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Enhancement of Vitamin E Levels in Corn

Department of Crop Sciences, University of Illinois, Urbana, Illinois

Address reprint requests to: T. Rocheford, PhD, Department of Crop Sciences, AW-101 Turner Hall, 1102 South Goodwin Ave., University of Illinois, Urbana 61801. E-mail: trochefo{at}uiuc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION BACKGROUND DESCRIPTION OF SUBJECT CONCLUSION REFERENCES

 
Vitamin E is the common name that describes eight naturally occurring compounds possessing -tocopherol activity. These eight vitamin E compounds are collectively termed tocols, and all have antioxidant activity. There is natural variation among different corn breeding lines for levels of tocols. The two predominant isomers present in corn grain are -tocopherol and -tocopherol. -tocopherol is considered more desirable for human and animal consumption because it has higher biological activity than -tocopherol. Most corn breeding lines naturally have much more -tocopherol than -tocopherol. Therefore a breeding goal is to increase levels of -tocopherol relative to -tocopherol. However, recent research suggests that -tocopherol and compounds metabolized from it have properties important to human health that are unique from properties of -tocopherol. Therefore it may be desirable to not only increase levels of -tocopherol in corn grain, but also levels of -tocopherol. Determination of levels of tocopherols in corn grain is very laborious, requires HPLC analysis and is too time consuming for use in routine commercial corn breeding programs. Therefore we are performing biotechnology enabled molecular marker mapping of chromosomal regions with genes that control levels and ratios of - and -tocopherol. Breeders can use molecular markers we have identified to expediently select for desirable alleles of genes that will improve levels of - and -tocopherol in corn grain, without having to perform laborious HPLC assays. Another biotechnology strategy we have initiated is genetic transformation of corn with the -tocopherol methyl transferase gene to enhance conversion of -tocopherol to -tocopherol and thus increase levels of -tocopherol. This transgenic strategy has been demonstrated in the model plant Arabidopsis, and we are now applying this approach to corn.

Key words: alpha ()-tocopherol, corn, gamma ()-tocopherol, quantitative trait loci (QTL), transgenics, vitamin E

Key teaching points:

• Vitamin E is the common name that describes eight naturally occurring compounds possessing -tocopherol activity; all vitamin E compounds have antioxidant activity.

• There is natural variation for levels of vitamin E compounds in corn grain, with -tocopherol in highest abundance followed by -tocopherol.

• Selecting for enhanced levels of -tocopherol and -tocopherol with HPLC analysis in commercial corn breeding programs is too labor intensive and time consuming to be practical.

• Molecular marker mapping has identified chromosomal segments with desirable genes controlling levels of - and -tocopherol. These markers are being used to indirectly select for higher levels of - and -tocopherols in corn grain.

• Transgenic technology has been used in the model plant Arabidopsis to convert most -tocopherol to -tocopherol, similar efforts have been initiated in corn.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION BACKGROUND DESCRIPTION OF SUBJECT CONCLUSION REFERENCES

 
Vitamin E is the common name that describes eight naturally occurring compounds possessing -tocopherol activity [1,2]. The eight compounds can be divided into two distinct groups, tocopherols and tocotrienols. The two groups differ in the saturation of the side chain. Both the tocopherols and tocotrienols have four derivatives alpha (), beta (ß), delta () and gamma (), which differ in the number and location of methyl groups (Fig. 1). Collectively these eight compounds are called tocols. Vitamin E was first discovered in 1922 as a macronutrient essential to reproduction in rats [3]. Since the 1922 study, other important roles of the vitamin E compounds in plants and animals have been demonstrated.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Chemical structures of tocopherols and the order of conversions between forms. Basic structure of all tocopherols is the same, the differences are in the number and location of the methyl groups on the benzene ring.

Natural -tocopherol has a higher biological activity than other tocopherols as well as the synthetic form of -tocopherol [3,4]. -tocopherol has the highest activity in mammalian tissues; one -tocopherol molecule is effective in the protection of 2000 phospholipids [1]. One reason for the high level of -tocopherol activity may be due to the method of absorption of vitamin E by the body. Vitamin E is absorbed with the lipids in the intestine and transported to the liver. In the liver -tocopherol is specifically selected for absorption into the body by the hepatic -tocopherol transfer protein. Because of the efficiency of the transfer protein, -tocopherol is detected in the plasma at ten times the level of -tocopherol.

Most research on vitamin E has concentrated on -tocopherol. Recent efforts have started looking at the potential benefits of -tocopherol in the diet. Initial studies showed -tocopherol levels in plasma were lower in subjects with coronary heart disease [5]. This low plasma level of -tocopherol could be overcome with a supplementation of corn oil, which increased the serum -tocopherol levels in healthy women [6]. The -tocopherol in corn oil has also been suggested to possess a higher antioxidant capacity as compared to -tocopherol [7]. Research on -tocopherol has also examined the role of compounds that are derived from -tocopherol [8]. -tocopherol is metabolized to 2,7,8-trimethyl-2-ßeta-carboxyethyl)-6-hydroxychroman (-CEHC), which is mainly excreted in the urine. -CEHC has natriutetic activity that may be of physiologic importance, inhibiting cyclooxygenase activity and providing anti-inflammatory properties. Finally, one possibly critical role of -tocopherol is its ability effectively to quench peroxynitrite, an electrophilic mutagen capable of damaging lipids, DNA and proteins [3,9].

There are differences in the retention of -tocopherol in rat tissues and human tissues. Relatively more -tocopherol is retained in human tissues, particularly skin, muscle, vein and adipose tissue. The biological activity of vitamin E has traditionally been based on the rat fetal resorption assay, which shows -tocopherol has the highest biological activity. This finding is probably related to the large differences in retention of -tocopherol and -tocopherol in rats. Rats metabolize -tocopherol differently than humans, leading to lower concentrations of -tocopherol in rats; this suggests the rat fetal resorption assay may not be the best estimator of human vitamin E requirements. Furthermore, -tocopherol supplementation in humans depresses plasma and tissue -tocopherol levels, whereas -tocopherol supplementation leads to a marked increase in both tocopherols [10].

Research studies have continued to support the concept that vitamin E in diets provides diverse health benefits. Corn, a major food source in the US and throughout the world, has considerable variation for levels of vitamin E compounds. However, the assays required to measure tocopherols in corn grain are too labor intensive and costly for use in commercial breeding programs that may wish to select for higher levels of vitamin E in corn grain. Advances in biotechnology have provided new tools that should facilitate increasing levels of vitamin E in corn grain. In this article we discuss some of the relevant biology of tocopherols, efforts enabled by biotechnology that are designed to improve vitamin E levels in corn grain and some practical and economic considerations relevant to achieving this goal.

	BACKGROUND

TOP ABSTRACT INTRODUCTION BACKGROUND DESCRIPTION OF SUBJECT CONCLUSION REFERENCES

 
Plants are the only organisms that produce tocopherols, which have both photosynthetic and non-photosynthetic functions. In photosynthetic tissues, tocopherols are stored mainly in the chloroplasts, involved in free radical scavenging [11], protecting the photosynthetic apparatus from lipid peroxidation. In non-photosynthetic tissues the tocopherols are indispensable for protection of the polyunsaturated fatty acids [12] and improve stability of stored lipids by protecting from autoxidation. -tocopherol is frequently the predominant form of vitamin E in plant seeds and in products such as vegetable oils that are derived from seeds.

-tocopherol equivalents supplemented in livestock rations have been shown to affect meat quality. High tocopherol levels in the tissues of poultry, hogs and beef cattle have been correlated with positive affects that have economic ramifications. For poultry, vitamin E supplementation increased the stability of poultry meat [13,14]. For hogs, vitamin E protects against rancid flavor, odor and discoloration and plays a part in increasing shelf life of packaged meat [15,16]. In beef, vitamin E supplements increased the color stability of beefsteaks, which increased visual acceptance [17]. In the beef industry discoloration of meat leads to a loss of approximately $1 billion annually [18].

Other than economic benefits to the meat industry, tocopherols, in particular -tocopherol, have been demonstrated to prolong shelf life of oils [7]. Due to the relationship between oil (fatty acid profile) and tocopherols, a recent report concluded that improving oil quality by increasing vitamin E content is highly advisable [12].

Maize kernels have been studied for their different levels and isoforms of tocopherols. Early studies focused on three isoforms, -tocopherol, -tocopherol and -tocopherol with -tocopherol generally regarded as being in the highest concentration [19,20]. The corn kernel consists of two parts, the endosperm which is mostly starch and some protein, and the germ. The germ of the kernel is a combination of the reproductive organ or embryo and the scutellum. The scutellum is the non-germinating tissue surrounding the embryo where most of the oil is stored. The distribution of the tocopherols in the kernels has been evaluated by hand dissection of the kernel in different corn lines. The germ of the kernel contains 70% to 86% of the tocopherols, with the endosperm having 11% to 27%, although the levels of tocopherol storage is genotype dependent. For individual tocopherols, 94% to 96% of -tocopherol and 93% to 96% of -tocopherol are found within the germ; for -tocopherol it is only found in the germ [19]. Considerable variation is present among different inbreds for tocopherol levels, as well as different ratios of -tocopherol to -tocopherol [20,21]. The nature of inheritance, as calculated by heritability values for - and -tocopherol as well as total tocopherols, indicate effective selection for levels of tocopherols should be attainable [21].

Measuring tocopherols in corn grain requires a series of steps and procedures that are very labor intensive, time consuming and require expensive equipment and some expertise. The grain is first ground, followed by a series of extractions, and finally the extracted samples are run on a high performance liquid chromatographer (HPLC). The extraction process requires the most time, with 16 to 24 samples extracted by one person in about three hours depending on the experience of the person. This is followed by the running of the sample on the HPLC which requires six minutes per sample. In total, 16 to 24 samples can be analyzed by one person in about 4.5 to 5.5 hours. Typical commercial corn breeding operations, which produce the hybrids that are planted on approximately 99% of the corn acreage in the US, do not have the time to perform these expensive assays. The major reason they do not have the time is that presently there is not enough of an economic incentive.

One recent technical development, molecular markers, is making selection for desirable traits that are expensive to measure much more feasible. Molecular markers reveal variation in DNA that can be linked to genes of unknown identity that control desirable traits. By using this method, analysis of the trait does not need to be performed all the time; instead, DNA is isolated from samples. These DNA samples are then assayed for a molecular marker to select plants with the desirable genes. This process is rapid and has been scaled up to accommodate large numbers of samples. Parts of the process can be automated to further increase throughput. The power of the molecular marker assay process is that it can be used for selection of genes that control very different traits, since they all involve DNA and molecular marker assays.

In this article we describe how we have linked molecular markers to desirable genes that control levels of - and -tocopherol in corn grain. These markers enable us to select desirable alleles of genes from different genetic stocks and pyramid them together through selection and genetic crossing to develop new improved breeding lines. Progress will be more rapid than by selection based on HPLC assays alone. Combining different favorable genes together from different genetic sources should enable elevating tocopherols to levels that are higher than presently available.

Molecular markers have been identified that are linked to genes that influence the ratio of - to -tocopherol [22]. Selection of the desirable alleles of genes linked to these markers should enable us to modify the ratio of - to -tocopherol. However, we do not expect to be able to make dramatic changes in the ratio of - to -tocopherol. We expect that -tocopherol would likely still be predominant in the grain, but that relative proportion of -tocopherol would increase. To create seeds with levels of -tocopherol significantly higher than -tocopherol, transgenic approaches would need to be taken. This involves the introduction of a gene into corn plants that expresses at a higher level than the same gene already in the plant. We will describe transgenic efforts for elevated -tocopherol levels later in this paper.

There are important economic and practical considerations that impact on commercial efforts aimed at increasing levels of vitamin E in maize grain. Commercial seed companies need eventually to get a return on their additional research costs in order to justify embarking on a new or additional effort. Although corn hybrids with higher levels of vitamin E would have nutritional value, presently it may be difficult to capture that value in an economic manner that benefits the seed company producing the seed.

There are ongoing efforts to modify the chemical and nutritional composition of corn grain; the concept is termed value added corn grain. Levels of starch, protein and oil are being modified in corn grain through selection in order to meet more efficiently specific end usages. The greatest effort involves increasing the level of oil in corn grain to make the grain more desirable as a feed for swine and poultry which require high calorie diets. Research efforts are also ongoing to modify amino acid, fatty acid, phytic acid, carotenoid and tocopherol profiles [23]. However it is not likely that modification of just one of these components will provide enough value to justify the additional research costs and perhaps more importantly the additional cost of identity preserving or keeping grain with particular modifications separate. Identity preserving is required to capture the value that comes from unique grain compositions, in comparison to typical commodity grain corn that is sold in open markets and channels [24]. Strategies are being developed to concurrently modify a number of kernel chemical and nutritional traits to add value worth identity preserving. This type of effort will likely receive more attention for animal feed first since 80% of corn grain is fed to animals [25]. Efforts to modify grain for animal feed may provide indirect benefits to humans through consumption of meat and derived products. However, once these nutritionally modified types of corn hybrids are grown extensively for animal feed, it will then be easier to move high tocopherol materials into processing of products such as corn oil that will have direct benefits in human diets.

	DESCRIPTION OF SUBJECT

TOP ABSTRACT INTRODUCTION BACKGROUND DESCRIPTION OF SUBJECT CONCLUSION REFERENCES

 
Variation for Vitamin E Compounds in Corn Lines
Initial research established that -tocopherol and -tocopherol were the predominant vitamin E compounds in corn grain and that other tocol compounds were in relatively low levels [19–21,26]. Consequently most research has focused on -tocopherol and -tocopherol. Two tocotrienol forms are detected in corn, - and -tocotrienol, and their concentration is generally lower than their respective tocopherol form [20]. -tocotrienol ranged from 2% to 28% of the total tocol concentration of 15 corn inbreds, with a mean of 9.35 µg g^-1, and -tocotrienol ranged from 4% to 21% with a mean of 5.63 µg g^-1. -tocopherol is also present in corn grain, but at a much lower concentration than either - or -tocopherol. In our research, we observed -tocopherol accounted for 2% to 10% of the total tocopherol concentration with a mean of 1.6 µg g^-1 in one mapping population and 1.0% to 5.33% of the total tocopherol concentration with a mean of 4.28 µg g^-1 in a second population.

Forgey [27] evaluated twenty maize inbreds (which are used to make hybrids) and found a range of 9.1 to 64.6 µg g^-1 for -tocopherol and 13.6 to 128.7 µg g^-1 for -tocopherol. However, these lines are no longer in use in breeding programs, and the technologies for measuring tocopherols [28,29] have improved since that time.

We performed a study to measure levels of - and -tocopherol in more contemporary public inbreds and experimental inbreds at the University of Illinois [30]. For corn, two inbreds are crossed to make the F₁ hybrid seed that farmers grow. We evaluated F₁ hybrids created from public inbreds and experimental inbreds since this may provide results more relevant to grain grown in farmers’ fields. -tocopherol ranged from 11.8 µg g^-1 to 66 µg g^-1 and -tocopherol from 43 µg g^-1 to 229 µg g^-1 in a set of 45 interrelated hybrids. We did not detect significantly higher concentrations of -tocopherol in the grain of these hybrids than reported previously for inbreds. Notably we did detect hybrids with levels of -tocopherol in the grain much higher than previously reported (229 µg g^-1 vs.128 µg g^-1). Averaged over all 45 hybrids, -tocopherol was present at a concentration of approximately 80% and -tocopherol 20%. The combined - and -tocopherol values ranged from 40 µg g^-1 to 286 µg g^-1. The ratio of - to -tocopherol ranged from 0.06 to 1.04; thus, we identified a hybrid with more -tocopherol than -tocopherol. This is noteworthy except that the hybrid with more - than -tocopherol had 58.4 µg g^-1 -tocopherol and 56.1 µg g^-1 -tocopherol, whereas the hybrid with the highest level of -tocopherol had 66.3 µg g^-1 of -tocopherol and 165.4 µg g^-1 of -tocopherol. Therefore, if selection for -tocopherol is the primary objective, then hybrids should be selected for the highest absolute level of -tocopherol, not the best ratio of - to -tocopherol.

The recent findings on the potential unique benefits of -tocopherol may prompt a different selection strategy for relative levels of - and -tocopherol. For example, the hybrid with the highest level of combined - and -tocopherol, 286 µg g^-1, had 57.2 µg g^-1 of -tocopherol and 229.1 µg g^-1of -tocopherol. The -tocopherol level in this hybrid was not much lower than in the hybrid with the best ratio of to or the hybrid with highest absolute level of -tocopherol, yet this hybrid had considerably more -tocopherol than the other two hybrids. Thus, future selection strategies may need to consider -tocopherol levels carefully.

Our observations show how plant breeding strategies need to stay informed with recent developments in the nutritional sciences. Until recently, we were interested in increasing -tocopherol in corn grain and less interested in -tocopherol levels. Increasing -tocopherol is technically much more challenging from a genetic standpoint because corn generally has higher levels of -tocopherol. However it appears that levels of - and -tocopherol should be considered separately and collectively in breeding strategies for vitamin E. Furthermore it may be that different hybrids could be developed with different ratios of - and -tocopherol depending on the end use nutritional objective and what we continue to learn about the relative health benefits of - and -tocopherol.

Quantitative Trait Loci Analysis for Tocopherols in Corn Kernels
Corn grain shows continuous variation for tocopherol concentration [20,21, 30] similar to variation for height or hair color in humans. Traits that show continuous variation are termed quantitative traits and are controlled by a number of genes as opposed to a single gene [31]. Each gene or locus with an affect on the trait is called a quantitative trait locus (QTL). In plant breeding programs that deal with quantitative traits like grain yield, disease resistance and plant height, the role of the plant breeder is to select those QTL with the largest positive affect on the trait of interest, but that also do not have negative affects on other traits.

By assaying a large number of molecular markers on a segregating population, such as an F₂, it is possible to create a linkage map at the molecular marker level. A molecular marker map can be used with statistical software packages to detect QTL controlling traits with continuous variation. We have been able to associate variation in tocopherol concentrations with molecular markers in segregating populations using linkage and statistical analysis. We have been able to identify QTL with the largest effects and narrow favorable QTL to regions flanked by two molecular markers. The markers flanking the QTL can then be selected in new breeding materials being developed, enabling selection of favorable genes without performing phenotypic analysis (Fig. 2).

View larger version (62K): [in this window] [in a new window]   Fig. 2. Tocopherol concentrations are determined by HPLC.(a) Chromatogram shows -tocopherol and -tocopherol and their respective retention times of 4.185 and 4.880. The concentrations are then associated with (b) a molecular maker genotype to determine if the genotype is associated with the phenotype.

Analysis with HPLC showed both corn mapping populations had considerable variation for levels of - and -tocopherol. The range in variation for the first population was 0.4 µg g^-1 - 59.5 µg g^-1 for -tocopherol and 3.3 µg g^-1 - 114.6 µg g^-1 for -tocopherol. For the second population the range of variation was 0.8 µg g^-1 - 192.3 µg g^-1 for -tocopherol and 4.37 µg g^-1 - 286.51 µg g^-1 for -tocopherol. Both populations were genotyped with molecular markers, including restriction fragment length polymorphism (RFLP) and microsatellite or simple sequence repeat (SSR) markers [32]. These molecular markers were used to create a linkage map of all ten chromosomes of corn. Using statistical software packages, several regions in the corn genome showed significant associations with - and -tocopherol (Fig. 3).

View larger version (21K): [in this window] [in a new window]   Fig. 3. Chromosome regions affecting tocopherol levels in two populations. Three regions are shared between populations, several are significant in only one of the populations.

We detected significant associations for - or -tocopherol on chromosomes 4, 6 and 7 in one of the populations. These regions are also important because they suggest that there may be a favorable allele of a gene for level of tocopherols in one of the inbred parents that is not present in one of the other inbred parents of these populations. In this case, through genetic crosses and molecular marker selection, the most favorable allele of a gene from one parent can be pooled with the most favorable allele of another gene from another parent. For example the favorable allele on chromosome 1 for -tocopherol comes from a different parent than the favorable allele on chromosome 7. The region on chromosome 1 accounts for 10% of the variation, and the region on chromosome 7 accounts for 8% of the total variation. Together these two regions from different inbred parents may account for 18% of the total phenotypic variation for -tocopherol.

The molecular markers flanking the QTL we have identified can be used for marker assisted selection, which should enable an increase in tocopherol levels in corn, without having to undergo the process of phenotyping every generation of selection. The molecular markers can be used intentionally to pool together favorable alleles of different genes from different chromosome regions and different inbred lines. The molecular markers will enable introgressing favorable genes into elite hybrids that are grown on farmers fields and the grain processed. The molecular markers will also be useful in pyramiding genes for tocopherols with genes for other traits such as fatty acid composition. Selection of very small chromosome segments flanked by markers reduces the possibility of decreasing grain yield by also bringing along unfavorable genes for grain yield (termed linkage drag). Some of the lines that have desirable genes for tocopherols nd genes for other aspects of nutritional composition), do not have the most desirable genes for agronomic performance such as grain yield. Therefore, in order for the economics to be in place to support new nutritionally enhanced hybrids, it must be done in a manner that the hybrids still perform at competitive levels in farmers’ fields.

Altering Ratio of - and -Tocopherol through Transgenic Approaches
The tocopherol biosynthetic pathway in plants has not yet been fully characterized, but a probable pathway has been formulated. Most of the work on the tocopherol biosynthetic pathway has been performed on Arabidopsis, a model plant system. Four of at least six proposed genes involved in the tocopherol biosynthetic pathway have been cloned in Arabidopsis. The earliest is geranylgeranyl diphosphate reductase [33], which sequentially reduces geranylgeranyl pyrophosphate to phytyl diphosphate through three steps. Phytyl diphosphate is then added to homogentisic acid by the cloned prenyltransferase [34], eventually producing 2-methyl-6-phytyl-plastoquinol. Homogentisic acid is produced from p-hydroxyphenyl pyruvate by the enzyme p-hydroxyphenylpyruvate dioxygensae [35]. At this point in the pathway there are some limits to the characterization of the pathway until the conversion of -tocopherol to -tocopherol by the -tocopherol methyl transferase gene [36]. Because of the higher level of -tocopherol relative to -tocopherol in corn, the -tocopherol methyl transferase gene may be very useful in efforts to convert - to -tocopherol.

Shintani et al. cloned the -tocopherol methyl transferase (-TMT) gene from Arabidopsis, which converts -tocopherol to -tocopherol. The -TMT gene was introduced into the Arabidopsis genome with transgenic technologies. The transgene construct was designed to cause a high level of expression of the -TMT gene. This transgenic event resulted in a change in the relative composition of - and -tocopherol from approximately 5% -tocopherol and 95% -tocopherol to 95% -tocopherol and 5% -tocopherol, causing an eightyfold increase in -tocopherol levels in the Arabidopsis line that was transformed. These results indicate that overexpression of -TMT most likely resulted in a direct conversion of -tocopherol to -tocopherol [36]. This study demonstrated that a single transgenic event can alter ratios of - to -tocopherol to an extent most likely not possible with traditional selection of natural variation or selection and pyramiding of desirable QTL with molecular markers.

We have initiated transgenic efforts in corn to overexpress the -TMT gene in maize. As opposed to the model species Arabidopsis, transformation of corn in the public sector presently is very time consuming and inefficient, and thus this is a long term project. The goal is to develop genetic materials with as high a level of -tocopherol as possible. Generally different genetic transformation events have different levels of efficiency in causing the intended outcome. This may result in transgenic corn materials with variable ratios of to -tocopherol. With the increasing knowledge about unique health benefits of -tocopherol, creating corn hybrids that are 95% -tocopherol and 5% -tocopherol may not be the most desirable. Therefore, transgenic events that are considered less efficient and convert lines to only 60% to 90% -tocopherol may be most desirable.

Integration of Conventional Breeding, Molecular Marker Selection, and Transgenic Efforts
The best lines from our evaluation of 45 hybrids will be selected for breeding use and molecular marker mapping studies. Some of these breeding lines are higher in oil concentration and also fit into plans for modification of multiple traits that influence nutritional composition of corn grain. We are beginning to use molecular markers to select for chromosomal segments with favorable affects on tocopherols. This should result in lines with higher levels of total tocopherols. We could then combine this with efforts to alter the ratio of alpha to gamma tocopherol, either by selecting for a molecular marker linked to QTL, which influence ratio of - to -tocopherol, or by crossing with transgenics that convert - to -tocopherol. This strategy uses natural variation and molecular marker assisted selection to develop what could be considered a large precursor pool of -tocopherol, which we may combine with transgenics to develop higher levels of -tocopherol than presently available. In the event that there are problems with genetically modified organism MO) acceptance, we can develop materials with improved - and -tocopherol levels without transgenics, using natural variation and molecular marker selection.

	CONCLUSION

TOP ABSTRACT INTRODUCTION BACKGROUND DESCRIPTION OF SUBJECT CONCLUSION REFERENCES

 
Corn breeders can use natural variation, molecular marker assisted selection strategies and transgenic technologies to alter overall levels of tocopherols and ratio of - to -tocopherol. Current nutritional research on the relative and unique benefits of - and -tocopherol should be considered in developing breeding strategies to alter levels of these important vitamin E compounds.

Received February 5, 2002.

	REFERENCES

TOP ABSTRACT INTRODUCTION BACKGROUND DESCRIPTION OF SUBJECT CONCLUSION REFERENCES

 

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