linolenate	Mutagenesis	-	-	10	5	23	60	2	-	-	-
High palmitate	Mutagenesis	-	-	17	3	17	55	8	-	-	-
High stearate	Mutagenesis	-	-	8	28	20	35	7	-	-	2
High oleate	Genetic engineering	-	-	7	4	85	1	2	-	-	1

* fatty acids: 12:0 = lauric, 14:0 = myristic, 16:0 = palmitic, 18:0 = stearic, 18:1n-9 = oleic, 18:2n-6 = linoleic, 18:3n-3 = α-linolenic, 20:1n-9 = eicosenoic, 22:1n-9 = erucic acid

Furthermore, relevant genes of Δ12 or Δ15 desaturases have been isolated and subsequently rapeseed has been genetically engineered leading either to high-oleic acid or high-linoleic acid profiles (cf. Hitz et al. 1995, Scheffler et al. 1997).

Vegetable oils with a high 18:1n-9 and/or low 18:3n-3 content are marketed for bottled salad oil and salad dressings as well as for food applications requiring high cooking and frying temperature stability including extended shelf-life products (such as snack foods). Increasing the 18:1n-9 content while reducing PUFA levels decreases the development of unpleasant flavors and off-odors indicating oil rancidity (Warner and Mounts 1993). Providing oxidative stability without extensive hydrogenation high-oleic and low-linolenic oils are being developed primarily to reduce trans-fatty acids formed during the hydrogenation of vegetable oils. Nutritional research suggests that these geometrical isomers of cis-fatty acids may have negative nutritional effects. Trans-fatty acids appear to increase serum low-density lipoprotein (LDL) cholesterol levels and may reduce serum high-density lipoprotein (HDL) cholesterol levels to a greater extent than saturated fatty acids (Mensink and Katan 1993). These concerns led to the recommendation to reduce their amount in the diet. Since trans-fatty acids are unavoidable during oil-processing, there will be an increased need for MUFA oils which do not require extensive hydrogenation (Hu et al. 1997).

Certain food products requiring oils with specific uses, such as baking shortenings and margarines, high-saturated fatty acid oils are offering a slight advantage to the potentially negative health impact of hardened oils, specifically trans-fatty acids. Canola and soybean oils with high saturated fatty acid levels are being developed to replace animal fats and tropical oils in margarines and confectionery products (cf. Lühs and Friedt 1994a). High palmitic lines of B. napus Canola have been developed by the introduction of a 16:0-ACP thioesterase gene isolated from Cuphea hookeriana (Jones et al. 1995). The introduction of the ClFatB4 gene from C. lanceolata resulted in transgenic rapeseed with a high sum (more than 40%) of 14:0 and 16:0 (cf. Rudloff and Wehling 1998). High-laurate Canola was developed by Calgene Inc. using the acyl-ACP thioesterase isolated from the California Bay Laurel (Umbellularia californica). It was the first transgenic oilseed crop produced commercially in the world in 1995. This specialty rapeseed oil contains about 40 wt-% lauric acid and is - due to its specific triacylglycerol composition - suitable for use in the confectionery industry, food coatings, simulated dairy products, bakery fillings and icings, dressings and spreads (cf. Friedt and Lühs 1998).

NUTRITIONAL VALUE OF CANOLA MEAL

Extracted rapeseed meal contains about 40% protein with a well balanced amino acid composition. It can supplement beef and swine feed rations, but is not preferred as a sole source of animal feed. Along with glucosinolates the quality of rapeseed meal is influenced by other anti-nutritional factors, including sinapine, phenolic acid and tannins as well as phytic acid. In the case of sinapine the production of trimethylamine off-flavor by susceptible hens limits the usability as poultry feed. Tannins exist mainly in the seed coat and are more abundant in dark than yellow seed coats. These compounds are known to interfere with digestive enzymes, especially those affecting protein hydrolysis. Phytic acid is found mainly in the embryo and is of interest in its role of binding phosphorus as well as other essential minerals (Uppström 1995, Griffiths et al. 1998, Matthäus 1998, Naczk et al. 1998, Velasco and Möllers 1998). Besides these anti-nutritional compounds, crude fiber and substances enclosed by them affect adversely the usability of rapeseed meal in the nutrition of monogastric animals. Regarding the reduction of crude fiber content and a concomitant increase of digestible energy and the proportion of protein, the introgression of the yellow seed trait into B. napus would have a significant impact on the quality of the Canola crop and the processed products from the seed as well as on the competitiveness compared with soybean meal (Bell and Shires 1982, Bell 1993, Slominski et al. 1994).

Research and breeding programs are underway to improve the nutritional value of rapeseed meal through the development of yellow-seeded rapeseed varieties. The combination of genetic and environmental factors gives rapeseed B. napus a range of seed colours varying from black to yellow. Regarding the introgression of genes encoding seed pigmentation, for example from related Brassica species, the situation in B. napus can be complex due its polyploid genetic constitution, predominantly maternal inheritance as well as multiple gene control, respectively (Rashid et al. 1994, Tang et al. 1997, Meng et al. 1998). Depending on the source of yellow seediness in most of the cases three genes are involved in the genetic control of testa colour (Shirzadegan 1986; Henderson and Pauls 1992, Van Deynze and Pauls 1994; Van Deynze et al. 1995). In general, only pure lines being completely homozygous for recessive alleles at all three loci breed true for yellow seed colour. That means, individuals possessing mottled yellow seeds, which are very common in B. napus, display segregation in descending generations. The genetic stability of the yellow seed trait is being established but selection for yellow seediness is still difficult due to pronounced environmental effects, such as temperature (cf. Van Deynze et al. 1993).

SPECIALTY RAPESEED OIL WITH UNCOMMON FATTY ACID FUNCTIONALITIES

Oils and fats now available for the oleochemical industry and other non-food applications are derived in large part from those usually used for food purposes. But these are not always the best starting materials for the production of oleochemicals. From an industrial point of view, the usability of a seed oil is targeted towards only one of its constituent fatty acids. A maximum content of the desired fatty acid will not only decrease the amount of waste, but it can result in considerable savings in downstream processing costs, too. Despite the unique role of lauric oils the majority of fatty acids used for industrial purposes consists of unbranched hydrocarbon chains with a range from 16 to 18 carbons. They are further transformed by chemical reactions involving either the carboxyl group or the hydrocarbon chain. On a commercial scale nearly all, i.e., 96% of these reactions are directed to the derivatization of the carboxyl group, only 4% deal with the modification of the hydrocarbon side chain (cf. Lühs and Friedt 1994b). On the other hand, hundreds of seed oils containing unusual fatty acids have been described so far, indicating a broad flexibility with regard to storage lipid biosynthesis in the plant kingdom. Thus, chain length is actually just one fatty acid property that can be altered by metabolic engineering. Functionalities such as the degree of desaturation or the positions and stereochemistry of double bonds also have the potential to be altered by recombinant DNA technology including sense or antisense suppression and overexpression of heterologous enzymes. Furthermore, increased flexibility and new raw materials for industrial purposes have to come from genetically engineered plant oils providing fatty acids with unusual chain lengths and/or functionalities, such as unique double bond positions or functional groups, e.g. hydroxy, epoxy, acetylenic, or keto groups, respectively. A prominent example in this context is the aim to develop rapeseed with unsaturated hydroxy fatty acids in its seed oil, which are usually found in castor beans (Ricinus communis) or cruciferous desert plants belonging to the genus Lesquerella. Recently, the expression of a cDNA encoding the oleate 12-hydroxylase from castor bean in transgenic Arabidopsis thaliana resulted in an accumulation of up to 17% hydroxy fatty acids including ricinoleic (18:1-OH), lesquerolic (20:1-OH) and densipolic acid (18:2-OH). Similar efforts in the area of epoxy fatty acids currently focus on cloning the responsible linoleate epoxidase gene from Euphorbia lagascae, a wild spurge native to Spain, with the goal of producing oil rich in vernolic acid (18:1epoxy) (cf. Friedt and Lühs 1998).

CONCLUSION AND PERSPECTIVES

One of the most important objectives in rapeseed breeding has been the genetic modification of seed quality by changing the proportion of fatty acids suitable for either nutritional or industrial purposes (Lühs and Friedt 1994a, Friedt and Lühs 1998). Nutritional concerns, functionality in food manifacturing, and the need for high stability and extended shelf life have had a tremendous impact on developing and commercializing modified oilseeds, so far. Commodity oilseeds now available include low-linolenic Canola and soybean oils as well as high-oleic sunflower oils. Food processors will soon have access to high-oleic soybean oils (Kinney and Knowlton 1998) as well as high-oleic/low-linolenic Canola oil. For products requiring oils with specific uses without hydrogenation, such as baking shortenings and margarines, high-saturated fatty acid oilseeds are an option. Genetically modified oilseed variants will gain more importance in the food sector in order to develop structured lipids which allow fat tailoring and functional food designing, to introduce or to improve desirable compounds, such as long-chain PUFA and tocopherol content, or to reduce anti-nutritional constituents like sinapine or phytate (Goddijn and Pen 1995, Knauf and Facciotti 1995, Fitch Haumann 1997, Shintani and DellaPenna 1998). Although market signals are often difficult to predict, one can expect the trend towards healthier fats and higher stability oils to continue.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge financial support of their projects dealing with industrial rapeseed breeding by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF), the Bundesministerium für Ernährung, Landwirtschaft und Forsten (BML), and the Gemeinschaft zur Förderung der privaten deutschen Pflanzenzüchtung e.V. (GFP), Bonn/Germany.

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