Identification of genetic variation in Brassica napus seeds for tocopherol content and composition using near‐infrared spectroscopy technique

Tocopherol is an essential fat‐soluble nutrient for humans. Increasing the tocopherol content in Brassica napus seeds can add value to rapeseed vegetable oil; this has become an important breeding target. However, there is no efficient and non‐destructive method for selecting rapeseed accessions with high tocopherol contents. Here, we report the first near‐infrared reflectance spectroscopy (NIRS)‐based technique for predicting rapeseed tocopherol content. Individual seed tocopherol compositions were estimated from 373 rapeseed genotypes of different origins. This method and chemical methods produced comparable predicted values of the tocopherol constituents in the seeds. Three equations were generated for the prediction of tocopherol content by using a modified partial least squares (MPLS) model. The total tocopherol content for the determination coefficient of cross‐validation (R²_cv) (0.74), determination coefficient (RSQ) (0.76) and one minus the ratio of unexplained variance to total variance (1‐VR) (0.65) values indicates a strong correlation between the calibration and validation sets. Overall, our model confirmed the NIRS method as feasible for predicting tocopherol content in rapeseed and as an efficient screening tool for future breeding programs.     

Selection for contrasting tocopherol content and profile in Ethiopian mustard

Tocopherols are compounds present in oilseeds with major effect on nutritional and technological properties of seed oils. Brassica spp. mainly contain varying concentrations of α‐tocopherol, with high vitamin E value, and γ‐tocopherol, which contributes to oil stability. The objective of this research was to select for contrasting tocopherol content and profile in Ethiopian mustard (Brassica carinata A. Braun). A collection of 211 F_4:5 lines was evaluated for tocopherol content and profile, and divergent selection was conducted till the S_3:4 generation. Selection resulted in two lines with increased tocopherol content (244 and 243 mg/kg seed as average of four environments), one line with reduced tocopherol content (117 mg/kg), two lines with reduced α/γ‐tocopherol ratio (0.22 and 0.28) and two lines with increased α/γ‐tocopherol ratio (3.74 and 3.34). Control line C‐101 averaged 196 mg/kg with α/γ‐tocopherol ratio of 1.10. Reduced α/γ‐tocopherol ratio was controlled by recessive alleles at a single locus. Seed to plant correlation was high (r = 0.92), which suggests the feasibility of selecting at the single‐seed level.     

Mapping quantitative trait loci (QTLs) underlying seed vitamin E content in soybean with main,epistatic and QTL × environment effects

Soybean (Glycine max (L.) Merr.) seed contains small amounts of tocopherol, a non‐enzymatic antioxidant known as lipid‐soluble vitamin E (VE). Dietary VE contributes to a decreased risk of chronic diseases in humans and has several beneficial effects on resistance to stress in plants, and increasing VE content is an important breeding goal for increasing the nutritional value of soybean. In this study, quantitative trait loci (QTLs) underlying VE content with main, epistatic and QTL × environment effects were identified in a population of F_5 : 6 recombinant inbred lines from a cross between ‘Hefeng 25’ (a low‐VE cultivar) and ‘OAC Bayfield’ (a high‐VE cultivar). A total of 18 QTLs were detected that showed additive main effects (a) and/or additive × environment interaction effects (ae) in different environments. Moreover, 19 epistatic pairs of QTLs were found to be associated with α‐tocopherol (α‐Toc), γ‐tocopherol (γ‐Toc), δ‐tocopherol (δ‐Toc) and total VE (TE) contents. The QTLs identified in multienvironments could provide more information about QTL by environment interactions and could be useful for the marker‐assistant selection of soybean cultivars with high seed VE contents.     

Genetic analysis of tocopherol content and composition in winter rapeseed

The improvement of the nutritional value and the stability properties of rapeseed oil is partly hindered by the lack of information on the genetic control of tocopherols. The objectives of this investigation were to characterize the inheritance of tocopherols by using two factorial mating designs (North Carolina Design II, NC_II). The first (NC_II‐A) was produced with two sets of nine parents each, one selected for high and the other for low total tocopherol contents. In the second (NC_II‐B), each set consisted of six parents, which were either high or low for the α/γ‐tocopherol ratio. Parents and F₁ hybrids from both experiments were tested under field conditions in 1998 and 1999 using a completely randomized design with two replications. Only small differences were detected between mean values of parents and F₁ hybrids. General combining ability (GCA) variation in both experiments was highly significant (P < 0.01) for all tocopherol traits. Significant specific combining ability (SCA) effects were only detected for a‐tocopherol in NC_IIA and therefore for the α‐/γ‐tocopherol ratio. These results indicate that tocopherol contents and composition are controlled mainly by genes with additive effects. Interactions of F₁ hybrids and GCA effects with the environment were significant for only tocopherol contents and not for tocopherol composition.     

Genetic variation of tocopherol content in a germplasm collection of Brassica napus L.

The genetic variation for tocopherol contents was investigated in a very divergent collection of 87 winter rapeseed genotypes grown in the greenhouse and in two years in the field. Genotypic and environmental effects were highly significant for alpha-, gamma- and total tocopherol contents and the alpha-/gamma-tocopherol ratio. Field and greenhouse environments differed significantly (p < 0.001) for tocopherol traits, with greenhouse means up to 19% higher than field means. Alpha-tocopherol content ranged from 63 to157 mg kg^-1 seed, gamma-tocopherol content from 114 to 211 mg kg^-1 seed, total tocopherol content from 182 to367 mg kg^-1 seed, and the alpha-/gamma-tocopherol contents ratio from0.36 to 1.23. The resynthesized lines often have a remarkably low alpha-tocopherol content and consequently a low alpha-/gamma-tocopherol contents ratio. The lines with altered fatty acid composition displayed the highest variation for both tocopherol content and composition. No significant differences were observed among groups with different seed quality types [00], [0+] and [++]. Total tocopherol content was not correlated with the alpha-/gamma-tocopherol ratio, indicating that total tocopherol content is independent from tocopherol composition. Alpha- and gamma-tocopherol contents were also not correlated. Gamma-tocopherol contents showed a significant positive correlation with oil content (r = 0.34^**). This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular mapping of soybean seed tocopherols in the cross ‘OAC Bayfield’ × ‘OAC Shire’

Soybean (Glycine max [L.] Merr.) is cultivated primarily for its protein and oil in the seed. In addition, soybean seeds contain nutraceutical compounds such as tocopherols (vitamin E), which are powerful antioxidants with health benefits. The objective of this study was to identify molecular markers linked to quantitative trait loci (QTL) that affect accumulation of soybean seed tocopherols. A recombinant inbred line (RIL) population derived from the cross ‘OAC Bayfield’ × ‘OAC Shire’ was grown in three locations over 2 years. A total of 151 SSR markers were polymorphic of which a one‐way analysis of variance identified 42 markers whereas composite interval mapping identified 26 markers linked to tocopherol QTL across 17 chromosomes. Individual QTL explained from 7% to 42% of the total phenotypic variation. Significant two‐locus epistatic interactions were identified for a total of 122 combinations in 2009 and 152 in 2010. The multiple‐locus models explained 18.4–72.2% of the total phenotypic variation. The reported QTL may be used in marker‐assisted selection (MAS) to develop high tocopherol soybean cultivars.     

Identification and genetic characterization of a safflower mutant with a modified tocopherol profile

Safflower (Carthamus tinctorius L.) seeds have a tocopherol fraction dominated by α‐tocopherol, which accounts for more than 95% of the tocopherols. α‐Tocopherol exerts a high vitamin E activity, but a low in vitro antioxidant action. For non‐food applications, replacement of high α‐tocopherol by γ‐tocopherol is preferred. Because of the limited variability found in germplasm of cultivated safflower, the objective of the present research was to search for variability for tocopherol profile in germplasm of wild safflower species. Bulk seed samples of 77 accessions from six species were analysed for total tocopherol content and profile. One accession of C. oxyacanthus showed increased γ‐tocopherol content (36%). Single‐seed analysis showed the existence of a bimodal distribution, which included high α‐tocopherol (>90%) and high γ‐tocopherol (HGT) seeds (>85%). Plants from the HGT seeds bred true for the trait and showed introgression of cultivated safflower, which allowed a rapid selection for cultivated safflower traits. Genetic analysis revealed that the HGT trait was controlled by partially recessive alleles at a single locus Tph1, which will facilitate the transference of the novel trait to diverse safflower germplasms.     

Genetic variability of tocopherol composition in sunflower seeds as a basis of breeding for improved oil quality

The variability of seed tocopherol content in wild sunflower species, the expressivity of tph1 and tph2 mutations in different lines and the oxidative stability of sunflower oil with altered tocopherol and fatty acid composition were objectives of this research. Near-isogenic lines for three genes, i.e. Tph1, Tph2, and Ol, were developed and investigated. Tocopherol content was determined with TLC and HPLC, as well as fatty acid composition with GC of methyl esters. Rancimat tests were used to estimate the oxidative stability of the oil. The seed tocopherol composition of wild sunflower species was shown to be uniform with a prevailing content of the α-homologue (90-99%). The genetic background of different near-isogenic lines was found to influence expressivity of mutations for tocopherol composition. High content of strong antioxidants, such as β-, γ-, and δ-tocopherols increased oil oxidative stability of linoleic and oleic types of oil by 1.2–3.0 times. The breeding model of sunflower hybrids should include antioxidant and vitamin parameters balanced for oils of different applications.     

Generation of tribenuron‐methyl herbicide‐resistant OsCYP81A6‐expressing rapeseed (Brassica napus L.) plants for hybrid seed production using chemical‐induced male sterility

Chemical‐induced male sterility (CIMS) is a method for hybrid rapeseed (Brassica napus L.) production. Some sulphonylurea herbicides such as tribenuron‐methyl (TBM) are used as chemical hybridization agents (CHAs) in CIMS systems. However, the male parents must be protected from herbicide injury with a shield during spraying of the female parents with CHAs to induce male sterility. Thus, using herbicide‐resistant rapeseed lines as the male parents can significantly simplify the seed production procedure and reduce the cost in hybrid seed production. A rice cytochrome P450 hydroxylase, OsCYP81A6, has been previously characterized to confer resistance to bentazon and sulphonylurea herbicides. We demonstrate here that the introduction of OsCYP81A6 renders rapeseed plants resistant to TBM. Compared with wild‐type plants, the transgenic plants displayed normal stamen development and male fertility when treated with 0.05 mg/l of TBM, the dose used for inducing male sterility in hybrid seed production. These results indicate that the OsCYP81A6‐expressing rapeseed plants can be used as the male parents for hybrid rapeseed production using CIMS.     

Genetics of the Erucic Acid Content in Interspecific Hybrids of Ethiopian Mustard (Brassies carinata Braun) and Rapeseed (B. napus L.)

Ethiopian mustard (Brassica carinata Braun) is a potential oil crop in which genes for low erucic acid content of the seed oil have not yet been found. In order to solve this problem the potential of rapeseed (B. napus L.) varieties as a source of these genes has been tested. Reciprocal F₁ hybrids between B. carinata and a low erucic acid variety of B. napus, F₂, and backcrosses with B. carinata were obtained. The fatty acid composition was determined in half seeds of F₁ and segregating generations from reciprocal interspecific crosses. The genetic analysis indicated that the erucic acid content of the seed oil of B. carinata is controlled by two genes with no dominance and additive in action.     

Expression of a cDNA encoding palmitoyl-acyl carrier protein desaturase from cat's claw (Doxantha unguis-cati L.) in Arabidopsis thaliana and Brassica napus leads to accumulation of unusual unsaturated fatty acids and increased stearic acid content in the seed oil

A cDNA encoding palmitoyl‐acyl carrier protein (ACP)‐desaturase from cat's claw (Doxantha unguis‐cati L.) was expressed in Arabidopsis thaliana and Brassica napus L. with the goal of decreasing the saturated fatty acid (FA) content of the seed oil. In general, transformation of Arabidopsis resulted in a greater change in the FA composition of the seed oil than for B. napus. An increase in palmitoleic acid (16:1cisΔ⁹) was obtained in transgenic lines, suggesting that the 16:0‐ACP‐desaturase cDNA was expressed in the manner originally intended. Other effects on lipid metabolism, however, were observed in the seed of transgenic plants. In Arabidopsis, there was a large increase in the proportions of cis‐vaccenic acid (18:1cisΔ¹¹) and cis‐13‐eicosenoic acid (20:1cisΔ¹³), possibly generated through elongation of 16:1cisΔ⁹. Elongation of 18:1cisΔ¹¹ to 20:1cisΔ¹³, however, was not observed in B. napus indicating that certain aspects of lipid metabolism in the model plant, Arabidopsis, may not apply to B. napus. As well, the appearance of 18:1cisΔ¹¹ was accompanied by a decrease in the proportion of oleic acid (18:1cisΔ⁹). Although the introduced ACP‐desaturase resulted in synthesis of some unsaturated FAs, the overall saturated FA content was maintained at similar levels to the control or was enhanced. Increased levels of saturation were mainly associated with an increase in stearic acid, which unlike 16:0, is considered non‐atherogenic. The results suggest that a mechanism exists further downstream in oil biosynthesis to counteract the decrease in saturation brought about by the 16:0‐ACP‐desaturase action.     

Quantitative trait loci mapping of seed colour,hairy leaf,seedling anthocyanin,leaf chlorosis and days to flowering in F2 population of Brassica rapa L.

A diversity arrays technology (DArT) map was constructed to identify quantitative trait loci (QTL) affecting seed colour, hairy leaf, seedling anthocyanin, leaf chlorosis and days to flowering in Brassica rapa using a F₂ population from a cross between two parents with contrasting traits. Two genes with dominant epistatic interaction were responsible for seed colour. One major dominant gene controls the hairy leaf trait. Seedling anthocyanin was controlled by a major single dominant gene. The parents did not exhibit leaf chlorosis; however, 32% F₂ plants showed leaf chlorosis in the population. A distorted segregation was observed for days to flowering in the F₂ population. A linkage map was constructed with 376 DArT markers distributed over 12 linkage groups covering 579.7 cM. The DArT markers were assigned on different chromosomes of B. rapa using B. rapa genome sequences and DArT consensus map of B. napus. Two QTL (RSC1‐2 and RSC12‐56) located on chromosome A8 and chromosome A9 were identified for seed colour, which explained 19.4% and 18.2% of the phenotypic variation, respectively. The seed colour marker located in the ortholog to Arabidopsis thaliana Transparent Testa2 (AtTT2). Two QTL RLH6‐0 and RLH9‐16 were identified for hairy leaf, which explained 31.6% and 20.7% phenotypic variation, respectively. A single QTL (RSAn‐12‐157) on chromosome A7, which explained 12.8% of phenotypic variation was detected for seedling anthocyanin. The seedling anthocyanin marker is found within the A. thaliana Transparent Testa12 (AtTT12) ortholog. A QTL (RLC6‐04) for leaf chlorosis was identified, which explained 55.3% of phenotypic variation. QTL for hairy leaf and leaf chlorosis were located 0–4 cM apart on the same chromosome A1. A single QTL (RDF‐10‐0) for days to flowering was identified, which explained 21.4% phenotypic variation.     

Construction of a molecular functional map of rapeseed (Brassica napus L.) using differentially expressed genes between hybrid and its parents

Brassica napus L. is an important oilseed and fodder crop with significant heterosis for seed yield and other agronomic traits, but very little is known about the molecular basis of heterosis. As an initial step towards understanding the molecular events associated with this phenomenon, a molecular functional map of rapeseed was constructed using differentially expressed genes in hybrid identified by microarrays. Single-strand conformational polymorphism (SSCP) analysis was applied for genetic mapping in an F ₂ population of 184 individuals resulting from crossing ‘`SI-1300 × Eagle’'. A total of 162 markers including 154 loci corresponding to 98 differentially expressed genes assigned to 17 functional categories and 8 SSR markers were grouped into 21 linkage groups (LGs), covering a total map distance of 2267.3 cM. Subsequently, this map was aligned with Arabidopsis thaliana in silico. Comparative mapping shows that genes localized on each Arabidopsis chromosome have orthologs dispreading in different B. napus LGs. Similarly, a majority of LGs were made of homologous genes from different Arabidopsis chromosomes. In addition, a total of 25 syntenic regions were identified in B. napus, in most of which the gene order was not consistent between the two species, and each of the conserved regions in the A. thaliana genome was homologous to 1--5 distinct regions in the B. napus genome. These results indicate that it is not easy to exploit A. thaliana information for B. napus based on synteny.     

Heterosis and combining ability analysis in Chinese semi-winter × exotic accessions of rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Fourteen accessions (spring and winter types) and four testers (semi-winter type) of rapeseed (Brassica napus L.) were crossed in line?×?tester mating design to estimate general and specific combining ability for seed yield, yield components, and oil content. The F₁ hybrids and their parents were evaluated in four environments in northern China. Highly significant differences were detected among the parents and hybrids for all the traits across environments. Plant height, setting position of first primary branch and length of terminal raceme were controlled by additive genes, whereas primary branches per plant, siliques on terminal raceme, siliques per plant, seeds per silique, seed yield per plant, 1000-seed weight, oil content, and seed yield were controlled by non-additive gene action. The accessions SP-Armada, 9E49, and CZ25 and the tester Zhong9 were good general combiners for seed yield. Among the 56 F₁ hybrids, four hybrids: Zhong9?×?CZ25, GZ1R?×?9E38, Zhong7?×?9E38, and Zhong7?×?CZ49 showed higher yield than the control and were the outstanding combinations for seed yield. These hybrids were recommended to be included in future breeding programs for development of new high yielding varieties with more desirable traits. Both winter and spring germplasm have potential in Chinese semi-winter rapeseed breeding program, for seed oil content improvement, more attention may be paid to spring germplasm.     

Genetic variability of seed-quality traits in gamma-induced mutants of sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) under water-stressed condition

Sunflower is one of the major annual world crops grown for edible oil and its meal is a potential source of protein for human consumption. It contains tocopherol that decreases potential risk of chronic diseases in human. The objectives of the current research are to assess the genetic variability and to identify AFLP markers and candidate genes associated with seed-quality traits under well-irrigated and water-stressed conditions in gamma-induced mutants of sunflower. Two mutant lines, M8-826-2-1 and M8-39-2-1, with significant increased level of oleic acid were identified that can be used in breeding programs for quality increase high oxidative stability and heart-healthy properties. The significant increased level of tocopherol in mutant lines, M8-862-1N1 and M8-641-2-1, is justified by observed polymorphism for tocopherol pathway-related gene; MCT. The most important marker for total tocopherol content is E33M50_16 which explains 33.9% of phenotypic variance. One of the most important candidate genes involving fatty acid biosynthesis, FAD2 (FAD2-1), is linked to oleic and linoleic acids content and explained more than 53% of phenotypic variance. Common markers associated with different seed-quality traits in well-irrigated and water-stressed conditions could be used for marker-assisted selection (MAS) in both conditions. Other markers, which are specific for one condition whereas linked to different traits or specific for a trait, could be useful for a given water treatment.     

Effects of high oleic acid soybean on seed yield,protein and oil contents,and seed germination revealed by near‐isogeneic lines

Typical soybean oil is composed of palmitic, stearic, oleic, linoleic and linolenic acids. High oleic acid content in soybean seed is a key compositional trait that improves oxidative stability and increases oil functionality and shelf life. Using a marker‐assisted selection method, near‐isogenic lines (NILs) of G00‐3213 for the high oleic trait were developed and yield tested. These NILs have various combinations of FAD2‐1A and FAD2‐1B alleles that were derived from the same backcrossing populations. The results indicated that G00‐3213 NILs with both homozygous mutant FAD2‐1A and FAD2‐1B alleles produced an average of 788 g/kg oleic acid content. The results also demonstrated that possessing these mutant alleles did not cause a yield reduction. Furthermore, seed germination tests across 12 temperatures (12.8–32.0°C) showed that modified seed composition for oleic acid in general did not have a major impact on seed germination. However, there was a possible reduction in seed germination vigour when high oleic seeds are planted in cold soil. The mutant FAD2‐1A and FAD2‐1B alleles did not hinder either seed or plant development.     

Characterization of genotype by environment interactions in soybean breeding programmes of southeast Europe

Southeast Europe is Europe's major production area of soybean of maturity groups (MG) 0, I and II, but genotype by environment interaction (GEI) for soybean traits in this region is still not characterized. The objective of this study was to characterize GEI in soybean grown in Croatia for seed yield, protein and oil content. Fourteen soybean cultivars of MG 0 and I were evaluated at 15 environments. In the combined anova , genotypes, environments and GEI were significant. All three seed traits were more affected by year than by location and seed yield and protein content were more sensitive to environmental changes than oil content. Genotype by environment interaction is generally of less importance than effects of genotypes and years, especially for oil content. High‐yielding genotypes had values of regression coefficient (b) close to 1, indicating that they were not responsive to extreme environments, either positive or negative. Means and b values were not correlated for yield and negatively correlated for protein and oil content, probably because of low‐input practice in the region. The absence of recognizable differences in means, b values and principal component scores of ammi analysis between two MGs for all traits indicated that soybean cultivars of MG 0 may be recommended for growing in the region because they responded similarly to environmental changes as do full‐season genotypes of MG I.     

Cytoplasmic male sterility with self-incompatibility,a novel approach to utilizing heterosis in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

The Polima cytoplasmic male sterility (CMS) system has been successfully used in three/two-line hybrid production in rapeseed (Brassica napus L.). However, the sterility of the Polima (pol) CMS lines is sensitive to temperature fluctuations. Also, traces of pollen can cause self-pollination within the CMS lines, which results in reduced levels of F₁ hybrid seed purity and leads to a significant yield loss. Self-incompatibility (SI) is another important approach for hybrid seed production in rapeseed. Despite having a wide range of restorers and being easily selected in a breeding program, SI system has some drawbacks. In this study, SI genes from a self-incompatible line of Brassica napus were transferred to a pol CMS line and S372A, a novel line of combined cytoplasmic male sterility with self-incompatibility was bred. Due to the SI genes, this line produced very few seeds when it was selfed at low temperature and no seeds at high temperature. This suggested that the line with CMS + SI had combined the advantages and overcome the disadvantages of both the pol CMS and SI systems. Furthermore, our results showed that most of the maintainers and all the restorers of the pol CMS system were also maintainers and restorers of the CMS + SI line, respectively. This indicates that the CMS + SI system can be easily used to establish three-line hybrids of rapeseed, and we believe this novel system could be extended to other species of Brassica.     

Genotype analysis of the wheat semidwarf Rht‐B1b and Rht‐D1b ancestral lineage

In wheat, semidwarfism resulting from reduced height (Rht)‐B1b and Rht‐D1b was integral to the ‘green revolution’. The principal donors of these alleles are ‘Norin 10’, ‘Seu Seun 27’ and ‘Suwon 92’ that, according to historical records, inherited semidwarfism from the Japanese landrace ‘Daruma’. The objective of this study was to examine the origins of Rht‐B1b and Rht‐D1b by growing multiple seed bank sources of cultivars comprising the historical pedigrees of the principal donor lines and scoring Rht‐1 genotype and plant height. This revealed that ‘Norin 10’ and ‘Suwon 92’ sources contained Rht‐B1b and Rht‐D1b, but the ‘Seu Seun 27’ source did not contain a semidwarf allele. Neither Rht‐B1b nor Rht‐D1b could be definitively traced back to ‘Daruma’, and both ‘Daruma’ sources contained only Rht‐B1b. However, ‘Daruma’ remains the most likely donor of Rht‐B1b and Rht‐D1b. We suggest that the disparity between historical pedigrees and Rht‐1 genotypes occurs because the genetic make‐up of seed bank sources differs from that of the cultivars actually used in the pedigrees. Some evidence also suggests that an alternative Rht‐D1b donor may exist.     

Novel variation for the tocopherol profile in a sunflower created by mutagenesis and recombination

α‐Tocopherol is the main tocopherol in sunflower seeds (>90%). Because it exerts a weak antioxidant action in vitro, its partial replacement by other tocopherols is an important breeding objective in this crop. The objective of this research was to develop novel tocopherol profiles in sunflower through mutagenesis and genetic recombination. Seeds of four ‘Peredovik’ accessions were used for chemical mutagenesis with ethyl methane sulfonate (EMS). Single‐seed screening in the M₂ generation resulted in two M₂ seeds, derived from different M₁ plants, with increased γ‐tocopherol contents of 19.2% and 96.7%, respectively. M₃ progeny from the M₂ seed with the 96.7% content bred true for high c‐tocopherol content, containing more than 90%γ‐tocopherol. M₃ progeny from the M₂ seed with only 19.2%γ‐tocopherol segregated in a range from 0 to 84.6%. Selection for high c‐tocopherol content produced an M_4:5 line, designated IAST‐1, with a stable high concentration of γ‐tocopherol. Crosses between IAST‐1 and T589, with an increased b‐tocopherol content, produced F₂ segregants with trans‐gressive levels of up to 77%β‐tocopherol or up to 68% d‐tocopherol. Both novel tocopherol profiles were confirmed in the F₃ generation.