Introduction

Brassica vegetables belong to the Brassica vegetables, which includes a wide range of horticultural crops, some of them widely consumed throughout the world due to its nutritional properties. Among them, Brassica rapa L. var. rapa, commonly known as turnips, is one of the oldest cultivated vegetables used for human consumption since prehistoric times. This species has been proposed to originate from two independent centers in Asia and Europe [1], and includes a great array of crops commonly used as leafy or root vegetables in the human diet, particularly in Asian cuisine, such as Chinese cabbage, pak-choi, among many other leafy vegetables, and turnip. In Europe, B. rapa is notably popular in Portugal, Italy and Spain. In Galicia (northwestern Spain) and Portugal, B. rapa includes two main crops, turnip greens and turnip tops as vegetable products. Turnip greens are the young leaves harvested trough the vegetative period while turnip tops are the flower buds and their surrounding leaves, which are consumed before opening and while still green [2]. They are commonly consumed as a boiled vegetable, being used in the preparation of soups and stews.

Turnip leaves and buds are characterized by a peculiar bitter and pungent taste, which has been related to the content of the glucosinolate (GSL) degradation products [3]. These compounds are one of the most characteristic metabolites produced by Brassica plants. GSL are β-thioglucoside N-hydroxysulfates containing a side chain and a β-D glucopyranose moiety. The glucosinolate-myrosinase system is a characteristic defense of the Brassicaceae and related plant families [4]. GSL release different biologically active breakdown products, including nitriles, isothiocyanates, thiocyanates, oxazolidine-2-thiones, and epithionitriles after plants are wounded by insects, harvesting or other disturbances [5]. Both GSL and deriving compounds are known to have a wide range of important biological activities. In vitro and in vivo studies have reported that isothiocyanates affect many stages of cancer development including modulation of phase I and II detoxification enzymes [6–11]. Variation on the amount and pattern of GSL has been attributed to both genetic and environmental factors including growing conditions, harvest and storage, processing and meal preparation. Furthermore, GSL vary greatly with plant variety and species, tissue type (roots, leaves, flowers, seeds), and the developmental stages of the tissue [12–14]. Brassica crops are characterized by a high intraspecific morphological variation to create different morphotypes depending on local market and consumer preferences [15, 16]. This variability is reflected in many horticultural characteristics such as plant size, root formation, plant shape, plant habit or earliness. As a result, there is a large variation in the plant organ that can be consumed (roots, leaves, and shoots) and consequently, artificial selection may also have modified the profile and GSL content in each type or crop. The high genetic diversity described within B. rapa species would be useful to select highly productive varieties with an improved nutritional value. Different GSL evaluations have been performed in vegetable B. rapa crops [17, 18]. In these works, the GSL content was affected by both genetic and environment, including soil type, temperature, light, water stress, and mineral nutrient availability. As far as we know, no studies have been reported on the effects of plant attributes such as plant habit and plant earliness on that variation. This information should be useful for developing new cultivars with a desired GSL profile.

The objectives of this study were: (i) to determine the variation on GSL content and GSL profile in leaves of B. rapa varieties representing different crops, earliness, and geographical origins and (ii) to determine the effect of earliness and plant habit in the total and individual GSL content in leaves.

Materials and Methods

Plant Material

A collection of 45 populations of B. rapa subsp. rapa was evaluated in this study. These varieties were collected throughout northwestern Spain, kept at the Misión Biológica de Galicia (MBG), and they included turnips, turnip greens, and turnip tops types. The populations were evaluated at two locations in northwestern Spain: Salcedo (Pontevedra, 42º24′N, 8º38′W) and Barrantes (Ribadumia, 42º30′N, 8º46′W). Both locations are 50 m above sea level and have a humid climate with an annual rainfall of about 1,600 mm. Barrantes has a slightly alkaline soil, while Salcedo has an acid sandy loam soil type. The populations were planted in multipot-trays and seedlings were transplanted into the field at the five or six leaves stage. Planting dates were October 27th 2007 and November 7th 2007, in Salcedo and Barrantes, respectively. Varieties were evaluated in a randomized complete block design with three replications. Each experimental plot consisted of two rows with 15 plants per row. Rows were spaced 0.8 m out and plants within rows were 0.6 m apart. Cultural operations, fertilization and weed control were made according to local practices.

Varieties were classified according to their plant habit into four groups: turnip, turnip greens, turnip tops and a mix using turnip greens/turnip tops. Plant habit was established according to the field growth performance and considering the potential product that could be harvested. Those varieties developing enlarged roots as a reservoir organ and large rosette were classified as turnips (13 %), varieties without rosette growth habit and enlarged root and having a fast and vigorous growth of the leaves were classified as turnip greens (33 %), varieties without rosette growth and enlarged root habit, and having numerous and vigorous flowering stalks were classified as turnip tops (13 %) and finally, those varieties having desirable characteristics either for turnip greens or turnip tops production (40 %). On the other hand, varieties were classified according to their earliness into four groups: early, medium, late, and extra-late. Earliness was measured as days to flowering counted from planting until 50 % of plants had the first flower. Thus, the early group comprised varieties with days to flowering shorter than 120 days after planting (DAP), the medium group comprised varieties with days to flowering between 120 and 140 DAP, the late group comprised varieties with days to flowering between 140 and 160 DAP, and finally the extra-late group comprised varieties with days to flowering higher than 160 DAP.

Glucosinolate Extraction and Purification

Data were recorded on individual and total GSL content. In each replication, a sample of healthy and fresh leaves was collected from three to five plants from each plot four months after planting. The five 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 taken immediately into the laboratory where they were stored at −80 °C. Then, the leaf samples were freeze-dried and milled to a fine powder for GSL extraction. For each leaf sample, 100 mg dw was weighed and ground in a blender Janke and Kunkel, Model A10 mill (IKA-Labortechnik Staufen, Germany) for about 20 s and a two-step GSL extraction was carried out in a water bath at 75 °C to inactivate myrosinase. GSL composition was determined by HPLC following the procedure described by Padilla et al. [2].

Statistical Analysis

A combined analysis of variance across locations was made. Populations were considered as fixed factor whereas locations, replications, and the location × population interaction were considered as random factors. Comparisons of means were performed for each trait using Fisher’s protected least significant difference (LSD) at P = 0.05 [19]. The sums of squares for populations were orthogonally divided into i) components according to earliness groups (early, medium, late, and extra-late) and ii) components according to main plant habit (turnips, turnip tops, turnip greens, and a mix using turnip greens/turnip tops). For those traits for which population × location interactions were significant, individual analyses of variance at each location were accomplished. Analyses were made using the GLM procedure of SAS [20].

Results and Discussion

Variation of GSL Concentration

Seven GSL were identified in the leaves belonging to the three chemical classes: four aliphatic, two indolyl and one aromatic. Aliphatic GSL were predominant, representing 95.5 % of the total GSL content, followed by indolyl GSL (2.4 %) and aromatic GSL (2.1 %). The global mean of total aliphatic GSL was 27.74 μmol g−1 dw and the global mean of total indolic and aromatic GSL were 0.68 and 0.61 μmol g−1 dw, respectively.

Varieties were significantly different for the total GSL content as well as for all the individual GSL content, which is in agreement with other works where GSL content in B. rapa varieties was dependent upon the crops and genotypes [2, 17, 18, 21]. Differences among locations were significant for gluconapoleiferin, which is a minor GSL in this species. Total GSL content, progoitrin, gluconapoleiferin and glucobrassicin showed a significant variety × location interaction (data not shown). Therefore, individual analyses of variance for these compounds were made at each location (data not shown). Comparing the results from the individual analyses, varieties showed a similar performance across locations, i.e., varieties with the extreme values were the same at different locations for most traits. Then, magnitude changes rather than rank changes contributed to these interactions. Moreover, the variety × location interaction was not significant for gluconapin and glucobrassicanapin (data not shown), which were found to be the two main GSL. For this reason, data are presented as means of the two locations.

The total GSL content of leaves ranged from 9.7 μmol g−1 dw (for the variety MBG-BRS0471) to 47.2 μmol g−1 dw (for the variety MBG-BRS0429). This last variety along with MBG-BRS0550 and MBG-BRS0438 had the highest total GSL content and could be used as a good source of these beneficial bioactive compounds. The identities of the main GSL compounds in B. rapa reported here are consistent with those found in previous studies for this species [2, 17, 21–23] although relative content of individual GSL could change. Both gluconapin and glucobrassicanapin occurred in leaves of all varieties evaluated in our work. Gluconapin was the most abundant and represented about 84.4 % of the total GSL content. The mean value of gluconapin was 24.7 μmol g−1 dw and ranged from 7.0 to 41.3 μmol g−1 dw. The second GSL in abundance was glucobrassicanapin which represented 7.2 % of the total GSL content and had a mean value of 7.2 μmol g−1 dw. These values are consistent with those previously found by Padilla et al. [2] but they are higher than those reported by Francisco et al. [21] in a set of turnip greens varieties and lower that those presented by [17] in different cultivars of turnip rape.

GSL Variation, Earliness and Plant Habit

Varieties were grouped into four groups (early, medium, late and extra-late) according to their flowering date. The combined analysis of variance showed significant differences among groups for total GSL content and for gluconapin, glucobrassicin and gluconasturtiin (data not shown). Total GSL content of leaves in extra-late group was approximately 2-fold higher (37.3 μmol g−1 dw) than that of the early varieties (19.0 μmol g−1 dw). Early varieties also showed the lowest gluconapin and glucobrassicin contents (Table 1). The leaves of all varieties were harvested at the same date (between 120 and 140 DAP), independently of their earliness. The main reason to harvest all varieties at the same time was to reduce the effects due to different environmental factors, mainly those due to temperature, rainfall or light, which notably affect to the final glucosinolate content. Moreover, turnip greens are used by their leaves which can be harvested several months after planting and therefore they are suitable to be eaten by consumers. At this period, early varieties are close to the flowering stage, medium varieties are at the pre-flowering stage with floral buds formation starting, and late and extra-late varieties are at the vegetative stage. Two varieties, MBG-BRS0471 (early group and turnip tops habit) and MBG-BRS0498 (medium group and turnip tops habit) had the lowest GSL content with values less than 14 μmol g−1 dw (9.67 μmol g−1 dw and 13.2 μmol g−1 dw, respectively). In contrast, MBG-BRS00429 (extra-late group and turnip greens), MBG-BRS0550 (extra-late group and turnip greens) and MBG-BRS0438 (late group and turnips) had the highest content of total GSL with values higher than 43 μmol g−1 dw (47.2 μmol g−1 dw, 44.8 μmol g−1 dw and 43.6 μmol g−1 dw, respectively). MBG-BRS00429 and MBG-BRS0550 were the latest varieties (177 days and 168 days from planting to flowering, respectively). For both varieties, leaf samples were taken at the vegetative stage, about 50 days before the flowering. Differences among accessions for individual indolic and aromatic GSL quantities even though significant, were not as dramatic as that observed for most individual aliphatic GSL.

Table 1 Total and individual glucosinolate content (μmol g−1 dw) in leaves of 45 Brassica rapa varieties grown at two locations in northwestern Spain grouped by earliness and by plant habit
Full size table

Different turnip edible parts (enlarged roots, fleshy leaves, and floral buds) can be used as vegetables depending upon the consumer preferences. According to this, the B. rapa varieties evaluated were grouped into four groups according to their main plant product. Plant habit groups differed significantly for the GSL content and for gluconapin content (data not shown). The total GSL content ranged from 19.5 μmol g−1 dw for turnip green group to 36.3 μmol g−1 dw for turnip group and these differences were consistent to values found for gluconapin content, where the turnip group had the highest values (31.8 μmol g−1 dw) and the turnip top group the lowest ones (15.7 μmol g−1 dw). No significant differences were found for other aliphatic GSL and for indolic and aromatic GSL although the same tendency can be noted, with the highest values in varieties grouped into the turnip group. The Fig. 1 summarizes means for gluconapin content at each location for both earliness groups (Fig. 1a) and plant habit groups (Fig. 1b). The same trend was noted at both locations since it was previously discussed the variety × location interaction was not significant for this GSL. Turnip tops and early groups showed the lowest gluconapin content although means for Salcedo were slightly lower than those found for Barrantes.

Fig. 1
figure 1

Gluconapin content (μmol g−1 dw) in leaves of 45 Brassica rapa varieties grouped by four earliness groups (early, medium, late and extra-late) (Fig. 1a) and by four plant habit groups (turnip, turnip greens, turnip greens/turnip tops, and turnip tops) (Fig. 1b) at each location

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The relative proportion of gluconapin to the total GSL content was higher than that of glucobrassicanapin in all groups, independently their earliness or plant habit. However, Kim et al. [17] found a higher proportion of glucobrassicanapin than gluconapin to the total GSL content in medium or late types of turnip rape varieties. Glucobrassicanapin comes from the elongation of gluconapin by an elongase enzyme in the biosynthesis GSL pathway. The enzymatic activity of this enzyme is highly variable and genetically dependent, which could explain the differences between both works.

In this study, significant differences among groups according to their plant habit were found for days to flowering (data not shown). Those varieties grouped into the turnip top group were the earliest (120 days from planting to flowering) whereas those varieties classified as turnips were the latest, although they did not significantly differ from turnip greens and turnip top/turnip green groups. Differences among GSL contents found into these four groups could be related not only to plant habit but also to earliness as it was previously discussed. GSL concentrations can be influenced by various factors such as genotypic differences, pre-harvest conditions, cultural practices, stage of maturity, harvesting methods and the interactions among these factors [13, 16]. Thereby, it is important to understand the factors which determine the occurrence and GSL concentrations not only among species and crops, but also between individual parts of the same plant. In B. rapa, it has been demonstrated that GSL content is affected by different environmental factors [2, 21] and postharvest handling procedures [24]. Results reported in this work strongly suggest that variation on GSL concentrations in leaves of turnip greens is also affected by other plant attributes that were not described to date like earliness and plant habit. This information will be helpful to decide the harvest time and the plant part most recommended for consumption since it contains the highest GSL content.

In conclusion, the results obtained in this work are very promising indicating that turnip may be an easily accessible dietary source of GSL even if the choice of the harvest time and the genotype related to the main use of the crop seems to be essential to profit all their beneficial properties. Elucidation of genetic diversity among crops can provide useful information to assist plant breeders to design improved breeding strategies in order to obtain varieties rich on GSL.