ABSTRACT

To study the genetic variability for tocopherol contents in rapeseed, 72 different lines were cultivated under greenhouse conditions (1996) and under field conditions (1997 and 1998). Total tocopherol contents ranged from 206 to 315 mg/kg seed. The alpha-/gamma-tocopherol ratio was in the range of 0.43 to 1.42. Broad-sense heritabilities for alpha-, gamma- and total tocopherol contents and for the alpha-/gamma-tocopherol ratio were 0.81, 0.80, 0.82 and 0.79, respectively. In addition, two complete diallel cross programs with six parents each (Diallel I and II) were carried out. The parental lines differed in both total tocopherol content and composition (p<0.01). For the alpha-/gamma-tocopherol ratio, GCA effects were statistically significant (p<0.01), indicating that additive gene action is of major importance in the inheritance of the tocopherol composition. For total tocopherol contents, only SCA effects were detected in Diallel II (p<0.05), whereas in Diallel I only GCA effects were significant (p<0.01). No reciprocal effects were detected for the analysed traits.

INTRODUCTION

An important group of natural antioxidants with biological activity in vegetable oils are tocopherols. They occur in four derivatives (alpha-, beta-, gamma- and delta-tocopherol; the alpha-derivative is also know as vitamin E), differing in the methylation of the tocol head group (Pongracz et al., 1995). The main biochemical function of the tocopherols is believed to be the protection of polyunsaturated fatty acids (PUFA) against peroxidation (Kamal-Eldin and Appelqvist, 1996). The vitamin effectiveness of the tocopherols is very different, being alpha-tocopherol the best of the four tocopherol-derivatives. However, it shows the lowest antioxidant effect in vitro, being gamma-tocopherol the most efficient of the four tocopherols (Pongracz et al., 1995).

In rapeseed (Brassica napus L.), total tocopherol content ranges from 300 to 800 mg kg^-1 oil (Appelqvist, 1972; Goffman and Becker, 1998). These values represent a medium to low total tocopherol content compared to that of other oil plants. Rapeseed oil contains on average 64% of gamma-tocopherol, 35% of alpha-tocopherol and a very low percentage (<1%) of delta-tocopherol (Appelqvist, 1972; Goffman and Becker, 1998). The ratio of the content of alpha- to gamma-tocopherol could be therefore used to describe the tocopherol composition in rapeseed. Goffman and Becker (1998) have found a wide variation for this ratio, which ranged from 0.32 to 1.40. Since gamma-tocopherol exerts a 25-fold lower biological activity than that of alpha-tocopherol (Grela and Günter, 1995), an increase on the alpha-tocopherol fraction in rapeseed could be desirable to balance the tocopherol complex in both the antioxidant and vitamin activities. For all these reasons, it appears to be interesting to breed for both higher total tocopherol contents and high alpha-/gamma-tocopherol ratio in rapeseed.

Estimates of genetic variance and heritability provide useful guidelines for answering many questions which arise in a plant breeding program. The aim of the present study was to investigate (i) the genetic variability for tocopherols and (ii) the inheritance of tocopherols in a diallel mating system.

MATERIALS AND METHODS

(i) Variability for tocopherol contents

Seventy-two different breeding lines and cultivars obtained from a broad B. napus germplasm collection, were evaluated in greenhouse and field experiments in Göttingen, Germany. The genotypes represented to a very divergent genetic material including old breeding varieties, glucosinolate and/or erucic acid containing lines, resynthesized B. napus and others. In the greenhouse experiment (1996), the genotypes were planted in multipots and after emergence vernalized (5-10 ⁰C) for 8 weeks. Then, they were transplanted to 11 cm-pots in a greenhouse and grown at 14-h daylength. Temperatures averaged 24 ⁰C during the day and 20 ⁰C at night. Four self-pollinated plants of each genotype were harvested and seed samples were analysed for tocopherols. The field experiments were performed over two years (1997 and 1998). The genotypes were cultivated in observation plots (3.2 m²) using a complete randomized design with one replication. Four plants of each plot were selfed and sampled for analyses of tocopherol contents.

(ii) Diallel Cross experiments

The material for two complete diallel cross programs (Griffing, 1956) were produced in 1997 using two sets of six genotypes each previously identified by the greenhouse experiment of 1996. In the first diallel (Diallel I), the parents selected were high or low for total tocopherol contents and in the second diallel cross (Diallel II), the parents used were high or low for the alpha-/gamma-tocopherol ratio. In 1998, the parental lines and the resulting F₁ plants of the two diallels were tested in a completely randomized design with 4 single plants representing each cross or parental line. The plants were conduced up to vernalization as described above and then carried to a vegetation room. They were self-pollinated and after maturity, they were sampled for tocopherol determination. The mean value of the four plants was used in the statistical analysis (Griffing, 1956, method III), which was performed with SAS-program.

Analysis of tocopherol contents

Tocopherol contents were determined by HPLC as described by Thies (1997), using a fluorescence detector (l_ex=295 nm and l_em=330 nm), a C-18 diol column (250 x 3 mm I.D.), and isooctane/tert-butyl-methylether (94/6) as mobile phase at a flow rate of 0.7 ml/min.

RESULTS AND DISCUSSION

(i) Variability for tocopherol contents

Figure 1 shows the distribution of the mean values of alpha-, gamma-, total tocopherol contents and the alpha-/gamma-tocopherol ratio over three different environments. Total tocopherol contents ranged from 206 to 315 mg/kg seed. The alpha-/gamma-tocopherol ratio was in the range of 0.43 to 1.42. Considering the genetic range of variation an increase in both the total tocopherol contents and the alpha-/gamma-tocopherol ratio appears to be possible by classical breeding. Correlations between tocopherol contents and the alpha-/gamma-tocopherol ratio are shown in Table 1. Total tocopherol content was not correlated with the alpha-/gamma-tocopherol ratio, indicating that total tocopherol content is independent from the tocopherol composition. Although gamma-tocopherol has been identified as a precursor in the synthesis of alpha-tocopherol (Soll and Schultz, 1980), the contents of both tocopherols were not correlated. A possible explanation of this result is the different localization of both tocopherols in the plant cells. Whereas alpha-tocopherol is found inside the chloroplast, gamma-tocopherol is mainly outside (Booth, 1963). In conclusion, these results suggest the possibility to breed for higher alpha- or gamma-tocopherol content without a decrease in the content of the other tocopherol-derivative, respectively. Broad-sense heritabilities for alpha-, gamma- and total tocopherol contents and for the alpha-/gamma-tocopherol ratio were 0.81, 0.80, 0.82 and 0.79, respectively.

Figure 1. Distribution of: (A) alpha-, (B) gamma- and (C) total tocopherol contents (mg

kg^-1 seed) and (D) alpha-/gamma-tocopherol ratio of 72 genotypes (mean values over three environments).

(ii) Diallel cross experiments

The genotypes selected as parental lines differed extremely (p<0.01) in both total tocopherol content and composition (Table 2). Estimates of general and specific combining ability (GCA and SCA, respectively) and reciprocal effects are presented in Table 3. The parental lines differed in both total tocopherol content and composition (p<0.01). For the alpha-/gamma-tocopherol ratio, GCA effects were statistically significant (p<0.01), indicating that additive gene action is of major importance in the inheritance of the tocopherol composition. For total tocopherol contents, SCA effects were only detected in Diallel II (p<0.05), whereas in Diallel I only GCA effects were significant (p<0.01). No reciprocal effects were detected for the analysed traits.

Table 1. Correlation coefficients between alpha-, gamma- and total tocopherol contents (mg kg^-1 seed) and the alpha-/gamma-tocopherol ratio.

* significant at the 0.01 probability level

Table 2. Total and individual tocopherol contents (mg kg^-1) and the alpha-/gamma-tocopherol ratio of the 12 parental lines used for the two diallel experiments.

WR: winter rapeseed line; DH: double haploid line; Resyn: resynthesized rapeseed; M high C18:1: mutant with high oleic acid content; M very low GSL: mutant with very low glucosinolate content; 00: low glucosinolate and erucic acid content; ++: medium to high glucosinolate and erucic acid content.

Table 3. Mean squares (MQ) and variance components of the analysis of variance for total tocopherol contents (mg kg^-1 seed) and alpha-/gamma-tocopherol ratio of the two diallels.

*, ** significantly different at p=0.05 and 0.01, respectively.

ACKNOWLEDGEMENTS

The authors thank Dr. Wolfgang Grüneberg for providing assistance in the statistical analysis.

REFERENCES

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