Euphytica (2008) 159:111122 DOI 10.

1007/s10681-007-9463-x

Variation of glucosinolates and nutritional value in nabicol (Brassica napus pabularia group)
a Elena Cartea V ctor Manuel Rodr guez Mar s Antonio de Haro Pablo Velasco Amando Orda

Received: 30 January 2007 / Accepted: 14 May 2007 / Published online: 8 June 2007 Springer Science+Business Media B.V. 2007

Abstract Glucosinolate levels in leaves were determined in a collection of 36 varieties of nabicol (Brassica napus pabularia group) from northwestern Spain grown at two locations. Crude protein, acid detergent bre, and sensory traits were also assessed by a consumer panel. The objectives were to determine the diversity among varieties in total glucosinolate content and glucosinolate prole and to evaluate their sensory attributes in relation to glucosinolate content for breeding purposes. Eight glucosinolates were identied, being the aliphatic glucosinolates, glucobrassicanapin, progoitrin, and gluconapin the most abundant. Glucosinolate composition varied between locations although the glucosinolate pattern was not signicantly inuenced. Differences in total glucosinolate content, glucosinolate prole, protein, acid detergent bre, and avour were found among varieties. The total glucosinolate

content ranged from 1.4 mmol g1 to 41.0 mmol g1 dw at one location and from 1.2 mmol g1 to 7.6 mmol g1 dw at the other location. Sensory analysis comparing bitterness and avour with variation in glucosinolate, gluconapin, progoitrin, and glucobrassicanapin concentrations suggested that other phytochemicals are probably involved on the characteristic avour. The variety MBG-BRS0035 had high total glucosinolate, glucobrassicanapin, and gluconapin contents at both locations and could be included in breeding programs to improve the nutritional value of this vegetable crop. Keywords Brassica napus Bitterness Flavour Glucosinolates Nabicol Taste panel

Introduction Brassica napus L. includes several crops grown as fodder, oilseed, and vegetables. The most important crops are oilseed rape (B. napus L. oleifera group DC.), swede (B. napus napobrassica group L. Reichenb.), and leaf rape (B. napus L. pabularia group (DC.) Reichenb). Crops of the last group take the common name of nabicol in Spain or couve-nabic a in Portugal, where are grown as fresh green vegetables during the winter season (Cartea et al. 2005). In these areas, crops of B. napus pabularia group are highly appreciated for human nutrition.

guez M. E. Cartea (&) V. M. Rodr s P. Velasco A. Orda n Biolo gica de Department of Plant Genetics, Misio Galicia, Spanish Council for Scientic Research (CSIC), Apartado 28, 36080 Pontevedra, Spain e-mail: ecartea@mbg.cesga.es A. de Haro Department of Agronomy and Plant Breeding, Instituto de Agricultura Sostenible, Spanish Council for Scientic Research (CSIC), Alameda del Obispo s/n, 14080 Cordoba, Spain

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Most research on the chemical composition of Brassica species has been focussed on both the negative and positive effects of glucosinolates, a group of compounds derived from amino acids, which are largely responsible for the specic avour of Brassica crops (Rosa 1999). The relation between some glucosinolates with bitter taste has been already studied in Brussels sprouts (Van Doorn et al. 1998) and turnip greens (Jones and Sanders 2002; Padilla et al. 2007). Some glucosinolates and their derived products have been reported to have benecial properties on human health. Among their breakdown products, isothiocyanates have been found to have anticarcinogenic and anti-mutagenic effects (Rosa et al. 1997; Farnham et al. 2004; Smith et al. 2005). Besides these benecial effects, other glucosinolates have been associated to goitrogenic effects in animals but the evidence for goitrogenic effects in humans is scarce (Mithen et al. 2000). Detailed reviews of glucosinolate content occurring in the major Brassica crops have been reported by several authors (Fenwick et al. 1983b; Rosa et al. 1997; Rosa 1999). Concerning B. napus crops, the glucosinolate composition in oilseed rape and swedes has been reported (Carlson et al. 1981; Grifths et al. 1991; Rosa et al. 1997). However, little information is available on the nutritional quality and the glucosinolate composition of the leaves of B. napus vegetable crops, such as nabicol or couve-nabic a, widely used for human consumption. Brassica vegetables are relatively high in protein and bre contents compared to other vegetables of high water content (Rosa 1999). There is renewed interest in the use of Brassica as crops for forage because of their high protein content and high dry matter digestibility. In several European countries, such as Great Britain, France or Germany, they are used in autumn as grazing either for sheep or, occasionally, for dairy cows (Rosa 1999). In this regard, the vegetable types of nabicol grown in Galicia might be also used for fodder with a mixed use of the crop. However, it would be necessary to know other quality attributes, such as bre and protein content for this eventual use. This study has two aims: (i) to determine the composition of glucosinolates, acid detergent bre, and crude protein of nabicol varieties and (ii) to study the relation of some glucosinolates with avour and bitterness.

Material and methods Plant material The trials comprised 35 local populations from Galicia (northwestern Spain) and one commercial variety. The morphological description of these nabicol varieties have been recently described (Rodguez et al. 2005). Trials were performed during two r years (2002 and 2003) in two locations in northwestern Spain: Pontevedra (42824 N, 8838 W) and Fornelos de Montes (42820 N, 8826 W). Both locations have a humid climate with an annual rainfall of about 1600 mm in Pontevedra and about 3500 mm in Fornelos de Montes. The soil type is acid sandy loam. Varieties were evaluated in a 6 6 lattice design with three replications. Each experimental plot consisted of two rows with 10 plants per row. Rows were spaced 0.9 m apart and plants between rows 0.6 m apart. Cultural operations, fertilization and weed control were made according to local practices. Leaves were harvested four months after transplanting from the experimental elds guez et al. (2005) for glucosinodescribed by Rodr late, bre and protein analyses and the consumer panel test. Glucosinolate analysis A sample of ve healthy and fresh leaves was collected from ve plants from each variety at each location. The ve upper leaves per plant (the two next to the apical leaf along with the adjacent three leaves) were sampled because they are the tender leaves used for human consumption. Leaf samples were frozen in situ and were taken immediately into the laboratory where they were stored at 808C. Then, they were ground in liquid N2, freeze-dried and milled to a ne powder for the glucosinolate extractions. Glucosinolate composition was determined by HPLC according to Font et al. (2005). For each leaf sample, 100 mg dry wt was weighed and ground in a Janke and Kunkel, Model A10 mill (IKA-Labortechnik) for about 20 s and a two-step glucosinolate extraction was carried out in a water bath at 758C to inactivate myrosinase. In the rst step, the sample was heated for 15 min in 2.5 ml 70% aqueous methanol and 200 ml 10 mM sinigrin (2-propenyl glucosinolate) as an internal standard. A second extraction was

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conducted after centrifugation (5 min, 5000 g) by using 2 ml of 70% aqueous methanol. One ml of the combined glucosinolate extracts was pipetted onto the top of an ion-exchange column containing 1 ml Sephadex DEAE-A25 in the formate form. Desulphation was carried out by the addition of 75 ml of puried sulphatase (E.C. 3.1.6.1, type H-1 from Helix pomatia) (Sigma) solution. Desulphated glucosinolates were eluted with 2.5 ml (0.5 ml 5) Milli-Q (Millipore) ultra-pure water and analysed with a Model 600 HPLC instrument (Waters) equipped with a Model 486 UV tunable absorbance detector (Waters) at a wavelength of 229 nm. Separation was carried out by using a Lichrospher 100 RP-18 in Lichrocart 125-4 column, 5 mm particle size (Merck). HPLC solvents and gradient followed the ISO protocol (ISO Norm 1992). The HPLC chromatogram was compared to the desulpho-glucosinolate prole of three certied reference materials recommended by U.E. and ISO (CRMs 366, 190 and 367). The amount of each individual glucosinolate present in the sample was calculated with an internal standard, and expressed as mmol g1 of dry wt. The total glucosinolate content was computed as the sum of all the individual glucosinolates present in the sample. Data were corrected for UV response factors for different types of glucosinolates (ISO Norm 1992). Protein and bre analysis For the protein and bre analysis, a sample of ve fresh leaves was collected from ve plants from each variety at each location. Leaves (lamina and petioles) were detached from the stems and both were ovendried at 608C for at least 48 h to determine dry matter concentration. Dried samples were ground (1 mm screen) for subsequent laboratory analysis. The ADF content was analysed by Van-Soest method and leaf protein concentration by Kjedhal method following the protocols of AOAC (2000). Consumer panel

The taste panel consisted of 12 members, all of them regular consumers of vegetable brassica crops. Hardness, bitterness, brousness and avour were scored on a continuous scale from 1 to 5. For bitterness and brousness, a rating of 1 was considered slight and 5 high; for hardness a rating of 1 was considered tender and 5 hard. Finally, for avour, a rating 1 was very bad and 5 very good. The consumer panel evaluation lasted 36 days, as no more than 6 samples per day could be tasted. Statistical analyses The experiment was set up in a completely random design and glucosinolate content per plant analyzed by individual analysis of variance. Varieties were considered as xed effects. Comparisons of means among varieties were performed for each trait using the Fishers protected least signicant difference (LSD) at P = 0.05 (Steel et al. 1997). A combined analysis of variance across locations was carried in a randomized block design with each location as a block and using the location variety interaction as the experimental error. Varieties were considered as xed effects and locations as random effects. For sensory characteristics, analyses of variance were performed for each trait and combined over locations according to a completely randomized block design and using the GLM procedure of SAS (SAS Institute 2000). Replications and locations were considered as random factors while varieties were considered as xed factors. Comparison of means among varieties were made by Fishers protected signicant difference (LSD) at P = 0.05 (Steel et al. 1997). All analyses were performed with the SAS statistical package (SAS Institute 2000). Phenotypic correlation coefcients were estimated among some glucosinolates with bitterness and avour (Johnson et al. 1955).

Results and discussion A sample of healthy and fresh leaves was collected from ve plants from each plot at each location. Twenty ve to thirty tender leaves from each plot were harvested according to the maturity cycle of each variety at the optimum time for consumption and boiled for 2 min in 1 dm3 of water with 5 g salt. Variability of glucosinolates in B. napus collection Eight glucosinolates belonging to the three chemical classes were identied in the leaves of nabicol

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114 Table 1 List of glucosinolates found in leaves of 36 varieties of nabicol evaluated at two locations in northwestern Spain Glucosinolate Common name Abbreviation

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Aliphatic glucosinolates (derived from methionine) 4-Pentenyl 2-Hydroxy-3-butenyl 3-Butenyl 5-Methylsulnylpentyl Indol-3-ylmethyl 1-Methoxyindol-3ylmethyl 4-Methoxyindol-3ylmethyl 2-Phenylethyl Glucobrassicanapin Progoitrin Gluconapin Glucoalyssin Glucobrassicin Neoglucobrassicin 4-Methoxygluco brassicin Gluconasturtiin GBN PRO GNA GAL GBS NGBS MeGBS

Indolyl glucosinolates (derived from tryptophan)

Aromatic glucosinolates (derived from phenylalanine) GST

varieties: four aliphatic, three indolyl, and one aromatic (Table 1) Three glucosinolates were detected in all varieties: glucobrassicanapin, progoitrin, and glucobrassicin. Aliphatic glucosinolates were predominant, representing the 90% of total glucosinolate content. Glucobrassicanapin was the most abundant (41% of total glucosinolate content in location 1 and 54% in location 2) followed by progoitrin and gluconapin (Fig. 1). Smaller amounts of glucobrassicin, glucoalyssin, gluconasturtiin, neoglucobrassicin, and 4-methoxyglucobrassicin were also detected. Gluconapoleiferin and 4-hydroxyglucobrassicin were found in some varieties analysed in location 2 although in low concentrations. Despite differences on glucosinolate content between locations, the glucosinolate pattern found at each location was very similar. Previous studies have

reported a similar glucosinolate pattern in leaves of fodder rapes (Rosa 1999) and vegetable types (Rosa et al. 1996) and in seeds of oilseed rape (Fenwick et al. 1983b, Li et al. 1999) and nabicol varieties (De Haro et al. 1995). The combined analysis of variance showed significant differences for total glucosinolate content among locations, varieties, and the location variety interaction (Table 2). The average glucosinolate content detected in leaves collected from location 1 was higher (17.1 mmol g1 dw) than those collected from location 2 (3.4 mmol g1 dw) (Table 3). Environmental factors, such as temperature, water stress and soil type are known to exert a signicant effect on glucosinolate content (Fenwick et al. 1983b; Rosa et al. 1996; Rosa et al. 1997). Soil differences across locations could be the cause of the signicant differences between locations and location variety interaction for all glucosinolates. Differences in the soil parameters were proved by edaphic analyses showing that the soil pH was strongly acid in location 2. The highest glucosinolate content occurred in location 1, with the highest soil pH, suggesting some type of relation between glucosinolate content and soil effect. Besides the inuence of soil composition, Brassica crops grown under cool temperatures and abundant rainfall seem to have lower glucosinolate content (Rosa et al. 1997). In location 2, the temperature was low during the early development stages of growth and total rainfall was more abundant than in location 1. Differences between locations were found for aliphatic, indolyl, and aromatic glucosinolates (Table 2). In our case, and under stress conditions for nabicol crop such as previously described in location 2, the pathway for glucosinolate biosynthesis

Fig. 1 Percentage of glucosinolates in leaves of nabicol varieties sampled at two locations. GBN: Glucobrassicanapin; PRO: Progoitrin, GNA: Gluconapin, GAL: Glucoalyssin, GBS: Glucobrassicin, NGBS: Neoglucobrassicin, MeGBS: 4Methoxyglucobrassicin, GST: Gluconasturtiin

60 50 40 30 20 10 0 GBN PRO GNA GAL GBS

location 1

location 2

NGBS

MeGBS

GST

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Table 2 Mean squares of the analysis of variance within each site and mean squares of the combined analysis of variance for the total glucosinolate content in the nabicol varieties evaluated at two locations in northwestern Spain Traits Location 1 Variety Total glucosinolate Aliphatic GBN PRO GNA GAL GNL Indolyl GBS NGBS MeGBS Aromatic GST Degrees of freedom 0.30** 35 0.03 134 0.04 32 0.03 79 0.45** 1 0.15** 35 0.15** 32 3.09* 0.24** 0.04* 0.37 0.04 0.01 0.10* 0.06** &0 0.05 0.01 &0 30.62* 1.68** 1.08* 1.38** 0.12* 0.02** 1.35** 0.17** 0.02* 103.61** 57.01** 14.99** 7.37** 11.92 5.51 2.06 1.15 2.44** 0.66* 0.24** 0.20* 0.06** 0.64 0.35 0.05 0.04 0.02 2206.32** 1067.61* 280.05** 53.47* 46.48** 24.26** 5.81** 3.04** 33.87** 19.58* 5.21* 2.27** 548.8** Error 39.3 Location 2 Variety 8.5** Error 3.1 Combined analysis Location 14452.8** Variety 243.0** Location Variety 181.7**

*, ** signicant at P  0.05 or 0.01, respectively GBN: Glucobrassicanapin; PRO: Progoitrin; GNA: Gluconapin; GAL: Glucoalyssin; GNL: Gluconapoleiferin; GBS: Glucobrassicin; NGBS: Neoglucobrassicin; MeGBS: 4-Methoxyglucobrassicin; GST: Gluconasturtiin

could have been modied. The biosynthesis of the individual glucosinolates in Brassica is under genetic control by means of enzymes via different side chain elongation. These enzymes could be modied depending on specic environmental conditions, increasing or decreasing some glucosinolates (Giamoustaris and Mithen 1996). The total glucosinolate content ranged from 1.4 mmol g1 to 41.0 mmol g1 dw in location 1 and from 1.2 mmol g1 to 7.6 mmol g1 dw in location 2 (Table 3). These contents are lower than those found in other B. napus crops (Carlson et al. 1981; Grifths et al. 1991; Rosa et al. 1997). Glucobrassicanapin, the main glucosinolate, showed an average of 7.08 and 1.87 mmol g1 dw in locations 1 and 2, respectively while the other glucosinolates had values lower than 1 mmol g1 dw in the last location (Table 3). Varieties were signicantly different for the total glucosinolate content and for all individual glucosinolates, except for gluconasturtiin and 4-methoxyglucobrassicin at location 2 (Table 2). Due to benecial effects of isothiocyanates derived from glucobrassicanapin and gluconapin on human health

(Mithen et al. 2003; Rose et al. 2005), the most promising varieties for future breeding purposes would be those with the highest total glucosinolate content. The variety MBG-BRS0029 had the highest total glucosinolate content (41 mmol g1 dw) in location 1 (Fig. 2A, Table 4) and the highest progoitrin and gluconapin contents (16.5 mmol g1 dw and 8.8 mmol g1 dw, respectively) at this location (Table 4). Other varieties with high total glucosinolate content at location 1 were MBG-BRS0035 and MBG-BRS0374 (Fig. 2A). This last variety also had the highest glucobrassicanapin content (16.3 mmol g1 dw). In location 2, varieties showed a low glucosinolate content. The variety MBG-BRS0329 had the highest total glucosinolate content (7.6 mmol g1 dw) (Fig. 2A, Table 3) and the highest glucobrassicanapin and progoitrin contents (4.1 mmol g1 dw and 2.3 mmol g1 dw, respectively) (Table 4). Other varieties with high total glucosinolate content at this location were MBG-BRS0035 and MBG-BRS0337 (Fig. 2A). Regarding both locations MBG-BRS0035 had the highest total glucosinolate content and MBG-BRS0092 the lowest glucosinolate content. The commercial variety MBG-BRS0373 had

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116 Table 3 Mean (mmol g1 dw) and range for total and individual glucosinolate content in leaves of 36 varieties of nabicol evaluated at two locations in northwestern Spain Location 1 Mean Range t-GSL Aliphatic GBN PRO GNA GAL GNLa Indolyl GBS NGBS Aromatic GST 0.23 00.86 0.16 00.36 0.04 0.94 0.24 0.261.09 0.041.09 00.38 0.23 0.06 &0 0.030.98 0.12 00.61 0.04 7.08 4.94 2.45 1.09 &0 0.6216.26 1.87 0.3816.48 0.74 0.058.79 05.12 0.26 0.10 0.08 1.014.09 0.67 0.012.32 0.46 01.39 01.41 00.67 0.28 0.21 17.1 1.441.0 Location 2 Mean Range 3.4 1.237.55 1.22 LSD (5%)

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MeGBSa 0.14

t-GSL: total glucosinolate content, GBN: Glucobrassicanapin, PRO: Progoitrin, GNA: Gluconapin, GAL: Glucoalyssin, GNL: Gluconapoleiferin, GBS: Glucobrassicin, NGBS: Neoglucobrassicin, MeGBS: 4-Methoxyglucobrassicin, and GST: Gluconasturtiin Values were negligible for Gluconapoleiferin at location 1 and for 4-methoxyglucobrassicin at location 2
a

content is considered inconvenient for food purposes. However, nabicol varieties included in this study displayed low progoitrin contents. Among the glucosinolates surveyed in Brassica crops, of particular interest is glucoraphanin, which has benecial effects for human diet because of its role as a cancer protecting agent (Rosa et al. 1997; Farnham et al. 2004; Smith et al. 2005). The glucoraphanin was absent in the collection evaluated whereas progoitrin and gluconapin were abundant which could offer future perspectives to modify the glucosinolate composition because glucoraphanin, progoitrin and gluconapin are in the same pathway of the biosynthesis of the aliphatic glucosinolates (Giamoustaris and Mithen 1996). Biosynthesis of gluconapin requires a functional allele at the Gsl-alk locus that converts glucoraphanin to gluconapin. Down-regulation of Gsl-Alk could produce Brassica varieties lacking the antinutrient progoitrin and would simultaneously produce plants accumulating glucoraphanin as a source of anticarcinogens. Li and Quiros (2003) obtained transformed Arabidopsis plants with reduced concentration of glucoraphanin, which was converted into gluconapin. Nutritional and sensory traits Regarding the nutritional value, crude protein and acid detergent bre are important parameters in traditional farming systems, where residual postharvest leaves are used as feeding. Signicant differences (P  0.01) were found between locations for both traits. In location 1, varieties had more protein (28.6% dw) and less ADF (16.7% dw) than in location 2 (13% dw of protein and 19.6% dw of ADF) (Table 5). Previous works agree with these results, since high temperatures and low soil humidity increase protein content and reduce bre content in Brassica fodder crops (Wiedenhoeft and Barton 1994; Rosa and Heaney 1996). Since the location variety interaction was not signicant for crude protein and ADF and varieties differed significantly for both traits, means for each variety are shown across locations. The commercial nabicol variety showed intermediate values for both ADF and crude protein. The average crude protein found in the nabicol leaves ranged from 16.5% to 25.5% dw (Table 5) and this content was similar to the values

low total glucosinolate content in both locations. Seeds of commercial variety came from Portugal where they were bought as couve-nabic a but they presumably correspond to a rapeseed variety. Because of the adverse effects of glucosinolates on animal nutrition, current commercial varieties of oilseed rape have very low glucosinolate levels in seeds, less than 30 mmol g1. However, no recommended values for glucosinolates have been described in vegetable Brassica crops. Varieties displayed the standard glucosinolate prole, except for MBG-BRS0029, MBG-BRS0037, MBG-BRS0041, MBG-BRS0048, MBG-BRS0061, MBG-BRS0113, and MBG-BRS0356 in location 1, which had relatively moderate progoitrin levels (Fig. 2B, Table 4). High levels of progoitrin have been implicated on goitrogenic effects in animals causing thyroid hypertrophy (Rosa et al. 1997) although no evidence on its possible involvement in goitre have been demonstrated in humans (Mithen et al. 2000). The use of varieties with high progoitrin

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mol/g

117

A 45
40 35 30

Loc 1

Loc 2

flavour

bitterness
4

3 25 20 2

15
10 5 0 BRS0014 BRS0028 BRS0029 BRS0034 BRS0035 BRS0037 BRS0039 BRS0041 BRS0044 BRS0048 BRS0054 BRS0056 BRS0061 BRS0063 BRS0065 BRS0068 BRS0073 BRS0079 BRS0085 BRS0087 BRS0090 BRS0092 BRS0105 BRS0107 BRS0110 BRS0113 BRS0131 BRS0134 BRS0329 BRS0333 BRS0337 BRS0346 BRS0356 BRS0373 BRS0374 BRS0378 0 1

B mol/g
18 5

Loc 1
16 14 12

Loc 2

flavour

bitterness
4

3 10 8 2 6 4 2 0 BRS0014 BRS0028 BRS0029 BRS0034 BRS0035 BRS0037 BRS0039 BRS0041 BRS0044 BRS0048 BRS0054 BRS0056 BRS0061 BRS0063 BRS0065 BRS0068 BRS0073 BRS0079 BRS0085 BRS0087 BRS0090 BRS0092 BRS0105 BRS0107 BRS0110 BRS0113 BRS0131 BRS0134 BRS0329 BRS0333 BRS0337 BRS0346 BRS0356 BRS0373 BRS0374 BRS0378 0 1

Fig. 2 (A) Bitterness, avour and total glucosinolate content expressed as (mmol/g dw) in 36 varieties of Brassica napus evaluated by a consumer panel and sampled at both locations (B) Bitterness, avour and progoitrin content expressed as (mmol/g dw) in 36 varieties of Brassica napus evaluated by a

consumer panel and sampled at both locations. Bitterness expressed as rating scale from 1 = slight to 5 = high. Flavour expressed as rating scale from 1 = very bad to 5 = very good. Varieties MBG-BRS0056, MBG-BRS0061, and MBGBRS0378 were not sampled at location 2

reported on other Brassica forage crops such as kales (Rosa 1999). Brassica crops are relatively low in bre and are readily digested, providing good concentrations of energy for ruminants. The combined mean over locations was 18.1% dw with a range between 14.9% and 21.31% dw (Table 5). The average ADF content was similar to the values found in different B. oleracea crops (Rosa 1999) and it would be consider appropriate for fodder use. Additionally, dietary bre and protein quality are some of the arguments used to increase Brassica consumption. In this context, values obtained in this work for ADF and crude protein concentration would allow us to use this crop for edible leaves either as food and/or feed. For sensory traits, varieties grown in location 1 had a best acceptability by taste panel (best avour,

less brousness, and less hardness) than those grown in location 2. Average variety values were 4.1, 2.1, and 2.5 in location 1 and 3.2, 3.6, and 3.1 in location 2 for avour, hardness, and brousness, respectively. Location 1 represents the environmental conditions where crop is well adapted while location 2 is an inland location where climate is more severe and environmental conditions are not suitable for nabicol crop. Bitterness was the only sensory trait for which locations showed no signicant differences (data not shown), suggesting that this trait is more related to other factors such as glucosinolate content than to the climatic conditions (Van Doorn et al. 1998). Varieties were signicantly different for avour (P < 0.05), being MBG-BRS0056, MBG-BRS0041, MBGBRS0337, and MBG-BRS0333 the best varieties with a value near to 4 in a scale from 1 to 5. The

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Table 4 Total and individual leaf glucosinolate content (mmol g1 dw) of 36 varieties of nabicol grown at two locations in northwestern Spain Variety (MBG-) BRS0014 BRS0028 BRS0029 BRS0034 BRS0035 BRS0037 BRS0039 BRS0041 BRS0044 BRS0048 BRS0054 BRS0056 BRS0061 BRS0063 BRS0065 BRS0068 BRS0073 BRS0079 BRS0085 BRS0087 BRS0090 loc 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 t-GSL 20.26 1.48 16.28 3.84 40.99 2.04 7.83 2.99 34.55 6.18 8.04 5.84 22.58 2.59 4.56 3.63 22.61 3.28 11.37 3.29 26.72 1.23 4.02 -a 3.21 -a 24.05 2.97 4.62 3.25 18.00 1.59 7.53 1.51 7.42 4.15 11.99 2.59 20.00 2.75 15.55 4.26 GBN 7.88 1.06 8.71 1.75 11.92 1.24 3.49 1.29 16.13 3.71 2.12 2.97 8.74 1.46 1.27 2.03 8.63 2.00 4.40 1.50 9.41 1.01 1.92 0.83 9.69 1.95 2.11 2.26 9.81 1.15 2.84 1.28 6.62 1.95 5.12 1.17 9.85 1.78 8.07 2.43 PRO 5.39 0.14 3.83 1.02 16.48 0.30 2.70 0.52 11.23 1.20 3.68 1.83 6.69 0.56 1.93 0.48 5.95 0.52 4.45 1.30 6.26 0.01 0.80 1.78 5.89 0.60 1.29 0.50 4.64 0.19 2.56 0.11 0.38 1.23 3.54 0.32 5.49 0.28 3.43 1.15 GNA 3.16 0 2.62 0.19 8.79 0.08 1.26 0 4.78 0.67 1.49 0.77 3.57 0.10 0.85 0.09 4.01 0.31 1.80 0.09 4.12 0 0.96 0.05 3.39 0.21 0.91 0.25 2.66 0 1.56 0.07 0.16 0.17 1.48 0 2.81 0.25 2.58 0.53 GAL 2.27 0 0.34 0.08 1.91 0 0.09 0 0.36 0 0.34 0 1.77 0 0 0.24 1.75 0.08 0.00 0.07 5.12 0 0 0.03 2.74 0 0 0 0.09 0 0.25 0 0 0 1.07 0 0.69 0 0.11 0 GBS 0.94 0.18 0.35 0.43 1.17 0.16 0.19 0.28 1.69 0.13 0.30 0.05 1.29 0.16 0.25 0.44 1.63 0.37 0.15 0.20 1.09 0.06 0.15 0.18 1.80 0.08 0.14 0.08 0.56 0.04 0.20 0.04 0.13 0.61 0.53 0.29 0.56 0.19 0.33 0.07 GST 0.46 0.10 0.28 0.30 0.23 0.13 0 0.29 0 0.20 0 0.17 0 0.22 0.08 0.32 0 0.11 0 0.22 0.14 0.15 0.05 0.05 0.09 0.11 0 0.11 0 0.17 0 0 0 0.16 0.02 0.36 0.40 0.23 0.73 0.07 NGBS 0.18 0 0.15 0.07 0.42 0.12 0.04 0.61 0.20 0.09 0.04 0.05 0.31 0.08 0.07 0.01 0.37 0 0.45 0 0.41 0 0.08 0.06 0.32 0.02 0.10 0.05 0.13 0.05 0.06 0.01 0.04 0.03 0.11 0.45 0.10 0.03 0.20 0 MeGBS 0 0 0 0 0.07 0.01 0.06 0 0.20 0 0.07 0 0.21 0.01 0.10 0.01 0.26 0 0.12 0 0.16 0 0.08 0.23 0.12 0 0.08 0 0.11 0 0.06 0 0.09 0 0.13 0.01 0.10 0 0.10 0.01

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Euphytica (2008) 159:111122 Table 4 continued Variety (MBG-) BRS0092 BRS0105 BRS0107 BRS0110 BRS0113 BRS0131 BRS0134 BRS0329 BRS0333 BRS0337 BRS0346 BRS0356 BRS0373b BRS0374 BRS0378 LSD (5%) loc 1 LSD (5%) loc 2 loc 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 t-GSL 3.36 1.59 28.48 3.84 26.81 3.22 26.88 3.47 10.66 3.23 22.80 1.67 25.90 2.52 24.33 7.55 2.21 3.78 6.49 6.24 26.31 5.91 22.08 4.79 4.16 1.92 32.34 3.89 19.97 -a 8.14 3.12 GBN 1.37 1.21 13.70 2.28 14.75 1.69 9.03 1.59 2.91 1.18 7.60 1.19 8.76 1.76 9.45 4.09 0.62 1.98 2.17 3.26 12.34 2.12 6.40 2.58 2.40 1.36 16.26 1.31 7.54 4.49 1.42 PRO 1.10 0.20 7.70 0.62 5.55 1.10 7.97 1.20 4.07 0.95 6.89 0.38 8.31 0.42 6.67 2.32 0.54 0.26 1.44 1.14 6.69 1.09 9.75 1.00 0.62 0.37 7.31 1.20 4.79 3.05 1.04 GNA 0.41 0.04 2.79 0.21 3.87 0.16 4.16 0.13 1.68 0.09 3.00 0.07 4.49 0.30 1.92 0.55 0.20 1.40 0.63 0.80 2.32 0.35 1.84 0.45 0.40 0 4.11 0.25 3.31 1.87 0.42 GAL 0 0 1.25 0 0.97 0 3.04 0 1.15 0.39 0.95 0 1.85 0 1.88 0 0 0 0.11 0.71 2.86 1.41 0.54 0.45 0 0 2.41 0 2.98 1.39 0.31 0.79 0.38 GBS 0.20 0.07 2.35 0.23 0.92 0.03 1.60 0.29 0.45 0.37 3.26 0.03 1.62 0.06 2.18 0.24 0.29 0.04 0.80 0.35 1.13 0.77 2.81 0.19 0.26 0.12 1.33 0.98 0.83 GST 0.13 0.07 0.58 0.35 0.36 0.21 0.60 0.17 0.20 0.18 0.61 0 0.43 0 0.86 0.29 0.13 0.10 0.27 0 0.63 0.19 0.45 0.13 0.03 0.06 0.56 0.13 0.19 0.21 NGBS 0.08 0 0.05 0.15 0.28 0.02 0.10 0.08 0.14 0.04 0.22 0 0.14 0.02 1.09 0.06 0.22 0 1.00 0 0.15 0 0.14 0 0.24 0.01 0.24 0.02 0.21 0.25 0.16

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MeGBS 0.08 0 0.06 0 0.12 0 0.38 0.01 0.07 0 0.27 0 0.30 0 0.32 0 0.21 0 0.08 0 0.19 0 0.15 0 0.21 0 0.12 0 0.12 0.12 -

t-GSL: total glucosinolate content, PRO: Progoitrin, GAL: Glucoalyssin, GNA: Gluconapin, GBN: Glucobrassicanapin, GBS: Glucobrassicin, GST: gluconasturtiin, NGBS: Neoglucobrassicin, MeGBS: 4-Methoxyglucobrassicin
a b

Varieties no evaluated in location 2 Commercial nabicol variety

location variety interaction was signicant (P < 0.01) for bitterness and brousness. Accessions were signicantly different for these traits in the location 1, with MBG-BRS0028, MBG-BRS0037, MBG-BRS0374, and MBG-BRS0356 signicantly less bitter.

Bitter taste foods are frequently disliked and this is one reason for the low acceptability of some Brassica crops. Most nabicol varieties showed less bitterness (less than mean value of 3.0 in a scale from 1 to 5) than other B. brassica crops as Brussels sprouts, turnip greens or turnip tops (Padilla et al. 2007) along with a

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120 Table 5 Means and standard deviation for acid detergent bre (ADF) and crude protein of 36 varieties of nabicol grown at two locations in northwestern Spain Varieties ADF (% of dry weight) 19.50 1.85 17.56 1.90 15.97 2.60 19.01 3.56 16.43 2.86 19.22 2.57 16.97 0.61 18.57 3.92 18.69 2.24 21.28 0.50 16.01 1.70 17.47 0.75 16.89 2.10 16.08 2.01 18.12 1.72 16.84 2.78 17.19 2.53 16.97 3.63 16.49 0.86 14.94 0.76 19.33 4.43 17.95 0.79 19.52 0.87 17.12 4.13 16.95 0.92 16.81 0.30 16.57 0.52 16.99 0.01 17.18 0.03 16.98 2.23 17.10 0.70 17.99 0.16 15.41 1.85 16.21 1.42 17.83 0.24 17.16 2.07 4.025 Crude protein

Euphytica (2008) 159:111122

MBG-BRS0014 MBG-BRS0028 MBG-BRS0029 MBG-BRS0034 MBG-BRS0035 MBG-BRS0037 MBG-BRS0039 MBG-BRS0041 MBG-BRS0044 MBG-BRS0048 MBG-BRS0054 MBG-BRS0056 MBG-BRS0061 MBG-BRS0063 MBG-BRS0065 MBG-BRS0068 MBG-BRS0073 MBG-BRS0079 MBG-BRS0085 MBG-BRS0087 MBG-BRS0090 MBG-BRS0092 MBG-BRS0105 MBG-BRS0107 MBG-BRS0110 MBG-BRS0113 MBG-BRS0131 MBG-BRS0134 MBG-BRS0329 MBG-BRS0333 MBG-BRS0337 MBG-BRS0346 MBG-BRS0356 MBG-BRS0373 MBG-BRS0374 MBG-BRS0378 LSD (5%)

20.41 9.61 24.73 8.58 20.75 15.34 20.94 12.25 22.98 15.59 21.54 10.97 23.02 12.13 20.40 8.06 24.22 9.74 18.24 8.58 18.18 13.47 21.12 7.89 19.19 9.77 18.91 14.56 18.22 12.13 21.99 12.88 20.50 8.07 17.28 9.08 22.11 13.42 19.80 12.45 20.77 9.09 20.74 13.10 19.78 12.91 21.81 14.13 21.44 14.66 20.29 10.91 22.03 15.24 25.53 7.74 17.92 14.83 21.11 8.20 18.39 9.35 21.35 13.22 23.63 3.35 20.29 2.56 22.14 11.40 16.53 10.14 6.382

vegetables while the bitterness associated with progoitrin is due to its decomposition product, the 5vinyl-oxazolidine-2-thione (OZT) (Fenwick et al. 1983a). Descriptive sensory analysis comparing avour attributes with variation in glucosinolate concentration have been performed in turnip greens (Padilla et al. 2007), broccoli and cauliower (Schonhof et al. 2004), and in Brussels sprouts (Van Doorn et al. 1998) but not in nabicol varieties. The content of sinigrin and progoitrin was positively correlated with bitterness and negatively correlated with taste preference in Brussels sprouts (Van Doorn et al. 1998). The relation between avour and bitterness with the total glucosinolate and progoitrin contents are shown in Figs. 2A and B, respectively. Total glucosinolate content and progoitrin content appears to be related to avour. MBG-BRS0029 with the highest glucosinolate and progoitrin contents in location 1 had a bad avour in a scale from 1 to 5 whereas MBG-BRS0333 with the lowest levels of glucosinolates in location 1 had a good avour. However, no relation was found between bitterness and the levels of glucosinolates and progoitrin; varieties with high glucosinolate and progoitrin contents were as bitter as varieties with low glucosinolate and progoitrin contents (Figs. 2A and 2B). Because gluconapin, glucobrassicanapin, and progoitrin were the major glucosinolates found in this work, simple correlation coefcients between these three glucosinolates with avour and bitterness were calculated (Table 6). Flavour was related to high glucosinolate content, but no to high gluconapin, glucobrassicanapin or progoitrin contents. Bitterness was not related to high glucosinolate contents, suggesting that these compounds and their breakdown products are not the primary determinants of the bitter taste and aroma of this vegetable and
Table 6 Simple correlation coefcients among total and some specic glucosinolates and two quality traits on 36 varieties of nabicol grown in two locations in northwestern Spain t-GSL Flavour Bitterness 0.47 ** 0.11 PRO 0.35 * 0.13 GBN 0.36 * 0.10 GNA 0.21 0.13

good avour. Isothiocyanate compounds derived from sinigrin, glucobrassicanapin, and gluconapin contribute to the bitter avour of cruciferous

*, ** signicant at P  0.05 or 0.01, respectively t-GSL = Total glucosinolate content; PRO = Progoitrin; GBN = Glucobrassicanapin; GNA = Gluconapin

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Euphytica (2008) 159:111122

121 fellowship from the Ministry of Science and Technology from Spain.

therefore, other glucosinolates or other phytochemicals are involved in the characteristic bitterness of this Brassica species. Because both low palatability and bitterness are not such critical in nabicol crops compared with other Brassica vegetables, it is probably difcult to detect relations between bitterness and high glucosinolate contents. Although nabicol varieties look quite uniform on the basis of molecular data (Cartea et al. 2005; Soengas et al. 2006) and agronomical characteristics guez et al. 2005), variation on glucosinolate (Rodr levels have been observed. With the application of appropriate breeding procedures, new nabicol varieties of acceptable agronomic type could be developed with higher glucosinolate contents than those found in varieties currently grown as food. The effect on other plant characters such as disease and pest resistance should be monitored to ensure that they are not adversely affected because leaf glucosinolate content can inuence feeding behaviour of insect pest (Giamoustaris and Mithen 1996). Because the attack by insect pests to Brassica crops is important in northwestern Spain (Picoaga et al. 2003), it would be interesting to manipulate the glucosinolate levels to improve pest and disease resistance. The interaction between plant glucosinolates and disease and pest resistance is complex and need considerable further study. As conclusion, nabicol varieties from northwestern Spain differed greatly in glucosinolate contents, ADF, crude protein and avour which enable us to select some interesting varieties to obtain improved nabicol varieties with different glucosinolate content that could be used for different purposes. The glucosinolate composition was signicantly inuenced by the environment although the glucosinolate pattern did not vary. The variety MBG-BRS0035 had high total glucosinolate content at both locations and could be included in breeding programs to improve the nutritional value of this vegetable crop. This is the rst study about the nutritional value of nabicol crops and it provides an interesting and valuable material for further breeding programs.
ndez, E. Santiago Acknowledgements We thank G. Ferna and R. Abilleira for work laboratory. This work has been supported by the Projects AGL 2003-01366 and AGL 2006n 04055 of the Spanish Government and Excma. Diputacio guez acknowledges a Provincial De Pontevedra. V.M. Rodr

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