Understanding developmental biology opens vastopportunities for designing novel feed and food:

A discussion of potential new plant products

HILDE-GUNN OPSAHL-SORTEBERG1, HEIDI RUDI1, STEIN ERIK LID1

AND ALAN H. SCHULMAN2,3

1Laboratory for Molecular Plant Biology, Department of Plant and Environmental Sciences,Agricultural University of Norway, P.O. Box 5003, N-1432 Ås, Norway

2Plant Breeding Biotechnology, MTT Agrifood Research Finland, Myllytie 10, FIN-31600 Jokioinen, Finland

3MTT/BI Plant Genomics Laboratory, Institute of Biotechnology, University of Helsinki,Viikki Biocenter, P.O. Box 56, Viikinkaari 4, FIN-00014 Helsinki, Finland

Abstract

Environmentally and economically sustainable food production remains a world-wide challenge, as does providing consumers with agricultural products that meettheir demands for food safety, quality and healthfulness. Three developments, how-ever, offer plant breeders help in meeting these challenges: increasingly effectivemolecular marker-assisted selection (MAS), improved understanding of biochemi-cal pathways through functional genomics and more efficient genetic transforma-tion. In addition to protein, carbohydrates and lipids are the major storage com-pounds of tubers and seeds. These provide both the basis of human nutrition andthe objects of attention for genetic engineering. We give examples here of the majorforces affecting plant breeding: consumer product development on the one handand sustainability on the other. First, we focus on the engineering of starch biosyn-thesis for improved quality and tailored properties. In addition, we discuss the useof plant transformation to alter the lipid complement of oilseeds in order to pro-vide a sustainable feed source for fish farming. As products of both types enter themarket, the public will be faced with the conundrum of crops that are “greener” inthe sense of environmentally safer and more healthful but which are simultaneous-ly opposed by some sectors of the environmentalist movement.

Tuberosa R., Phillips R.L., Gale M. (eds.), Proceedings of the International Congress “In the Wake of the Double Helix: From the Green Revolution to the Gene Revolution”,27-31 May 2003, Bologna, Italy, ......................., ©2004 Avenue media, Bologna, Italy.

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Introduction

An important challenge concerning all the earth’s inhabitants is to establish and main-tain environmentally and economically sustainable food production. Farmers needcrops and production strategies that are both environmentally friendly and that havethe quality and cost-effectiveness able to meet marketplace competition and chang-ing consumer demands (Lyson 2002). At the other end of the production chain, con-sumers are concerned about food safety, quality and healthfulness. Ultimately, plantbreeders are called upon to provide farmers with improved varieties so that these var-ied and sometimes conflicting requirements can be fulfilled. Increasingly, traditionalbreeding methods are not sufficiently rapid or efficient for such development. Newmethods to increase the speed and accuracy of selecting for recombination betweendesired segments of the genome, such as the use of molecular markers inmarker-assisted selection (MAS), promise to shorten the breeding cycle. Furthermore,a more thorough understanding of the genetic and biochemical basis of plant pro-duction will help breeders target the genes that need to be selected and to estimate thelikelihood of pyramiding particular traits (Koebner and Summers 2003).

At the same time, research into basic biological processes has led to an explosionof new methods, data and biological understanding over the last decade (Tuberosaet al. 2002; Hammond-Kosack and Parker 2003; McDowell and Woffenden 2003).Entire genomes have been sequenced and the new disciplines of genomics, pro-teomics and systems biology have emerged (Goff et al. 2002). Our knowledge ofhow genes contribute to and control plant development and how the componentsof metabolic pathways interact has expanded enormously (Chaudhury et al. 2001;Ye et al. 2002; Zik and Irish 2003). To illustrate the short distance between basicresearch and applied research, one can look at the aleurone cells surrounding thecereal starchy endosperm (Becraft et al. 2001). Unraveling the genetics of aleuronecells allows us to understand cell specification and development; at the same timethis information may be exploited to increase the number of aleurone cells and seedoil content (Lid et al. 2002; Shen et al. 2003). Moreover, functional genomics,where one seeks to analyse the function of all genes and gene products in an organ-ism, is greatly adding to this knowledge and providing new tools in genetics andbiotechnology (Morgante and Salamini 2003). Our interest in understanding thebiological basis of life has merged with the need for products, such as crops, derivedfrom living creatures. Basic research can no longer be readily separated from appliedresearch. Furthermore, application of biological processes to develop new products(i.e. biotechnology) will require the concerted efforts of a broad scientific commu-nity, together with experts in bioethics, regulatory authorities, policy makers, con-sumer groups and the general public.

Traditionally, gene function has been inferred from studying the phenotypiceffect of natural or induced mutations. The era of whole-genome sequencing pro-

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jects has brought complete or close-to-complete DNA nucleotide information foran increasing number of organisms, lifting us to a new level of understandingregarding life processes (Arabidopsis Genome Initiative 2000; McPherson et al.2001; Yu et al. 2002). Sequence information together with technological inventionsmake it possible to take a holistic view of life processes by putting all genes repre-senting an organism onto one or a few DNA chips (microarrays). Transcriptionprofiling on microarrays, combined with large-scale protein analysis (proteomics),allows for the monitoring of the activity of virtually all genes in a species, and hasthe potential to generate systematic maps of the networks connecting all the genesand gene products involved in the various biological pathways (Aharoni and Vorst2002; Tabata 2002). The biological significance of such information can be furtherexplored by so-called reverse-genetics technology, whereby the function of specificgenes is altered, allowing us to verify their role. Following the first report onhigh-throughput RNA profiling using a microarray (Schena et al. 1995), biologyhas moved from being an information-poor to an information-rich discipline. Thischange first affected microbiology and biomedicine, followed by model systemssuch as Arabidopsis, and is currently making an impact on plants of agricultural sig-nificance (Rhee et al. 2003). By using the data and tools now available, we are inthe position to achieve the level of understanding of the developmental genetics ofplants necessary to devise strategies for sustainable and healthful production of foodand feed.

We shall illustrate the new tools available in genetics and biotechnology byfocussing on two important storage compounds, oils and starch, produced in twomajor classes of crops, respectively oilseed crops and cereals. Here, we shall use theterms “transgenic” and “genetic engineering” due to their precision over the common“GM” and “GMO” for genetically modified organisms. All natural and human selec-tion, with or without biotechnology, involves genetic modification; this is a pointoften lost. The term “transgenic” is problematic when the gene transferred is derivedfrom the species to be engineered and not from a distantly related organism. Howev-er, we shall use the term also for these cases due to conventional practice.

Seeds for life

Human nutrition depends on the storage products of plants, those compounds plants“place in the bank account” for the next generation. These are found in roots, tubers,or seeds and include starch and carbohydrates, oils and lipids, and proteins, as well ascertain secondary metabolites, minerals and vitamins. Cereal seeds represent ourmajor renewable energy resource for food, feed and industrial raw material. In tem-perate regions, potato is an important source of starch and protein, whereas edibleand industrial oil is produced from rapeseed and turnip rape as well as from soy,maize, sunflower, cottonseed and safflower. Within seeds, the various storage prod-

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ucts and metabolites are produced in different cell types or tissues. An understandingof the basic biology of these tissues enables a targeted approach to be taken to theimprovement of the tuber or seed as a food or industrial feedstock. We shall take cere-al grains and rapeseed as our examples of this connection related to starch and oil pro-duction, respectively.

Starch is one of the most important plant products to man and the main sourcesof starch are the cereal crops, rice, maize, wheat and root crop potato (Hills 2004).One way of modifying the starch quantity and quality is to modify the committed,or terminal, segment of the starch biosynthetic pathway (Slattery et al. 2000). How-ever in order to do this we need a better understanding of the individual enzymes andtheir interactions with the other pathways of carbohydrate metabolism in storage tis-sues and the whole plant (Figure 1). The application of biochemistry, genetics, andtransformation technologies together have transformed our view of starch biosynthe-sis over the last decade (reviewed by James et al. 2003; Hills 2004). ADP-glucosepyrophosphorylase (AGP) is a key enzyme in the biosynthesis of starch in plants andglycogen in bacteria. The AGP enzyme has been examined in many different plantspecies, including lower plants, monocots and dicots, and is multimeric, consisting oftwo small and two large subunits encoded by distinct genes (Preiss et al. 1991). Inbarley there are two genes encoding the large subunits and at least two genes encod-ing the small subunits (SSUs; Thorbjørnsen et al. 1996; Johnson et al. 2003). Sever-al mutants, including Risø16 in barley, have been identified with reduced starch con-tent. This mutant has a deletion in one of the SSU AGP genes, and contains 31% ofthe normal AGP activity and less than 80% of the normal amount of starch (John-son et al. 2003). The relative contribution of the various SSU transcripts in differenttissues and developmental stages has been analysed in mutant and wild-type barleyusing Quantitative Real Time PCR (QRT-PCR) and transcriptional profiling (H.Rudi et al., unpublished results). The conclusion is that the distinct SSU transcriptshave different expression levels in different tissues and developmental stages and thatboth the plastidial and cytosolic located SSU isoforms are important for starch syn-thesis in seeds. The variation in AGP activity and starch accumulation rates seen inmaize and wheat grains during grain filling may be explained by a simultaneouschange in the subunit quantity of both AGP subunits, possibly giving the transcrip-tional control of AGP expression in grains. The AGP enzyme catalyses the rate limit-ing step in the starch biosynthesis. It is allosterically regulated by the ratio of 3-phos-phoglyceric acid (3-PGA) to inorganic phosphate (Pi) in photosynthetic tissues. Theregulatory properties in cereal endosperms are not fully clear and the AGP in wheat(Gomez-Casati and Iglesias 2002) and barley (Doan et al. 1999) is relatively insensi-tive to these regulators. Thus, the AGP isoform in the endosperm of barley has thepotential to increase starch quantity and maybe as well starch quality in importantcrops. This view results from expression analyses of the cytosolic AGP transcripts,showing that they follow the starch accumulation in developing grains (Doan et al.1999; H. Rudi et al., unpublished results).

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Figure 1. Simplified representation of the pathways for starch and lipid biosynthesis in seeds. Thediagram is generalized for grains and oilseeds, and summarizes our current understanding (James etal. 2003; Hills 2004). Sucrose (suc) enters into storage tissues via the phloem. In some plants, cellwall invertase (1) creates an influx gradient through cleavage of sucrose into glucose (gluc) and fruc-tose (fruc). Sucrose can then be resynthesized and translocated into vacuoles (transporters are shownas grey lozenges) where it can be polymerised to fructans by various transferases. In grains, fructansmay serve as a transitory storage pool that is turned over for starch biosynthesis as development pro-ceeds. Sucrose is converted to UDPglucose (UDPgluc) by sucrose synthase (2). UDPglucose is fur-ther converted to Glucose-1-phosphate (G-1-P) by UDPglucose pyrophosphorlyase (3), which canbe interconverted to other hexose or triose phosphates. Hexose phosphates are translocated into theplastid where glucose-1-phosphate is converted into ADPglucose (ADPgluc) by ADPglucosepyrophosphorylase (AGP, 4), representing the first committed step in starch biosynthesis. In cere-als, AGP can also be found in the cytoplasm, whereby ADPgluc is synthesised and then translocat-ed into the plastid. ADPgluc is the substrate for synthesis of amylose primarily by granule-boundstarch synthase (GBSSI, 5) and of amylopectin by the combined actions of soluble starch synthases(SS, 6) and starch branching enzymes (SBE, 7). Recent work shows that the hydrolytic debranch-ing enzymes (8) and isoamylase (9) play an important role in forming the final amylopectin struc-ture of clustered branch points and in starch granule initiation. Lipid biosynthesis proceeds via pyru-vate, which may be formed in plastids or translocated in. Pyruvate is converted to fatty acids throughthe combined actions of acetyl-Co-A (10) and fatty acid synthase (11). Fatty acids can be translo-cated out of the plastid to the cytoplasm, where their acyl-CoA derivatives, together with glyc-erol-3-phosphate (G-3-P), are converted into diacylglycerol (DAG). This is carried out within theendoplasmic reticulum (ER) by acyltransferases (12). The diacylglycerols serve either in the pro-duction of phospholipids (PC) for cell membranes or for synthesis of triacylglycerols (TAG) for stor-age in oil bodies. Other metabolites: phosphoenolpyruvate (PEP), malonyl-CoA (mal-CoA). Fattyacid biosynthesis and interconversions are detailed in Figure 3.

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Whereas quantitative control of starch biosynthesis affects crop yields and ulti-mately the amount of food produced, starch quality determines the uses of thecrop. Starch structure, formed by the starch synthases, starch branching enzymesand debranching enzymes (Figure 1), determines the crystallinity and thereby thegelatinization properties of starch. These in turn affect the performance of starch insuch diverse applications and parameters as bread baking and staling, gels, thicken-ings and puddings, the ease to which starch is converted to sugar during digestionand hence the strength of the hypoglycemic response, and the fermentability ofstarch to alcohol in beverage production. The subject has been extensively reviewed(Schulman et al. 2000; Ball and Morell 2003; Emes et al. 2003; James et al. 2003).

Starch is comprised of linear, alpha-1,4-linked chains of glucose. The linearchains are formed by the starch synthases, and can be linked to each other throughalpha-1,6 bonds, which are formed by the starch branching enzymes (Figure 1).The lengths of the chains and the position and frequency of the 1,6 bonds deter-mines the properties of the starch. The two major types of starch, amylose and amy-lopectin, differ in their degree of branching and enzymes responsible for theirbiosynthesis. The alpha-1,4 backbone of amylose, which is rarely branched, is madeby the granule-bound starch synthases, whereas that of amylopectin, which is moreextensively branched, is formed by the soluble starch synthases. A major differencebetween potato and cereal starch is that the former is phosphorylated (Blennow etal. 2002). This affects both its behaviour as a gel and its fermentability. For morethan a decade, intense efforts have been made to understand the role of the variousisozymes in producing the particular starch structures typical of different species,cultivars and mutants (Figure 2), with an eye to eventually engineering starch tohave specific properties. The investment made in basic research of starch biosyn-thesis has now begun to produce advances in the engineering of starch structure(Slattery et al. 2000; Schulman 2002; Kok-Jacon et al. 2003).

Feed for aquaculture: A global food chain

One tends to think of crops as destined for consumption by humans as food orby farm animals as feed. However, this ignores a large and growing industry, that ofaquaculture. The UN Food and Agriculture Organization (FAO) expects aquaculture to increase global food fish supplies and to further help to reduce glob-al povety and food insecurity over the next two decades (www.fao.org/fi/meetings/aq2000/tech_proc/third_mill.asp). The expansion of aquaculture is furthersupported by a growing demand for meat in parts of the world that previously sub-sisted on a largely vegetarian diet. In addition, the western world displays anincreasing demand for fish as a replacement for beef, and aquaculture is serving tocompensate for shortages in wild fish stocks. In part, the wide-range of health-pro-moting properties of the omega-3 fatty acids found in fish oils (Sayanova and Napi-

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er 2004) attracts a growing number of consumers. Taken together, the increaseddemand in aquaculture leads to a major challenge: the delivery of a stable, pre-dictable and high-quality feed supply able to sustain the industry in the future.

Lipids are the major energy substrate in feed for farmed carnivorous cold-waterfish species. From an energetic point of view, a 40% lipid content is optimal in thefeed. Plants are a rich source of C18 fatty acids, and the essential fatty acids linole-ic and alpha-linoleic can be synthesized by plant but not by animals (Sayanova andNapier 2004). Likewise, plants in general lack the capacity to convert C18 to C20fatty acids. Regarding the metabolism of the fish, it is important that a sufficientamount of eicosapentaenoic acid (EPA) or docosahexeanoic acid (DHA) be present(Figure 3). The C20 fatty acids enter the food chain of fish through the consump-tion of aquatic micro-organisms. For carnivorous fish, this is supplied mainly as fishoil rich in long-chain n-3 polyunsaturated fatty acids (PUFAs).

The demand for fish oil is increasing rapidly not only due to increased aquacul-

Figure 2. Starch granules in mature barley endosperm. The large (A-) and small (B-) granules arevisible in this scanning electron micrograph of barley endosperm 40 days post anthesis. The mutantshx in cv. Bomi background is shown, which despite smaller than normal A-granules still retains thebiphasic distribution typical of the Triticeae. Courtesy of Marko Jääskeläinen, University of Helsin-ki. Bar is 20 µm

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tural production, but also from new “consumers” such as poultry producers. Thisingredient is therefore expected to increase in price, and the demand to exceed thesupply, in the near future. As a consequence, the use of vegetable oils as a feed sup-plement for farmed carnivorous cold-water fish is increasing. Although beneficial insome aspects, this is not without complications. The increased demand for plantfeed sources for animals in general, together with new insights in plant genetics andgenomics, create, respectively, the motive and the means for exploring plants thatyield storage compounds such as lipids that are tailored for applications in feed(Thompson 2000). These tailored products will in many cases be derived fromgenetic engineering.

The viability of animal production rests on many factors, including accessibili-ty, costs, optimal effects of feed, and finally consumer demands. Aquaculture mightbe made more sustainable through the use of feed with fatty acids supplied fromtransgenic crops, rather than from harvesting wild sea resources. Furthermore,land-based fish production based on transgenic crops could both increase controlover the availability and quality of feed and reduce biological contaminants derivedfrom ocean fish. Thus, consumers could choose fish products, raised on feed that isbased on transgenic plants, that offer both environmental and health benefits over

Figure 3. Fatty acid metabolism in plants and animals. Fatty acid interconversions and the keyenzymes involved are shown on the left. Plants generally only produce C18 fatty acids, which arethe dietary source of fatty acids for herbivorous animals. See text for further details. The Figure hasbeen modified from Opsahl-Ferstad et al. (2003).

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products currently on the market (Opsahl-Ferstad et al. 2003).Lipid digestibility by rainbow trout remains fairly constant (≈ 90%) for lipid

sources with melting points < 10 °C, but drops dramatically at higher meltingpoints (Austreng et al. 1979). As a consequence, lipids with low melting points(oils) are preferred in diets for salmonids, and probably other cold-water fish aswell. This favours vegetable oils from oilseeds, which generally have melting pointswell below 0 °C. Vegetable oils are also rich in C18 fatty acids, while the longer“marine” n-3 PUFAs (20:5, 20:6, 22:6) are absent. In rainbow trout, C18 fattyacids are oxidised for energy production at a much higher rate than C20 and C22fatty acids (Henderson and Sargent 1985), as in mammals (Leyton et al. 1987).Thus, vegetable oil should be a good energy source in feeds for cold-water fish, bothbecause of its low melting point as well as its favourable fatty acid compositionregarding oxidation potential.

Fish, like other animals, do not possess the metabolic enzyme systems (desat-urases) that are necessary for the formation of 18:2 n-6 and 18:3 n-3 from 18:1 n-9fatty acids (Figure 3). Those fatty acids are therefore considered essential con-stituents of the diet. Freshwater and temperate fish species, including salmonids,generally possess the ability to desaturate and elongate 18:2 n-6 or 18:3 n-3. How-ever, several strictly marine fish species lack a ∆-6 desaturase, and thus the abilityto desaturate and elongate 18:3 n-3 to “marine” fatty acids with 20 or 22 C-atoms(20:4 n-6, 20:5 n-3 and 22:6 n-3), which are biologically active as polar membrane(phospho) lipids and eicosanoid precursors. In consequence, such species must havetheir fatty acid requirement satisfied by dietary n-3 PUFAs.

Vegetable oils for fish should contain a minimum of 18:2 n-6. Furthermore, ahigh level of 18:1 n-9 is desirable, whereas the level of 18:3 n-3 should be moder-ate. These criteria are currently best met by oil from rapeseeds (Brassica napus),although even rapeseed oil contains 16-25% 18:2 n-6. Bell and co-workers (Bell etal. 2001) showed that rapeseed oil could be used successfully as a substitute for fishoil in diets of Atlantic salmon at a level of < 50% of dietary lipid. A fish oil alter-native for carnivorous fish should furthermore contain some n-3 PUFAs. To obtaina more suitable vegetable oil for fish, available rapeseed cultivars must be screenedand novel cultivars of oil seeds will have to be bred. The Brassica genus and otherplants lack the necessary endogenous genes to produce the oils typical of ocean fish.However, the appropriate genes can be introduced by recombinant DNA technol-ogy. Genes altering the enzyme systems for fatty acid desaturation and elongationcan be introduced into the genome of oilseed plants so that oils of the type neededby aquaculture can be produced from plant sources.

Initially, as many of the genes that are implicated in fatty acid synthesis in rape-seed as possible should be identified. Alleles linked to differences between geno-types in oil content and quality must also be found. This might be achieved bycharacterising different varieties first by comparing transcription profiles onmicroarrays and then determining the association of single nucleotide polymor-

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phisms in the candidate genes with differences in oil quality. This will give anoverview of genes expressed in different rapeseed cultivars having the preferred fattyacid profile. However, microarray studies alone will not reveal changes that are notdirect consequences of transcript abundance. Consequently, a systems approach,incorporating proteome and metabolome data, might be necessary to tailor plantoil production for specific applications such as fish feed. Such data should also becombined with classical genetics, molecular biology, mutant studies and breeding.The approach to achieve an improved fatty acid composition in rapeseed to meetthe demands of fish farming requires distinct segments. After achieving a globaloverview of the genes involved in rapeseed oil biosynthesis, genetic modificationand germplasm screening would be carried out next, together with the solving oftechnical issues in feed design. Genes from other species conferring the appropriatefatty acid profile would be introduced. The advantage of using a gene from a dis-tantly related organism, such as a bacterium, is that its expression is unlikely tointerfere with that of native plant genes.

Genetic engineering: An important tool for new options

An important technical challenge before us is to apply the results of basic researchto devise strategies for improving agronomical and nutritional traits in cultivars.This may be achieved by developing tools for marker-assisted breeding (Maccafer-ri et al. 2003), by generating transgenic varieties, or by a combination of bothapproaches Genetic transformation and recombinant DNA technology representimportant tools for basic biological science in the elucidation of gene function, evenwhen not directly applied to create new varieties. Improvement of B. napus in theproduction of rapeseed or canola oil became an early goal of genetic engineering.Transgenic canola was reported (Radke et al. 1988) some five years after the firstsuccessful plant transformations. Already by 1992, field trials of transgenic canolahad been carried out (Arnoldo et al. 1992). Adoption of transgenic B. napus intocultivation has been rapid, not only driven by increased returns for the farmer butalso followed by benefits to the environment. According to statistics(www.canola-council.org) collected in Canada, conventional canola had droppedfrom nearly 100% of the area planted in 1995 to some 10% by 2003. Virtually allof the transgenic material planted consists of herbicide-resistant varieties. Farmersplant the transgenic B. napus primarily for weed control, reduced costs, higherreturns and better yield. The Canadian data(www.canola-council.org/produc-tion/gmo_main.html) indicate up to a 40%increase in net cash return with transgenic varieties, and a cumulative benefit overthe 1997 to 2000 growing seasons of CAD $144 million adjusted to dollars of theyear 2000. For an overview of transgenic crop production see the International Ser-vice for the Acquisition of Agri-biotech Applications SEAsiaCenter (ISAAA:

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www.isaaa.org/). The Pew Initiative on Food and Biotechnology (http://pewag-biotech.org/) has the mission to offer unbiased information to the public, mediaand policy makers.

Although herbicide resistance offers benefits to the farmer, improved nutrientquality of seeds will ultimately offer greater direct advantages to the consumerand to intermediate users such as fish farmers and processing industries. Eventu-ally, the tailoring of oil components to specific applications will lead to segmen-tation both of the oil market and of the cropping strategies and productionschemes. This has, of course, already emerged for oils aimed at the industrial andfood sectors as such. At the moment, the target for food-grade oils is high oleicand low linolenic acid content. Both transgenic and mutation breeding has beenused to reduce linolenic acid levels, but the emphasis here will be on the trans-genic approaches.

Success in applying appropriate promoters and targeting expression to oilbodies was reported in 1991 (Lee et al. 1991). Soon thereafter, antisense tech-nology was used to successfully modify the degree of fatty acid saturation incanola oil. Antisense reduction of a stearoly-acyl carrier protein desaturase wasthe first approach taken (Knutzon et al. 1992). The transgenic plants had a con-siderably increased level of stearate in the seeds, up to 40%, in addition toincreased 18:3 fatty acids. An E. coli gene for 3-ketoacyl-acyl carrier protein syn-thase III (KAS III) was subsequently expressed in B. napus and the product tar-geted to plastids, decreasing the amounts of 18:1 and increasing the amounts of18:2 and 18:3 fatty acids.

Following these earlier efforts, various researchers have succeeded in increasinglaurate content (Eccleston and Ohlrogge 1998; Stoutjesdijk et al. 2000; Wiberg etal. 2000), in elevating total oil content and the proportion of very-long-chain fattyacids in seed triacylglycerols (Zou et al. 1997), and in raising linolenic acid content(Hong et al. 2002). Others (Shewmaker et al. 1999; Ravanello et al. 2003) haveproduced carotenoids in transgenic canola, to approach the need for colorants inmargarine, poultry feed, and feed for farmed salmonoid fish. Further improvementshave been made by reducing the synthesis of sinapine (Nair et al. 2000), an unde-sirable component because it makes the oil taste bitter and confers a fishy odor tothe eggs of some chickens fed on canola. For industrial applications, transgenicapproaches have been used to raise the content of erucic acid content andvery-long-chain fatty acids (Katavic et al. 2000). Despite the successes representedby these efforts, analyses indicate that the transgenes used heretofore to modifyfatty acid biosynthesis may not be used as efficiently in B. napus as in their nativeplants (Larson et al. 2002), an indication that more basic biology, together withbiochemistry and protein engineering, may be necessary to achieve all the objectivessought through genetic engineering.

Given the amount of research on transgenic approaches to oil improvement incanola, it is no surprise that products based on these strategies have appeared on the

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market. The first to find their way to farmers’ fields have been high laurate varieties,including Monsanto’s ‘LauricalTM’, aimed at the confectionary industry and dairyproducts containing vegetable oil as a dairy fat replacement. Cargill introduced‘Clear Valley 75’, which is 75% oleic acid. The general view is that the main limi-tation to increased production of new modified oils is two-fold. First, de facto tradebarriers, particularly in the EU, on import of products containing or derived fromtransgenic plants have tended to inhibit commercialisation where the production ismainly export-oriented. Second, there is currently an insufficient premium paid onthe modified oils to encourage contract planting and the risk-taking associated witha new commodity. Factors such as agronomic performance and protein profile, aswell as oil properties and strategies for parallel production of transgenic andnon-transgenic varieties, will all play a role in the adoption of transgenic materialswith improved oils.

Genetic engineering and the public

Why is the use of transgenics still so controversial in some countries? The currentdebate is very complex and anything but clear; thus this question does not havea simple answer. As scientists in the fields of genetics and biotechnology, we feelthat the debate is often obscured by too many different concerns being broughtinto it at the same time resulting in blurring of the issues. A number of differentissues typically mingle within the debate. One major concern regards whethertransgenics are safe for consumers and for the environment. This is expressed interms of risks, for example the risk of developing allergies from transgenic prod-ucts or the risk and consequences of transgenes, such as for herbicide resistance,spreading to natural ecosystems. The approval procedures put in place by nation-al governments assess risks, environmental impact, equivalence to conventionalcrops and many other factors. Nevertheless, public evaluation of risks, generallyin a way very different from that of statisticians, becomes mixed with deeper mis-givings. Some people see genetic transformation as “unnatural”, a tampering withthe intrinsic and inviolable integrity and value of nature. While some wouldargue that all human technology is in any case “unnatural” and therefore permit-ted providing it is safe, others would argue that all plant breeding, especially thatinvolving modern tools such as tissue culture or marker-based selection, is unnat-ural and therefore morally suspect.

In the middle ground between the pragmatic issues of safety and the deeply heldvalues regarding man and nature are questions regarding the role of transgenics inbusiness, trade, economy, social equality and globalisation. While these topics are notsolely in the domain of the production of improved varieties, transgenics or not, theuse of genetic engineering is often used as a touchstone for much wider concerns. Arelated question regards the basic moral aspects of patenting genes, and of what con-

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stitutes an invention rather than merely a discovery. Furthermore, there is a concernthat patenting of biotechnological tools may effectively block competition, resultingin monopoly. It has also been argued that the patenting of genes and transgenic vari-eties only benefit large corporations and that farmers, especially in the developingworld, will not be able to take advantage of potential improvements. Alternatively, itcan be argued that patents lead to diversification of technology, and that third-worldfarmers in particular stand to benefit from the disease resistance, increased valueadded, and post-harvest stability only achievable through genetic transformationstrategies. These are ultimately political questions that need to be solved by interna-tional treaties. It has further been argued that various countries, such as memberStates of the EU, that seek to suppress the use of transgenics hide behind the precau-tionary principle in order to protect their markets from competing transgenic prod-ucts. As a consequence of these issues and partly, in the end, to let each consumerdecide, the issue of labelling products containing transgenes arose.

A deeper discussion of these issues is beyond the scope of this review. However, aswe see, the issues described above are by no means unique to recombinant DNA tech-nology; they represent very important concerns with a range of new technologies andproducts. Most importantly, several if not all of these issues cannot be satisfactorilyanswered by science alone because they are also matters of politics and ethics as wellas of international and domestic trade and economics. Therefore, to address the var-ious concerns and draw some democratic conclusions, individuals from many differ-ent disciplines, including both the natural sciences and the social sciences, as well asfrom other interest groups and other sectors of society, should strive to reach a con-sensus. To do so, all parties must work to untangle the threads of the debate, to refrainfrom demagoguery, and to realise that needs and solutions may differ in differentparts of the world.

Much, but not all, can be achieved without using recombinant DNA technol-ogy. Some achievements, however, must rely on gene transfer between species andyet others are too labour-intensive to be performed with traditional breeding.Considering fish feed, recombinant DNA technology is the only way we candevelop plants from which we will be able to harvest marine oils. This, ultimate-ly, will lead to reduced pressure on marine resources, cleaner feed, and therebyhealthier fish products. Regarding the future, if we could achieve homologousrecombination in plant transformation, we would be able both to predict and totarget where the transgenes are inserted and in what copy number as well as tooptimise expression levels. This would greatly improve the efficiency of the tech-nology and its application. We believe we cannot afford not to explore the poten-tial of the technology to achieve, for the future, the elimination of hunger andthe sustainable utilisation of global resources. Louis Pasteur once stated, “Thereare no such things as applied sciences, only applications of science”. Human andglobal well-being will increasingly depend on the best science, combined withglobally sustainable technologies.

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