A time-course study of glucosinolates in the ontogeny of forage rape (Brassica napus L)

One cultivar and two breeding lines of forage rape, grown in a glasshouse, were sampled at weekly intervals from 9 to 22 weeks after sowing. Separate determinations of glucosinolate content were made on leaves and stems by HPLC. There were differences in the proportion and concentration of individual glucosinolates between the rapes and between leaves and stems, showing that levels of individual glucosinolates cannot be predicted from total glucosinolate content. Of particular interest was the variation with harvest date in the levels of individual glucosinolates. This has considerable implications for the comparison of levels of individual glucosinolates between cultivars and crops, and suggests that detailed agronomic field studies are required to determine whether the intake by animals of potentially toxic glucosinolates, in particular progoitrin, can be minimised by exploiting these seasonal differences in glucosinolate concentrations. Progress has been made in lowering the level of this glucosinolate, and the possibility of breeding forage rape with very low or zero levels of progoitrin is indicated.     

A time-course study of the effect of sulphur on glucosinolates in oilseed rape (Brassica napus) from the vegetative stage to maturity

Samples consisting of the whole above-ground part of the oilseed rape plant (Brassica napus L), were taken every 2 weeks from before stem extension to maturity. The plants were separated into vegetative tissue, floral tissue, pods and seeds (when these components appeared), and the individual glucosinolates present were quantified by high performance liquid chromatography. A high glucosinolate variety (Rafal) and a low glucosinolate variety (Cobra) were compared. The effect of sulphur (32 kg ha^?1 elemental sulphur applied at the beginning of stem extension) on the various parts of the plant was investigated. Total glucosinolate concentration in all plant parts was higher in Rafal than in Cobra. Glucosinolate concentration in the vegetation declined over time, most noticeably when the flowers and seeds were produced, and glucosinolate concentration of the pods also fell as that of the seeds rose. It is suggested that there may have been some redistribution of glucosinolates or glucosinolate precursors within the plant as maturity approached. Changes occurred in the proportions that individual glucosinolates contributed to the total glucosinolate content, and this may be relevant to plant/pathogen relationships. Sulphur application increased the glucosinolate concentration of the vegetative tissue by mid April and also increased the glucosinolate concentration of the flowers. It is suggested that this could affect the plant's resistance to disease.     

Effect of harvest date and pod position on glucosinolates in oilseed rape (Brassica napus)

Pod samples from four varieties of winter oilseed rape (Brassica napus L) were taken twice weekly from 26 to 3 days before harvest from upper, middle and lower third portions of the main raceme. Seeds were analysed for individual glucosinolate concentration by HPLC. Total glucosinolate content increased suddenly during the sampling period. The timing of the increase depended on variety but occurred before harvest would have been practical. Proportions of individual glucosinolates in Rafal, the only “high” glucosinolate variety examined, did not change over the sampling period. In the other varieties, the proportion of 2-hydroxy-but-3-enyl (progoitrin) increased and the proportion of 2-hydroxy-pent-4-enyl decreased as sampling progressed, so that the concentration of individual glucosinolates could not be predicted from total glucosinolate content. The ‘low’ glucosinolate varieties contained a significantly smaller concentration of progoitrin and summed aromatic glucosinolates than Rafal. Variations in glucosinolate content with pod position were smaller than those due to sampling date, but there was a greater concentration of glucosinolates in seeds from pods lower on the terminal raceme. The results would indicate that care should be taken if seed samples from the plant are taken.     

Determination of total glucosinolates in oilseed rape by X-ray spectrometric analysis for oxidised sulphur (S6+)

A rapid X-ray spectrometric (XRS) method has been developed for the determination of the total glucosinolate content of oilseed rape and other Brassica oilseeds. The method is based on analysis for fully oxidised sulphur (S⁶⁺), which includes half the sulphur (S) in the glucosinolate molecule, and the S in sulphate. Results are highly correlated with glucosinolate content determined by glucose release, a standard method widely used in Australia. The relationship is total glucosinolates = (23·97 S⁶⁺ -9·43) r² = 0·987, where the glucosinolate content is expressed as μmol g^?1 and the S⁶⁺ content in mg g^?1. The relationship is applicable to seed of any glucosinolate content and to meal, and is unaffected by changes in protein sulphur content. The correlation of glucosinolates with S⁶⁺ is shown to be closer than the correlation with total S. The latter correlation forms the basis of the existing XRS method, used within the European Community in recent years. The advantages of S⁶⁺ derive from the linearity of the regression and the elimination of errors caused by variation in protein content. The method should be valuable to the Australian oilseed industry because it allows the rapid screening of breeding lines to ensure low glucosinolate content and the assessment of deliveries for crushing and of meal.     

Sulphur turnover in the developing pods of single and double low varieties of oilseed rape (Brassica napus L)

Changes in the sulphur (S) concentrations in pod walls and seeds of the double low variety Cobra and single low variety Bienveou of winter oilseed rape (Brassica napus L) were studied during pod development. The S concentration in the seeds of Cobra increased between 47 and 61 days after flowering and thereafter remained stable, whereas in Bienvenu seed S concentration was much higher initially and continued to increase throughout the sampling period (from 57 to 108 days after flowering). These differences in S concentration reflect different patterns of glucosinolate accumulation in the seeds. In contrast, there was a large and progressive build-up of S in the pod walls of Cobra, such that at maturity the S concentration in the pod walls of Cobra was approximately double that of Bienvenu. A large and progressive accumulation of inorganic sulphate was found to be responsible for the build-up of S in the pod walls of the double low variety. Short-term feeding experiments with terminal racemes showed that a smaller proportion of ³⁵S was translocated from the pod walls to the seeds in Cobra than in Bienvenu. However, the build-up of S in the pod walls of Cobra did not seem to result from a restricted translocation of glucosinolates, since in both varieties exogenously supplied allylglucosinolate was translocated rapidly from the pod walls to the seeds. These results are therefore consistent with the hypothesis that pod walls are a major site for the biosynthesis of glucosinolates present in the seeds, and a metabolic block in the pathway of glucosinolate biosynthesis is responsible for the low glucosinolate concentration in the seeds of double low varieties, which consequently results in a large build-up of sulphate in the pod walls.     

Glucosinolate profiles of international rapeseed lines (Brassica napus and Brassica campestris)

Seed samples from a range of international rapeseed lines (Brassica napus L and B campestris L) were analysed for glucosinolate content using high performance liquid chromatography (HPLC). This analytical method provided a total profile consisting of eight different glucosinolates, including those used to define the Canola standard. The data indicated that broad categories of distinct glucosinolate profiles existed among the seed lines analysed. Indole glucosinolate content, expressed as a percentage of total glucosinolate content, was higher in the low glucosinolate lines of B napus. The actual contents of all eight glucosinolates in the seed were variable, particularly gluconapin, glucobrassicanapin and progoitrin. The importance of this HPLC procedure in facilitating selection for high or low contents of individual glucosinolates is discussed.     

Influence of nitrogen and sulphur on the glucosinolate profile of rapeseed (brassica napus l)

Clear influence of N and S applications on the profile of individual glucosinolates in the seeds of winter oilseed rape (Brassica napus L) was found in field experiments. The major effect of N was on the relative abundance of the four alkenyl glucosinolates. Increasing the N rate increased the relative proportion of 2-hydroxybut-3-enyl at the expense of pent-4-enyl, and to a lesser extent, 2-hydroxypent-4-enyl, in the double-low (low erucic acid and low glucosinolates) variety Cobra, but at the expense of but-3-enyl in the single-low (low erucic acid) variety Bienvenu. The results strongly suggest that a high N supply favours the hydroxylation step from but-3-enyl to 2-hydroxybut-3-enyl. In contrast, the major effect of S appeared to be on the relative abundance of the alkenyl and indole groups as a whole. Sulphur deficiency decreased the concentrations of the alkenyl glucosinolates more than those of the indole glucosinolates, whereas S application to a S-deficient crop resulted in a larger response in the alkenyl group than in the indole group. The more sensitive response of alkenyl glucosinolates to the plant S status is probably due to the requirement of methionine in their biosynthesis, as compared with the indole glucosinolates which are derived from tryptophan. Implications for changes in the glucosinolate profile in rapeseed are discussed.     

Physiological role of glucosinolates in brassica napus. Concentration and distribution pattern of glucosinolates among plant organs during a complete life cycle

Concentrations and distribution of glucosinolates in various organs of rape (Brassica napus L) were examined during major stages of development. Fluctuations in the total content as well as in the individual levels of glucosinolates were investigated in double low Drakkar and high glucosinolate Chine 32 cultivars. The distribution pattern of aliphatic and aromatic glucosinolates between the various organs, as well as the variation in the concentrations of individual glucosinolates during plant development were quite similar between the two cultivars. Peculiar stages of accumulation during the vegetative growth period and the seed maturation period were identified. These stages were followed by periods of intense degradation during flowering, germination and early growth stages of seedlings. Fluctuations in the amount and structure of the glucosinolates in each of these periods suggest a storage function for these compounds which are required at specific stages of development. The involvement of some of these compounds in hormonal control of flowering is also discussed.     

The response of double-low winter oilseed rape to fertiliser sulphur

The effect of soil-applied ammonium sulphate (10, 20, 30, 50 and 80 kg S ha^?1) and agricultural gypsum (20 and 50 kg S ha^?1) and of foliar-applied elemental sulphur (20 kg S ha^?1) on the seed yield and chemical composition of double-low cultivars of winter oilseed rape was determined at 11 sites in England during 1989-1991. Significant (P ? 0.05) mean seed yield responses to applied sulphur (S) of 10 and 17% were obtained at two sites on sandy soils in northern England which showed symptoms of severe S deficiency. At a third site on a shallow calcareous soil in south-west England, which did not show S deficiency symptoms, seed yield was consistently but not significantly increased by an average of 8%. At one of the sites in 1989, application of ammonium sulphate caused leaf scorch and, at the maximum rate of S applied, seed and oil yield were decreased. No conclusions were reached regarding the amount of S required for maximum yield response. The S treatments decreased seed oil content by an average maximum of 9 mg g^?1. Seed glucosinolate content was increased by a greater amount at the two sites in northern England compared to other sites, where increases averaged only 3 μmol g^?1. Analysis of individual glucosinolates in the harvested seed from one of the sites in northern England showed that only the alkenyl glucosinolates were increased by S application. Yield responses were best predicted by total S concentrations and N : S ratio values in young fully-expanded leaves at flowering. Foliar-applied elemental S was consistently less effective in raising leaf S content than ammonium sulphate. Gypsum was occasionally found to be less effective than ammonium sulphate as an S fertiliser.     

Variation of glucosinolates in three edible parts of Chinese kale (Brassica alboglabra Bailey) varieties

Glucosinolates in Chinese kale (Brassica alboglabra Bailey) were analysed by liquid chromatography–mass spectrometry (LC–MS), and the variation of glucosinolates in three edible parts (sprout, rosette leaf and bolting stem) of 27 Chinese kale varieties was investigated. Thirteen glucosinolates, including eight aliphatic glucosinolates, four indole glucosinolates and one aromatic glucosinolate, were identified in Chinese kale. The contents of total glucosinolate varied extensively among the different edible parts, and the total glucosinolate contents in the sprouts were obviously higher than those in rosette leaves and bolting stems. Obvious differences in the total and individual glucosinolate contents were also observed in each edible part among the different varieties. Gluconapin was the most abundant glucosinolate among all the edible parts of the 27 varieties except for the sprout of JL-26. The JL-26, JL-25 and JL-16 varieties are good candidates for future breeding programs since they contain high levels of methylsulphinylalkyl glucosinolates (glucoiberin and glucoraphanin) in certain edible parts.     

Changes in the dry matter,sugar, plant fibre and lignin contents of swede,rape and kale roots in response to turnip root fly (Delia floralis) larval damage

Plants from two genotypes of swede (Brassica napus var napobrassica), kale (B oleracea var. acephala) and rape (B napus var napus) were each inoculated at the three to four true leaf stage with 10 eggs of the turnip root fly, Delia floralis. After 8 weeks the damage caused by the resultant larvae significantly reduced the weight of leaves, stems and roots compared with uninoculated plants, with the greatest reduction (26–46%) being found in the roots. Dry matter content, which did not differ between either crop type or cultivar, was unaffected by larval damage. Ethanol-soluble sugar content was reduced in all cases by larval damage, but only significantly in one rape and one swede cultivar. The effect of D floralis damage on the concentrations of individual sugars (glucose, fructose and sucrose) was crop type and cultivar dependent. The neutral detergent fibre and lignin contents of the undamaged swede roots were significantly lower than in either the undamaged kale or rape cultivars studied. In all crop types the neutral detergent fibre and lignin content significantly increased following D floralis damage. The six Brassica genotypes studied fell into two groups on the basis of host status for D floralis. The two kale cultivars and the forage rape Hobson were considered poor hosts for D floralis, with low percent age pupation (38–41%) and low pupae weight (12.5–14.4 mg). The two swedes and the oilseed rape Ariana were considered good host for D floralis, with both higher percentage pupation (51–59%) and pupae weights (19.8–20.0 mg). The possible relationships between the root composition of undamaged and attacked roots, and D floralis resistance are discussed.     

Theoretical principles for the indirect determination of the total glucosinolate content in rapeseed and meal quantifying the sulphur concentration via X-ray fluorescence (X-RF method)

The introduction of double low oilseed rape cultivars into common crop production will require the development of methods for a precise, rapid and simple determination of the total glucosinolate content in rapeseed. Recent methods are often inappropriate for plant breeding work, and also for the trade and oil mills in separating parcels of seed according to their glucosinolate contents. In contrast to the methods commonly used (chromatography, enzymic and colorimetric tests) an indirect method is described here which is based on the very close correlation between the total sulphur and glucosinolate content in rapeseed. The sulphur content of the samples can be determined precisely and non-destructively by means of X-ray fluorescence spectroscopy (X-RF). This new method, which is suitable for rapeseed and meal, requires simple preparation of the seed samples by grinding and compressing. The whole analytical procedure (sample preparation and measurement) takes 2-4 min. If necessary, moist seed samples can be quickly dried by microwave heating, because the method does not require intact glucosinolates. The steps of the X-RF method are fairly insensitive to errors. The method shows an excellent repeatability (r = 1.9 μmol g^?1) and reproducibility (R = 3.8 μmol g^?1). The results obtained with this X-RF method correlate closely with the conventional reference methods (r² = 96-99%).     

Glucosinolates in brassica vegetables. Analysis of 22 varieties of brussels sprout (Brassica oleracea var. gemmifera)

The glucosinolate content of 22 cultivars of Brussels sprout have been determined. The levels of total glucosinolates, determined by methods based on glucose release and gas chromatography, were substantially higher than those reported by other workers for cabbage and Chinese cabbage. The major glucosinolates in Brussels sprout leaf material were identified as gluconapin, progoitrin, sinigrin and glucobrassicin, with the latter two generally predominating. Detailed analysis of the material from five different growing sites has shown considerable variation in the total content but surprisingly little difference in the relative proportions of the individual glucosinolates within each cultivar. The glucosinolates of the corresponding seed material differ significantly from that in leaf; in particular glucobrassicin is found in trace amounts only, and glucoerucin, glucotropaeolin and neoglucobrassicin are all present, in addition to sinigrin, progoitrin and gluconapin.     

Enzymatische Bestimmung des Gesamtglucosinolat-Gehaltes in FutterrapsBrassica napus L.var. napus

Zusammenfassung Für die Bestimmung des Gesamtglucosinolat-Gehaltes in Futterraps wurde eine enzymatische Methode ausgearbeitet, die auf folgenden Analysenschritten beruht: Der Inaktivierung pflanzeneigener Enzyme, der Extraktion der zu bestimmenden Glucosinolate, der enzymatischen Zerstörung freier Glucose, der Hydrolyse der Glucosinolate durch Myrosinase und der enzymatischen quantitativen Erfassung nicht oxidierter freier sowie von Glucosinolaten stammender Glucose.In 23 Sorten von Rapspflanzen wurden 0,95 bis 1,96% Glucosinolate in der Trockensubstanz gefunden mit einer mittleren prozentualen Abweichung der Einzelwerte vom Mittelwert von 2,9. Zur Beurteilung von Futterraps ist es mit diesem Verfahren möglich, neben dem Ertrag und der Krankheitsresistenz auch den Gehalt an Glucosinolaten, die sowohl Tierfutter als auch Eier, Milch und Fleisch indirekt beeinträchtigen, mit heranzuziehen.

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Enzymatic determination of the total content of glucosinolates in the rape plantBrassica napus L.var. napus

Summary A method for the quantitative determination of the total content of glucosinolates in rape plants used as green forage was worked out. The analysis includes the following steps: Inactivation of the native plant enzymes, extraction of the glucosinolates to be determined, enzymatic oxidation of the free glucose, hydrolysis of the glucosinolates by myrosinase and enzymatic quantitative analysis of the unoxidized free glucose and finally the one originating from glucosinolates. In 23 rape varieties the glucosinolate content ranges from 0.95 to 1.96% of dry matter. The average deviation for single determinations from the mean amounts to 2.9%. With this procedure a selection of rape varieties for green forage is possible not only based on their yield and disease resistence but also on their content of glucosinolates which influence negatively the forage as well as indirectly eggs, milk and meat.

     

Genotypic variation of glucosinolates in broccoli (Brassica oleracea var. italica) florets from China

Five main cultivars from China and 143 parent materials grown in a greenhouse were used to investigate the glucosinolates in broccoli florets. Eight aliphatic glucosinolates, four indole glucosinolates, and one aromatic glucosinolate were identified and quantified using high-performance liquid chromatography (HPLC). The results showed that glucoraphanin, glucobrassicin, 4-methoxyglucobrassicin and neoglucobrassicin were present in all samples. However, the predominant type of glucosinolate was different among pure lines. The anti-cancer glucoraphanin concentration ranged from 0.06 to 24.17 μmol/g in pure lines and from 1.57 to 5.95 μmol/g in commercial cultivars. The progoitrin concentration in commercial cultivars varied from 1.77 to 6.07 μmol/g with a mean value of 3.20 μmol/g. Significant variations were observed in the concentration of individual glucosinolates and in each class of glucosinolates among broccoli populations. Dozens of specific lines with altered glucosinolate profiles, as well as ten good candidates for breeding high-chemoprotective glucosinolate cultivars, were obtained according to the putative glucosinolate pathway in broccoli.     

Variation in S-methyl cysteine sulphoxide concentration with harvest date in forage rape (Brassica napus)

Patterns of variation in glucosinolate concentrations of three forage rapes (Brassica napus L) over a 14-week period suggest that a high frequency of sampling the crop is necessary to determine when animals are likely to have a minimum intake of such anti-metabolites (Macfarlane Smith and Griffiths 1988). A study of S-methyl cysteine sulphoxide (SMCO), which causes anemia in animals (Greenhalgh 1969), in the same three rapes showed similar variation in concentration with time in both the leaf and stem. However, for any given sampling date, SMCO concentrations were very similar throughout the plant. Variation in nitrogen concentration within the plant did not follow the same pattern as SMCO.     

A simple spectrophotometric method for estimating total glucosinolates in mustard de-oiled cake

Glucosinolates are anti-nutritional factors present abundantly in the seed meal fraction of oilseed Brassica species. They are found in varying levels among different genotypes. Those genotypes containing less than 30 µmol/g are considered low/zero glucosinolate type and are preferred for edible purposes due to low pungency. Twenty two different genotypes were taken for the analysis of glucosinolates by spectrophotometry. A regression model was obtained using Ordinary Least Square technique which predicted a formula. Total glucosinolates (µmol/g) = 1.40 + 118.86 × A₄₂₅, where A₄₂₅ is the absorbance at 425 nm. The total glucosinolate content obtained by the prediction formula when compared with HPLC data showed a correlation coefficient of 0.942. This high correlation between the two data sets validated the developed methodology. This method also simplifies the estimation of total glucosinolates by excluding the use of HPLC or other sophisticated instruments.     

Effect of nitrogen and sulfur fertilization on glucosinolates in the leaves and roots of broccoli sprouts (Brassica oleracea var. italica)

Broccoli (Brassica oleracea L. var. italica cv. Marathon) sprouts are a rich source of glucosinolates, particularly 4‐methylsulfinylbutyl glucosinolate (glucoraphanin), the precursor of the chemoprotective isothiocyanate, sulforaphane. Sulfur and nitrogen fertilization have been shown to influence the pattern and levels of glucosinolates in mature broccoli, but little information is available on the fertilization of sprouts, a transient stage of broccoli growth, which have been recommended for salads. Therefore, an experiment was set up to evaluate the effect of N and S fertilization on the glucosinolate content of the aerial part and roots of broccoli sprouts. Nitrogen was tested at 0, 45.5, 91.0 mg L^?1 and sulfur at 0, 14.6 and 29.2 mg L^?1. The results showed that total glucosinolates in the aerial part were significantly higher (P < 0.001) than in the roots. The major glucosinolates in the aerial part were 4‐methylsulfinylbutyl and 3‐methylsulfinylpropyl whereas in the roots they were 2‐phenylethyl and 4‐methylthiobutyl. Fertilization of broccoli sprouts had a significant (P < 0.001) detrimental effect on the levels of aliphatic glucosinolates whereas the opposite was noted for indole and aromatic glucosinolates, for some of the fertilization combinations tested. Overall, the results indicate that broccoli sprouts do not benefit from fertilization. Copyright © 2006 Society of Chemical Industry     

The effect of cultivar,sample date and grazing on the concentration of s-methylcysteine sulphoxide in oilseed and forage rapes (brassica napus)

A study of the S-methylcysteine sulphoxide (SMCO) concentration in foliage from four double-zero, three single-zero cultivars of oilseed rape and four cultivars of forage rape revealed little inter-cultivar variation. Significant differences were however found with respect to harvest date, with the concentration of SMCO remaining fairly constant over the early winter period but then increasing as the plants commenced regrowth in the late winter-early spring. Continual exposure of plots to grazing wild rabbits produced small but statistically significant changes in SMCO concentration but compared with seasonal differences the changes due to grazing were small and inconsistant. These findings are discussed in relation to the recently reported harmful effects of double zero oilseed rape cultivars on browsing wildlife.     

Total and individual glucosinolate contents in inflorescences of eight broccoli cultivars grown under various climatic and fertilisation conditions

Total aliphatic, indolic and aromatic glucosinolates were evaluated in the edible portions of fresh harvested inflorescences of five commercial and three experimental broccoli (Brassica oleracea L var italica) cultivars grown under various climatic and agronomic conditions, ie early (winter) or late (spring) season with poor (15 kg ha^?1) or rich (150 kg ha^?1) sulphur fertilisation, in an attempt to identify differences due to genetic and agronomic factors. The predominant glucosinolates in all broccoli cultivars were 4‐methylsulphinylbutyl‐glucosinolate (glucoraphanin), 3‐indolylmethyl‐glucosinolate (glucobrassicin) and 1‐methoxy‐3‐indolylmethyl‐glucosinolate (neoglucobrassicin). The results showed no significance differences in total glucosinolates between rich and poor fertilisation, whereas total glucosinolates were detected more significantly in the late than in the early season. All broccoli cultivars showed a higher content of indolic glucosinolates than aliphatic glucosinolates. Clear advantages were detected in the analysed commercial cultivars, as the experimental cultivars yielded lower concentrations of these compounds. © 2003 Society of Chemical Industry