J Sci Food Agric 1989, 46, 319-324

The Use of Glucosinolates for Cultivar Identification in Swede, Brassica napus L var napobrassica (L) Peterm

Helen Adams,* John G Vaughan

Department of Food Science, King’s College (Queen Elizabeth College), Campden Hill Road, London W8 JAH, UK

and

G Roger Fenwick

Molecular Science Department, AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich NR4 7UA, UK

(Received 14 January 1988; revised version received 16 February 1988; accepted 11 March 1988)

Key words: Glucosinolates, Brassica napus, swede, high performance liquid chromatography, cultivar identification, chemotaxonomy.

A quantitative study of the glucosinolates in the seed, cortex and medulla (peeled root) fiom nine cultivars (cus) of swede, Brassica napus L var napobrassica (L ) Peterm using high performance liquid chromatography was undertaken to see whether such characteristics could be used as a biochemical index of cultivar identity. All nine cultiuars could be distinguished following an analysis of medulla glucosinolatzs, but the analysis of seed and cortex glucosinolates yielded no such usehl information.

With the introduction of Plant Breeders’ Rights and the increasing complexities of plant breeding, the unambiguous identification of individual cultivars is becoming more important. Within the genus Brassicu, methods have tended to rely on agronomic or botanical characters which, because they necessitate the examination of a growing plant, are costly and labour intensive.

The swede, Brassica napus L var napobrassica (L) Peterm, has the capacity to synt hesise glucosinolates, a class of sulphurcontaining glycosides (Fenwick et a1

Present address: Department of Applied Genetics, John Innes Institute and AFRC Institute of Plant Science Research, Colney Lane, Norwich NR4 7UA. UK.

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J Sci Food Agric 0022-5142/89/$03.50 0 1989 Society of Chemical Industry. Printed in Great Britain

320 H Adams, J G Vuughun, G R Fenwick

1983). This is an ability it shares with all members of the Cruciferae so far examined (Kjaer 1976; Robbelen and Thies 1980; Fenwick et al 1983). Glucosinolates coexist but are not in contact with an enzyme, myrosinase (thioglycose glucohydrolase, EC 3.4.3.1). Following any disruption or damage of plant material in the presence of moisture, glucosinolates are hydrolysed by myrosinase, and the products include sulphate, glucose, isothiocyanates, thiocyanates and nitriles (Fenwick et a1 1983).

Concern over the toxic effects of such glucosinolate hydrolysis products has led to detailed studies of both glucosinolates and their derivatives in cruciferous crops (Van Etten et a1 1976; Heaney and Fenwick 1987). As an indirect result of this work, significant differences in the proportions of glucosinolates synthesised by different cultivars of the same species have been noted with the suggestion that differences in these glucosinolate ‘patterns’ could be used to distinguish cultivars (Cole and Phelps 1979; Heaney and Fenwick 1980; Tookey et a1 1980). This present work forms part of a larger study initiated to assess the potential value of glucosinolates as chemotaxonomic markers for the identification of cultivars of the different agronomic forms of Brassica napus L: oilseed rape, forage rape and swede (Adams 1986).

2 MATERIALS AND METHODS

The material selected for analysis comprised the swede cultivars in the 1984 National List of Recommended Varieties (Anon 1984) published by the National Institute of Agricultural Botany, viz Acme, Angus, Doon Major, Magres, Marian, Melfor, Ruta Otofte, Sator Otofte and Seegold.

Swede seed and roots of the cultivars were obtained through the National Institute of Agricultural Botany at Cambridge; the roots, grown under cultivar trial conditions at Headley Hall, Tadcaster, Yorkshire, were analysed a maximum of 5 days after harvest.

2.1 Sample preparation and analysis

The seeds were defatted and the glucosinolates extracted from the airdried meal using the method of Thies (1976). Swede roots were peeled, and cortex and medulla extracts were prepared according to Sones et a1 (1984). All extracts were stored at - 20°C prior to analysis.

The method of analysis followed was as that of Spinks et a1 (1984), in which desulphoglucosinolates are separated and quantified by HPLC in the reverse-phase mode. Prop-2-enyl and benzyl glucosinolates were selected as internal standards for the seed and medulla, respectively.

2.2 Identification and quantification

Chromatographic peaks were monitored using a Perkin Elmer LC75 spectrophotometric detector at 230 nm, and integrated by a Fortran program developed for an IBM 1130 computer. Individual peaks were identified by comparison with available standards or by inspection of their GLC-MS fragmentation patterns (Eagles et a1 1981). Response factors were calculated using

Use of glucosinolates for cultivar identification in Swede 32 I

pure standards; where these were unavailable (pent4enyl and 2-hydroxypent-4- enyl glucosinolates), the response factors of the lower homologues were used. If this was not possible (for the methyl-4-thiobutyl-, 2-phenethyl-, 4-methoxyindolyl-3- methyl-, 1-methoxyindolyl-3-methyl glucosinolates), a response factor of 1 was utilised.

3 RESULTS AND DISCUSSION

Table 1 presents data on the major glucosinolates recorded in swede seed, cortex and medulla in this study. Glucosinolates found in swede seed are in general agreement with those recorded by Sang et af (1984), though indolyl-3-methyl glucosinolate was not identified. The glucosinolates identified in swede cortex and medulla support the findings of Sones et af (1984). Table 1 highlights the marked qualitative differences in the glucosinolates accumulated by both different organs and by different tissues of the same organ (Fenwick et al1983; Sang et al1984). This may result from an alteration of substrate availability or mayreflect different control mechanisms operating on the glucosinolate biosynthetic pathway in different tissues.

Analysis of swede seed provided two different glucosinolate profiles. In the majority of cultivars the glucosinolate profile was similar to that recorded for high- glucosinolate oilseed rape (Adams et af 1985), with but-3-enyl and 2-hydroxybut-3- enyl glucosinolates being approximately 20% and 65 % of the total, respectively. In three cultivars (Acme, Doon Major and Marian), but3enyl glucosinolate

TABLE 1 The relative abundance ( % total glucosinolate content) of glucosinolates in cultivars of swede

as determined by HPLC

Glucosinolate Glucosinolate content, %

Seed" Cortex Medulla'

But-3 enyl 17 (2-25y tr 4 (0-15) Pent-4-enyl 2 (0-4) tr tr 2-Hydroxybut -3enyl 72 (60-72) 12 (4-21) 28 (12-44) 2-Hydroxypent-4enyl 2 (0 -5) tr tr 4-Hydroxyindolyl-3-methyl 8 (3-11) tr 5 (0-8)

tr 4 (34) Methyl-4-thiobutyl - Indolyl-3-methyl - 5 (3-8) 11 (5-21)

7 (1-14) 4 (3-6) 4-Methoxyindoly l-3-methyl - 2-Phenethyl - 5 (2-12) 12 (3-21)

1 -Methoxyindolyl-3-methyl - 72 (60-84) 32 (1945)

" Figures represent the mean of 11 accessions. 'Figures represent the mean of 9 accessions. ' Range of contents. tr, Trace amount ( < 0 5 %).

322 H Adams, J G Vaughan, G R Fenwick

i 2 3 Glucosindates

20 I d L 10 1 2 3

Fig 1. Glucosinolate profiles. reflecting the relative abundance of three glucosinolates as a percentage of the total recorded in swede medulla. A (Acme), B (Angus). C (Doon Major), D (Magres), E (Marian), F (Melfort), G (Ruta OtoRe), H (Sator Otofte). 1 (Seegold). Glucosinolates I (4- hydroxyindolyl-3-methyl), 2 (2-phenethyl), 3 ( I -

methoxyindolyl-3-methyl). Glucoskroletes

accounted for approximately 5 % of the total, and 2-hydroxybut-3enyl glucosinolate made up 80% of the total. Gland et a1 (1981) reported similar results following an analysis of swede seed. Three different glucosinolate profiles were recorded following an analysis of swede cortex but the analysis did not suffice to differentiate between the individual cultivars.

Analysis of medulla extracts yielded the most valuable information, since all nine cultivars could be distinguished following a comparison of the relative proportions of the individual glucosinolates. Eight different glucosinolates were quantified (Table l), but on average over 70% of the total comprised 2-hydroxybut-3eny1, 2-phenethyl- and 1 -methoxyindolyl-3-methyl glucosinolates. The relative propor- tions of these three glucosinolates were found sufficient to identify each of the cultivars tested (Fig 1). Although the results illustrate the apparent value of medulla glucosinolate analysis for cultivar identification, further work is needed to determine site-to-site, year-to-year and intracultivar variation.

Absolute values for intracultivar variation do not seem to be available for swede roots, although Cole and Phelps (1979) found that this was not large enough to preclude the separation of swede cultivars following an analysis of glucosinolate hydrolysis products. Carlson et a1 (1981) carried out an analysis of glucosinolate hydrolysis products in swede roots, with results suggesting that the relative proportions of the glucosinolates were stable from year to year- though absolute amounts did vary. Although environmental factors are important determinants of total glucosinolate content, the relative proportions of the individual contributory

Use of glucosinolares for cultivar idenri/ication in Swede 323

glucosinolates have been found to be remarkably constant both in seed (Robbelen and Thies 1980; Gland et a1 1981; Adams et aZ1985) and vegetative tissue (Fenwick et a1 1983; Heaney et a1 1983). It is thus considered that the recorded differences in the glucosinolate profiles of swede medulla may be of value for cultivar identification.

ACKNOWLEDGEMENTS

The authors are grateful to staff at the National Institute of Agricultural Botany for supplying all material used in this study, which was supported by a SERC-CASE award to HA.

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